42nd Scientific MeetingBSRM-VFS Joint Scientific Meeting22-23 November 2019, Antwerp Programme & abstract book

BEL/NONF/1117/0058 (1) (Approved November 2019)

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BEL/NONF/1117/0058 (1) (Approved November 2019)

NOTE THESE DATES FOR YOUR DIARY

43th BSRM Scientific Meeting27 March 2020

Crowne Plaza Brussels – Le Palace

44th BSRM Scientific Meeting27-28 November 2020

Dolce La Hulpe, Brussels

Dear Colleague,

We are very pleased on behalf of the BSRM and VFS Board to welcome you in Antwerp at the occasion of the 42nd BSRM Scientific Meeting, BSRM-VFS Joint Scientific Meeting, Friday and Saturday, 22-23 November 2019.

Christophe Blockeel Bernard RoelenBSRM President VFS President

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10:00 - 10:30 Registration10:30 - 10:45 Welcome by the BSRM President, Christophe Blockeel, UZ Brussel Welcome by the VFS President, Bernard Roelen, Utrecht University, the Netherlands

10:45 - 12:30 Chairs: Carla Tomassetti, UZ Leuven - Kathrin Fleischer, Radboud UMC Nijmegen10:45 - 11:15 (Re)production, a never ending story… Peter Bols, UAntwerpen Free communications11:15 - 11:27 FC-01 Mechanically disconnected mouse preantral and antral follicles lead to developmentally competent oocytes following follicle culture Anamaria Cristina Herta, Vrije Universiteit Brussel, Follicle Biology Laboratory, Brussels11:27 - 11:39 FC-02 Heterozygous mutations in PLCZ1 are associated with fertilization failure after ICSI Ramesh Reddy Guggilla, Ghent-Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent11:39 - 11:51 FC03 Increase in live birth rates after non-invasive oocyte selection in a prospective interventional study in 108 ICSI patients Inge Van Vaerenbergh, Follicle Biology Laboratory, Vrije Universiteit Brussel, Brussels11:51 - 12:03 FC04 Single-blastocyst genome-wide bisulfite sequencing for assessing the impact of in vitro follicle culture, superovulation and age on mouse embryo development Laura Saucedo-Cuevas, Follicle Biology Laboratory (FOBI), UZ Brussel, Vrije Universiteit Brussel, Brussels12:03 - 12:15 FC05 Rebiopsy in preimplantation genetic testing (PGT) can provide a genetic result and maximizes the number of transferable blastocysts for patients Sofie Ellegiers, Department for Reproductive Medicine, Ghent Fertility And Stem cell Team (G-FAST), Ghent University Hospital, Ghent, Belgium12:15 - 12:27 FC06 Trophectoderm Specification: Compaction, Polarisation and Inner and Outer Cells occur simultaneously in the Human Preimplantation Embryo Andrej Demtschenko, Research Group Reproduction and Immunology and Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels

12:30 - 13:30 Lunch Letrozole in ART Chairs: Frauke Vanden Meerschaut, UZ Gent - Charlotte Cheruy, Hôpital de Libramont 13:30 - 14:00 Ovulation induction in PCOS Robert Casper, TRIO Fertility and University of Toronto, Toronto, Canada 14:00 - 14:30 The role of Letrozole in fertility treatment in breast cancer patients Ornit Goldrat, Hôpital Erasme

14:30 - 15:00 Sponsored symposium Gedeon Richter: The Future of fertility by age banking Christine Wyns, UCL Saint-Luc

15:00 - 15:30 Coffee break

How to choose the right one? Chairs: Bernard Roelen, Utrecht University, the Netherlands - Bjorn Menten, UGent15:30 - 16:00 Genetic screening prior to conception: to what extend? Saskia Bulk, CHU Liège, Genetics and cytogenetics - Aimé Lumaka, CHU Liège, Genetics and cytogenetics16:00 - 16:30 DNA methylation in preimplantation embryos Mellissa Mann, Magee-Womens Research Institute / University of Pittsburgh, USA16:30 - 17:00 Sponsored symposium Merck: Differentiating LH from hCG action: pathophysiology and clinical relevance Manuela Simoni, Università degli Studi di Modena e Reggio Emilia, Modena, Italy

Friday, 22 November 2019

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BSRM Honorary Member 201917:00 - 17:10 Honorary member 2019 Pierre Vanderzwalmen, IVF center CHIREC, Belgium - Nextclinics, IVF Center Prof. Zech, Austria - Faculty of Veterinary Medicine, Embryology Unit, University of Liège, Belgium Introduced by Jacqueline Greindl, CHIREC, Braine L’Alleud 17:10 - 17:30 Of animals and men: across species mammalian embryology for improving human IVF skills and knowledge Pierre Vanderzwalmen, IVF center CHIREC, Belgium - Nextclinics, IVF Center Prof. Zech, Austria - Faculty of Veterinary Medicine, Embryology Unit, University of Liège, Belgium

For those who like:17:30 - 18:15 Medicine and machines: will AI take over ART? Cristina Hickman, Imperial College London, UK

19:30 Reception & dinner (entrance Zoo - Marmeren zaal)

09:00 - 10:30 Free communications & poster presentations Chairs: Annick Delvigne, Clinique Saint-Vincent - Willem Verpoest, UZ Brussel

Free communications09:00 - 09:12 FC07 Towards clinical use of germline nuclear transfer to overcome mitochondrial diseases and female infertility Maoxing Tang, Ghent-Fertility And Stem cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium09:12 - 09:24 FC08 Mitochondrial DNA mosaicism in early human development Joke Mertens, Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels09:24 - 09:35 FC09 Does the Freeze-all strategy improves the cumulative live birth rate and the time to become pregnant in IVF cycles? Stephanie Johnson, CHC Saint-Vincent Rocourt, Liège09:35 - 09:47 FC10 Pre-implantation development is compromised in Pou5f1-null mouse embryos Panagiotis Stamatiadis, Ghent-Fertility And Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent09:47 - 09:59 FC11 Time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome? Lynn Vandenberghe, Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel, Centre for Reproductive Medicine, Brussels09:59 - 10:10 FC12 Rnasequencing of human trophectoderm cells reveal major pathways predicting implantation using a new three-dimensional in vitro model Wafaa Essahib, Reproduction and Immunology (REIM), Brussels

Poster presentations 10:10 - 10:15 P01 Morphokinetic parameters of embryos with numerical or structural anomalies are delayed in comparison to euploid embryos Klaas Declerck, Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Ghent10:15 - 10:20 P02 The role of macrophages and mast cells in testicular fibrosis seen in Klinefelter men Margo Willems, Research Group Biology of the Testis (BITE), Vrije Universiteit Brussel, Basic Research10:20 - 10:25 P03 Oocyte vitrification : How to go further by reducing the osmotic stress ? Déborah Desmet, CHIREC, Brussels

Saturday, 23 November 2019

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Session 5: Improvement of embryo culture systems? Chairs: Stefanie De Gheselle, UZ Gent - Sophie Debrock, UZ Leuven10:30 - 10:50 The added value of the time-lapse Cristina Hickman, Imperial College London, UK10:50 - 11:10 The future of embryo culture Etienne Van den Abbeel, University Ghent and Hôpital Erasme ULB 11:10 - 11:40 Coffee break

Chair: Willem Verpoest, UZ Brussel11:40 - 12:00 BELRAP 2017 Diane De Neubourg, President of the College of Physicians Reproductive Medicine12:00 - 12:30 BSRM - VFS Keynote lecture Reproduction in endangered species Thomas B. Hildebrandt, Leibniz Institute for Zoo & Wildlife Research, Berlin, Germany 12:30 BSRM-VFS Award

12:45 Farewell drink & lunch

“There are a few things

I’d like to do before becoming

a mum. Like finding the guy

who’ll be the dad ;-)”

KEDP/DADF5D/BE, date of creation 11/2019

Gedeon Richter launched the ‘Be ready, whenever

you’re ready’ campaign to encourage women to think

about fertility at an earlier stage in their lives

We want to enable women to be ready to make the right choices

for them when it comes to starting a family.

The campaign aims to:

• Encourage women to think about their fertility journey at an earlier stage in their lives

• Provide factual answers to questions that women considering fertility preservation may have

• Encourage women to speak with their local fertility specialist to discuss

fertility preservation and their options

Be ready, whenever you’re ready.www.unbebequandjeseraiprete.be - www.eenbabyalsikerklaarvoorben.be

/unbébéquandjeseraiprête - /eenbabyalsikerklaarvoorben

Gede_0063_Ad_Age_Freezing_A4_UK.indd 1 8/11/19 15:51

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Abstracts (Re)production, a never ending story…Peter Bols, UAntwerpen

Over the past decades, the toolbox of assisted reproduction techniques (ART) expanded dramatically,both in human as well as in the veterinary field. The most important driver on the veterinary side wasthe increased need of animal products such as meat and milk. The explosion of the world populationover the past 50 years combined to changing dietary habits and globalization of food markets boostedthe development and use of efficient assisted reproductive techniques. On the ‘pet animal’ and ‘horse’side, the exclusivity of certain genetic combinations similarly resulted in the application of the mostcomplicated reproductive technologies available on the market. While some developments in humanreproduction can be considered as reciprocal to veterinary developments, the basic driver was clearlynot economical but therapeutic as in vitro embryo production became a successful alternative forinfertile couples to fulfill their child wish. The current introduction will briefly elaborate on the interplaybetween production and reproduction and the consequences of the establishment of assistedreproductive technologies both in animal and human.

Ovulation induction in PCOSRobert Casper, Mount Sinai Hospital, Lunenfeld-Tanenbaum Research Institute, Toronto, Canada

Clomiphene citrate (CC) has been in clinical use for ovulation induction in PCOS patients for more than 5 decades. It works through estrogen receptor (ER) depletion in the hypothalamus/pituitary to remove estro-gen negative feedback on gonadotropin release. Side effects of ER antagonism included thin endometrium, decreased uterine blood flow and poor cervical mucous resulting in lower pregnancy rates than expected. We developed letrozole to decrease estrogen negative feedback centrally, similar to CC, but avoiding the side effects of ER antagonism. In 2014, Legro et al. published the results of an RCT comparing letrozole to CC in 750 women with PCOS. The ovulation rate with letrozole was higher than CC and the cumulative live birth rate with letrozole (25.5%) was higher than CC (19.1%; 95% CI 1.1 - 1.87). This study and a subsequent meta-analysis were instrumental in establishing letrozole worldwide as a primary treatment for ovulation induction in PCOS patients.

The role of Letrozole in fertility treatment in breast cancer patientsOrnit Goldrat, Hôpital Erasme, Brussels

Thanks to advances in systemic therapy, the survival of young breast cancer patients has greatly improved over the last decades. Chemotherapy is nonetheless gonadotoxic and may impair future fertility, hamper-ing access to motherhood. Oocyte and/or embryo freezing for fertility preservation require controlled ovar-ian hyperstimulation (COH). High estradiol level following COH is subject of debate regarding its potential impact on tumor growth in hormone responsive tumors. Therefore, a modified COH protocol associated to letrozole, an aromatase inhibitor, has been developed over a decade ago, and is now offered to patients in order to collect several oocytes while maintaining infra-physiological estradiol level. Several recent studies have shown that letrozole associated COH seems efficient in terms of mature oocyte yield, oocyte quality and pregnancy rate, and is safe regarding relapse risk.

The Future of fertility by age bankingChristine Wyns, UCL Saint-Luc

Planned egg cryopreservation is accepted as a preventive measure for age related fertility decline.While its efficiency and factors influencing its outcome are quite well documented in terms of live birth per oocyte, with modelization tools that may help the specialist to provide the patient with and individualized information, its utility is still under debate. A number of other open questions on physical and emotional long-term health of the child or on implications of the procedure for society still need to be addressed.

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DNA methylation in preimplantation embryosMellissa Mann, University of Pittsburgh, USA

Alarmingly, ~50 million couples worldwide are unable to conceive after 5 years of unprotected sex, with infertility rates still rising. This in part may be attributed to the average age of first-time mothers, which has risen steadily over the last several decades. Medically assisted reproductive technologies (ARTs) constitute important treatment modalities for infertile couples and represent their best chance to conceive. While generally considered safe, ARTs are associated with higher incidences of preterm birth, intrauterine growth restriction, and low birth weight. Moreover, there are more Beckwith-Wiedemann, Silver-Russell, Angel-man and Prader-Willi Syndrome children in the ART population harboring imprinted methylation errors compared to those in the general population. Genomic imprinting is an epigenetic mechanism that con-trols the expression of a small subset of genes by silencing either the maternally- or paternally-inherited allele, while the other parental allele is expressed. The relationship between ARTs and imprinting disor-ders is that ART usage coincides with important stages of imprinted DNA methylation programming during gamete and preimplantation development, raising the possibility that ARTs lead to imprinting errors. Our data from a mouse model indicates that superovulation does not alter imprinted methylation acquisition in mouse oocytes, but instead leads to imprinted methylation errors in early mouse and human embryos. Furthermore, when we analyzed the effects embryo culture, we found that embryos developing faster to the 8-cell stage exhibited a greater frequency of imprinted methylation errors than those developing at a slower rate. Finally, analysis of various ARTs revealed that the frequency of blastocysts with imprinted methylation loss was similar between the superovulation only and the embryo culture only groups, while the combination of superovulation and embryo culture resulted in a greater number of mouse blastocysts with maternal imprinted methylation perturbations than superovulation alone. With respect to the age of mothers, there has been little investigation on the effects of advanced maternal age, with or without ARTs, on genomic imprinting. We found that imprinted methylation acquisition and maintenance were unaf-fected by increasing maternal age, and that the frequency of imprinted methylation errors in blastocysts was unchanged when increasing maternal age was combined with ARTs. In conclusion, while ARTs, either singly or in combination, altered imprinted methylation in mouse blastocysts, advanced maternal age did not increase the burden of imprinted methylation errors when combined with ARTs. These results provide cautious optimism that advanced maternal age is not a contributing factor to imprinted methylation errors in embryos produced in the clinic. Furthermore, our data on the effects of ARTs strengthen the need to ad-vance clinical methods to reduce imprinted methylation errors in in vitro-produced embryos.

Differentiating LH from hCG action: pathophysiology and clinical relevanceManuela Simoni, Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Italy

The human species is naturally mono-ovulatory and the two gonadotropins FSH and LH are fundamental for the selection, growth, maturation and ovulation of a single oocyte. LH is the physiological stimulus for both androgen production by theca cells and final follicular maturation in granulosa cells. In assisted re-production overstimulation by FSH ensures multiple follicular growth and it is presently debated whether addition of so-called “LH-activity” may improve the yield of mature oocytes and pregnancy rate. LH-activi-ty may be provided by hCG, hMG or LH and these different hormone combinations exert different effects at the cellular level, demonstrated using different cell systems.hCG arose only recently evolution and is present only in primates. Apart from the different half-life and structure, LH and hCG can be differentiated on the basis of their action on the intracellular pathways me-diating their action. In general, hCG is more potent on the cAMP/PKA/steroidogenic pathway (required for massive progesterone production during pregnancy), while LH is more active on anti-apoptotic, prolifera-tive pathways, including stimulation of the ERK1/2 and AKT pathway. The addition of FSH reinforces further LH and hCG action, increasing both their potency and duration of action. An excess of cAMP production, however, as provoked by massive FSH and hCG stimulation has a pro-apoptotic effect on human granulosa cells and this effect is physiologically counteracted by estrogen. In conditions of insufficient “local” estro-genization, follicular growth may be impaired.

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A recent meta-analysis comparing the clinical effects in ART of FSH alone, vs. hMG, vs. FSH+hCG, vs. FSH+LH, demonstrated that LH increases significantly pregnancy rate, particularly in women of older age and in the GnRH agonist protocol, suggesting that LH may improve oocyte maturation. The role of estrogen, which derives from androgen produced by the theca cells under LH stimulation, in improving follicle maturation is currently being elucidated. Indeed, estrogen blocks the potentially pro-apoptotic effect of FSH by heter-odimerization with the FSH receptor, diverting pro-apoptotic toward proliferative signals.In conclusion, in the “artificial” setting of ART, the growth and maturation of a sufficient number of follicles and oocytes of good quality depends on the interplay between FSH, LH and estrogen. Understanding the molecular mechanism thereof is expected to provide novel therapeutic approaches to ovarian stimulation.

Of animals and men: across species mammalian embryology for improving human IVF skills and knowledge. Pierre Vanderzwalmen, CHIREC, Brussels, Belgium; Nextclinics IVF Centers Prof. Zech, Bregenz, Austria

Ever since the first IVF baby was born in 1978, assisted reproductive technologies to overcome infertility have advanced rapidly. Therapies that were considered as cutting-edge decades ago, are now an integral part of routine IVF practice. Multidisciplinary collaborations between human and veterinary medicines, bi-ology and bio-engineering fields are one of the main reasons for the overwhelming steady stream of knowl-edge, progress and technological advancement in assisted reproductive technology all over the world. My own experience in the field of animal and human reproduction confirms such statement.The past: With hindsight, I realize I was lucky and it was a privilege for me to integrate in 1978 a research program at the Veterinary Faculty of the University of Liege. Our research team focused on investigating different topics in the field of embryo transplantation, including: (i) bovine IVM-IVF and sperm preparation techniques, (ii) embryo culture and observation of the different patterns of bovine embryo development using time lapse technology (iii) micromanipulations to produce identical calves after either embryo split-ting or after nuclear transfer and electrofusion in enucleated oocytes (iv) vitrification of mice and bovine embryos.In a retrospective view, it is undeniable that such experience for 11 years provided the most comprehensive update on all different IVF laboratory aspects, both, theoretical and practical. In 1989, I joined the human IVF center of Prof. Schoysman. In the 1990’s, with my colleagues G. Bertin and M. Nijs we were able to initi-ate different aspects of ART laboratory techniques such as: (i) the sperm density gradient centrifugation, (ii) treatment of male infertility patients first with PZD and SUZI and following the success with ICSI in the VUB, we obtained the first baby using testicular sperm (iii) we implemented day 5 embryo culture and transfers and (iv) we developed vitrification to replace slow freezing. The present: Such “win-win” collaboration still continues with Dr. Fabien Ectors from the University of Liege. For example, project to investigate the different ways to achieve an intracellular amorphous solidification.In order to reach maximum effectiveness, current vitrification methods consist to expose or equilibrate oocytes or embryos to highly concentrated permeable cryoprotectants solutions (Cps) that will be concen-trated by dehydration in the final step just before cooling.The fear of exposing gametes and embryos to such high amounts of CPs was the central part of a debate initiated by advocates of the slow freezing procedure that involves low concentration of penetrating CP. We showed with the mouse model that intracellular concentration of cryoprotectants (ICCP) is far lower than in the vitrification solution itselfOur next question was if it is possible to reduce dramatically the ICCP by achieving only one step of dehy-dration. We demonstrate, with murine oocytes and embryos that (i) in vitro survival, (ii) development to the blastocyst stage and (iii) in vivo development to birth led to results equivalent or better with one-step dehydration method than after classical step-wise exposure to Cps. The hypothesis is that once dehydra-tion occurs, the osmotic removal of water from the cytoplasm, almost free of CP, leads to an increase in the packing density of macromolecules with an infinite increase of viscosity (gel) leading to solidification by colloidal vitrification. Such vitrification approach dramatically lowers if not suppresses ICCP and associ-ated toxicity, thereby challenging some commonly accepted concepts of cryobiology. In my opinion, BSRM reflects such multidisciplinary collaboration. BSRM succeed to the society BSGFS (Belgium Study Group for Fertility and Sterility) that was initiated by the Professors Leroy, Lhermite, Bros-ens and Beckers (DVM) with the aim to bring together scientists from different horizons.

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I would like to end by paying tribute to who made my work a great deal easier and particularly, the late Prof. Francis Ectors, Prof.Alban Massip Prof. Robert Schoysman and yet active Prof Jean Francois Beckers, Prof. Bernard Lejeune and Prof. Herbert Zech.

The future of embryo cultureEtienne Van den Abbeel, University Ghent and Hôpital Erasme ULB

With the improvement of culture conditions, with widespread application of new approaches including radical elimination of toxic components, introduction of sequential back-to-nature and single step let-the-embryo-choose culture systems and also decreasing the atmospheric oxygen level during the whole cul-ture period, in human IVF, embryo transfer at the blastocyst stage has become the strategy of choice to most clinics worldwide and single blastocyst transfer has been considered the method of choice to pre-vent multiple births. Extended static culture however, may trigger increased risk of preterm delivery birth-weight, childhood growth and long term disease including type II diabetes and cardiovascular problems. There exist fundamental differences between the oviduct and the drop in the culture dish. Exploration of the differential effects of physical and structural environment experienced by gametes and embryos in vitro versus in vivo may provide a means to further improve clinical IVF. In vivo gametes and embryos are exposed to the constricted “moist” environment of the female reproductive tract, surrounded by various oriented glycoproteins, as they are moved via ciliated epithelia to their destination. This is in stark contrast to the expansive static environment gametes and embryos are exposed to in vitro, resting on the inert syn-thetic polymers, bathed in a relative ocean of media (Swain et al, 2008, Swain et al, 2016). Furthermore in the oviduct embryos are subjected to mechanical and physical (shaking, rattling, rolling, vibrating) forces as they move through the reproductive tract in vivo and applying them in vitro may further offer a means of improving the culture microenvironment (Swain et al, 2011, 2013). Microfluidic technology offers a plat-form on which to further manipulate culture conditions in vitro in the hopes of creating an environment more suitable to gamete and embryo development and function. Microfluidic technology can be beneficial in the in vitro culture of mammalian embryos as it is a potential dynamic culture environment. However, this field of study is still in its infancy and further investigations regarding development and efficacy of im-plementation of microfluidic platforms are required.

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Free communicationsFC-01Mechanically disconnected mouse preantral and antral follicles lead to developmentally competent oocytes following follicle cultureA.C. Herta, N. Akin, K. Billooy, L. Saucedo-Cuevas, F. Lolicato, E. Anckaert, J. SmitzVrije Universiteit Brussel VUB, Follicle Biology Laboratory, Brussels, Belgium.

In vitro maturation (IVM) of oocytes was introduced in the fertility clinic as a mild-approach alternative to conventional IVF requiring minimal stimulation. Cumulus oocyte complex (COC) integrity secures cell-to-cell communications supporting growth, maturation and acquisition of oocyte competence. Since partially denuded COCs show low developmental competence, they are not considered for IVM. Improving oocyte-somatic connections could support proper oocyte quality. Our study investigates if mechanically discon-nected follicles in a mouse model are able to re-establish transzonal projections (TZPs) connections and acquire competence comparable to oocytes derived from intact cultured follicles. Mouse secondary follicle culture was performed for 10 days. On day 5, in half of the follicles, the oocyte was mechanically discon-nected from its granulosa cells and placed back in culture; meanwhile the other half of follicles remained intact. At this time point, most follicles are late preantral, while 30% of them score as early antral. Thus we assessed (i) the culture outcome of disconnected preantral and antral follicles versus their intact control groups and (ii) the influence of the follicle development stage at disconnection on the oocyte’s potential to re-connect to its somatic counterparts and its derived developmental capacity. MII oocytes from intact and disconnected follicles after culture underwent IVF. Two cell embryo and blastocysts rates were assessed. Gene expression analysis was performed on MII oocytes and corresponding cumulus cells for oocyte qual-ity markers (Gdf9, Bmp15), early embryo development (Zar1, Mater, Nmp2, Stella, Oct4) and cumulus ex-pansion (Has2, Ptgs2, Ptx3). Our preliminary results showed that disconnected groups attained the same morphological features as the controls, by the end of the follicle culture. By day 9 of culture, an increased density of TZPs was detected in both experimental groups, presumably due to de novo formation. Oocyte maturation and 2 cell embryo rate were not significantly different for both preantral and antral experimen-tal and their correspondent control groups. Significantly higher blastocyst formation was observed in the antral reconnected group compared to the intact. Stella, Npm2 and Has2 genes presented higher expres-sion in the preantral reconnected group. Meanwhile, Mater and Ptx3 showed higher levels in the recon-nected antral follicles. These results suggest that reconnection of disrupted mouse preantral and antral fol-licles, mediated by TZPs reestablishment, does not impair oocyte meiotic and developmental competence. Our approach provides information that could support future strategies for rescuing the competence of partially denuded human COCs undergoing IVM. FC-02Heterozygous mutations in PLCZ1 are associated with fertilization failure after ICSI R. Reddy Guggillaa, A.K. Boela, M. Ferrer-Buitragoa, D. Bontea, P. De Suttera, P. Couckeb, B. Heindryckxa

a Ghent-Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.b Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Introduction: The oocyte activation process is initiated by sperm-oocyte fusion and is characterized by a series of calcium oscillations in the oocyte cytoplasm. A sperm specific factor phospholipase C zeta (PLCZ1) is responsible for this series of calcium oscillations, which hallmarks the start of oocyte activation. To ob-tain more insight into the role of PLCZ1 in fertilization and to know the frequency of PLCZ1 gene mutations in patients experiencing failed or low fertilization after ICSI, PLCZ1 gene screening in a large cohort of pa-tients is required. Methods: Patients (n=38) with previously low or total failed fertilization after routine ICSI were enrolled in the study after obtaining written consent from 2014-present. Genetic screening for PLCZ1 mutations was performed, next to evaluation of the activation potential of the sperm. PCR was performed using primers designed to amplify all the 15 coding exons and the exon-intron boundaries. The amplified PCR products were sequenced using illumine MiSeq.

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A diagnostic test Mouse oocyte activation test (MOAT) was performed on the mouse oocytes by injecting patient sperm into mouse oocytes and determine the number of activated oocytes, in comparison to a positive control.Results: PCR amplification and Illumina sequencing of PLCZ1 gene revealed seven different mutations in thirteen patients, which include four missense mutations D46N, H233L, S500L and R141H, one splice site mutation c.136-1G>C and two protein truncating mutation K322* and Q94*. Three of the patients had ex-perienced partial hydatidiform moles (PHMs) with their partners in vivo and OAF after ICSI. Eight patients carried mutations in a heterozygous state, two patients carried mutations in a homozygous state and three patients carried two mutations in a compound heterozygous state. The activation potential of the sperm was classified in three groups according to the MOAT result: based on the percentage of mouse oocytes activated by patient sperm: MOAT1 group (0-20%), MOAT2 group (21-84%) and MOAT3 group (>85%). The patient with H233L and K322* mutations in a compound heterozygous state was classified in MOAT1 group and the patients with H233L, R141H and the patient with Q94*and S500L mutations showed normal activation potential (MOAT3 group). All the other patients were classified as MOAT2 group. Mouse and Human oocyte activation tests revealed a reduced or no activity when sperm with PLCZ1 mutations is injected into mouse and human oocytes respectively.Conclusions: From our study we show that most of the patient’s sperm with heterozygous mutations can-not initiate oocyte activation due to reduced amount of functional PLCZ1 protein in the sperm, mostly due to haploinsufficiency, confirming the crucial role of the PLCZ1 protein in the fertilization process. Since the frequency of PLCZ1 mutations in patients with OAF is as high as 34%, it is recommended to do PLCZ1 screening for better management of OAF.

FC-03Increase in live birth rates after non-invasive oocyte selection in a prospective interventional study in 108 ICSI patients I. Van Vaerenbergh1, T. Adriaenssens1, N. Akin1, W. Coucke2, I. Mateizel3, G. Verheyen3, M. De Brucker3, M. Ca-mus3, E. Van Hecke4, A. Rosenthal4 and J. Smitz1

1 Follicle Biology Laboratory, Vrije Universiteit Brussel, Brussels, Belgium, 2 Quality of Laboratories, Sciensano, Brussels, Belgium, 3 Centre for Reproductive Medicine, UZ Brussel, Brussels, Belgium, 4 Fertiga, Lede, Belgium

Objective(s): To compare clinical pregnancy and live birth rates by non-invasive cumulus testing using gene expression in combination with embryo morphology versus morphology alone selection in eSET patients treated by ICSI. Design and methods: A prospective blinded interventional clinical trial was performed with planned fresh transfers of day-3 ICSI eSET. Patients were stimulated with GnRH antagonist and HP-HMG. Oocytes un-derwent single denudation of cumulus-corona cells after pick-up. The cumulus cells were analysed with QRT-PCR for three predictive genes CAMK1D, EFNB2 and SASH1 (Corona Test) and two control genes. The analysis resulted in a single score for each oocyte. The score was used to select and transfer a single day 3 embryo with excellent or good morphology. The control group was matched (blinded for outcome) under the same conditions as the intervention group (same age, same number of embryos, same stimulation protocol).The primary outcome was clinical pregnancy (fetal heartbeat confirmed by endovaginal ultrasound at 7 weeks), with stratification for age and number of excellent/good quality embryos (GQE). Secondary out-come included live birth rate and cumulative pregnancies from frozen embryo transfers. Outcomes were compared among treatment arms using one-tailed chi-square test. Results: A total of 108 patients underwent the Corona Test and were matched with 108 control patients nearest in time to the treated cases. There was a significant increase in clinical pregnancy rate on a day 3 eSET from 34% in the control group to 61% in the Corona Test group (p < 0.0001). Live birth rate increased significantly from 29% in the control group to 49% in the Corona Test group (p = 0.0018). Extra-uterine preg-nancy and miscarriage rates were not significantly different between control- and Corona Test group (p = 0.224). The cumulative clinical pregnancy rate was 50% in the control group and increased significantly to 79% in the Corona Test group (p < 0.0001). Conclusions: Using Corona Test as a non-invasive test to select a day 3 embryo almost doubles the clini-cal pregnancy rates and significantly increases the live birth rates. These data indicate that morphology selection complemented by non-invasive cumulus testing could drastically increase the efficiency of eSET in ART.

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FC-04Single-blastocyst genome-wide bisulfite sequencing for assessing the impact of in vitro follicle cul-ture, superovulation and age on mouse embryo development L. Saucedo-Cuevas1, A.C Herta1, E. Ivanova2, K. Billooye1, J. Smitz1, G. Kelsey2,3 & E. Anckaert1

1 Follicle Biology Laboratory (FOBI), UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium, 2 Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK, 3 Centre for Trophoblast Research, University of Cambridge, Cambridge, CB2 3EG, UK

The recent advances in cancer diagnosis and treatment amongst girls and women have increased the focus on their future life quality, including safeguarding the ability to have biological children. Oocyte or embryo cryopreservation is offered as standard fertility preservation strategy to oncological patients before receiv-ing gonadotoxic treatments. However, these options are not suitable for prepubertal girls and may not be feasible for many women because of the urgency of starting cancer therapy. An alternative could be ovar-ian cortical tissue cryopreservation but it carries the risk of reintroducing cancer cells into the patient. An alternative option is using in vitro follicle culture (IFC) systems that support growth of oocytes from early stage follicles. IFC has proven successful in mouse to support the development of follicles from primordial or early pre-antral stages up to preovulatory stage. However, there is concern that it might affect normal embryonic development by interfering with the timely acquisition of correct methylation patterns in oo-cytes and the maintenance of genomic imprinting after fertilization, both required for normal embryonic development. Recent unpublished data from our group shows that both IFC and superovulation globally preserve the methylation landscape of oocytes and do not affect de novo methylation of imprinted genes. Interestingly, specific and consistent alterations in DNA methylation are found depending on both the age of the animals and the approach used to grow and mature the oocytes. To elucidate the extent to which IFC might alter DNA methylation after fertilization we have conducted whole-genome bisulfite sequencing on single blastocyst using a post-bisulfite adapter tagging approach (PBAT). We have generated genome-wide DNA methylation maps in blastocysts derived from in vitro cultured oocytes collected from pre-pubertal and adult mice and their age-matched superovulated and in vivo controls. Our preliminary results sug-gest a global loss of DNA methylation in IFC derived blastocysts. Detailed analysis of methylation levels at the regulatory regions in imprinted genes, and at other genomic features will define the effect of IFC and superovulation in relation to maternal age on maintenance of methylation and proper reprogramming of blastocyst.

FC-05Rebiopsy in preimplantation genetic testing (PGT) can provide a genetic result and maximizes the number of transferable blastocysts for patientsS. Ellegiers, I. De Croo, P. De Sutter and K. TillemanDepartment for Reproductive Medicine, Ghent Fertility And Stem cell Team (G-FAST ), Ghent University Hospi-tal, Ghent, Belgium

Objective(s): Trophectoderm biopsy is the method of choice in PGT and after biopsy, generally 44,5% blas-tocysts are euploid, 53,0% aneuploid and 2,5% show an inconclusive genetic diagnosis (Cimadomo et al.,2018). Little is known about the fate of these inconclusive blastocysts. The aim of this study was to in-vestigate whether these inconclusive blastocysts can provide an unambiguous genetic result after rebiopsy and what their implantation potential is.Design and methods: A retrospective descriptive monocentric analysis of 1431 blastocysts from 349 PGT-cycles was performed (01/01/2016 - 31/08/2019) derived from 254 couples (mean maternal age 32,38±4,52) and subdivided in 69 cycles PGT-SR, 113 cycles PGT-A and 167 cycles PGT-M. The outcome parameters for the rebiopsied blastocysts were survival rate, genetic status and live birth rate.Trophectoderm biopsy and vitrification was performed on D5 or D6. Whole genome amplification (SurePlex Illumina) and copy number variation sequencing (PGT-A and PGT-SR) or PCR amplification of the affected exon in PGT-M and Sanger sequencing (ABI Genetic analyzer) was executed. An inconclusive result was caused by DNA amplification failure (AF) or non-interpretable data (NI). Statistical analysis was performed by Fisher’s Exact Test (p≤0.05).Results: Of the 1431 biopsied blastocysts, 46,6%(667/1431) showed a normal chromosomal profile, in 47,2%(675/1431) chromosomal anomalies were observed and 6,2%(89/1431) presented an inconclusive

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result (AF 55,1%(49/89); NI 44,9%(40/89)). There was a statistical higher incidence for inconclusive results for PGT-M (6,9%(51/734)) compared to PGT-A (3,8%(15/397)) (p=0.03). For PGT-SR 7,7%(23/300) of the biop-sied blastocysts were inconclusive (not significant).There was a statistical difference in the amount of excellent quality blastocysts (cat A+B) related to the ge-netic result after PGT: 74,2%(495/667) were eligible for transfer, 68,7%(464/675) displayed genetic anoma-lies (p=0.03). The inconclusive blastocysts cat A+B (70,8%(63/89)) showed no statistical difference concern-ing embryo quality compared to blastocysts having (ab)normal genetic results.For all PGT-cycles together, 89 inconclusive blastocysts were thawed with a survival rate of 89,9%(80/89). A rebiopsy and vitrification could be performed in 51,7%(46/89) of the warmed blastocysts. After genet-ic analysis 34,8%(16/46) blastocysts were eligible for transfer, 45,7%(21/46) not eligible for transfer and 19,6%(9/46) again inconclusive. In the group of transferable blastocysts after rebiopsy, 11 blastocysts had already been warmed of which 9 had been transferred (81,8%(9/11)). Six transfers (66,7%(6/9)) resulted in positive hCG and 3 ongoing pregnancies (50%(3/6)).Conclusion: It is worthwhile to perform a rebiopsy and revitrification on blastocysts with inconclusive ge-netic status as this can result in transferable blastocysts and a healthy live birth.

FC-06Trophectoderm Specification: Compaction, Polarisation and Inner and Outer Cells occur simultane-ously in the Human Preimplantation EmbryoA. Demtschenko1,2, W. Essahib1, G. Verheyen3, K. Sermon2, H. Tournaye3, H. Van de Velde1,3

1Research Group Reproduction and Immunology and 2Research Group Reproduction and Genetics Vrije Uni-versiteit Brussel, Brussels 1090, Belgium, 3Center for Reproductive Medicine, Brussels University Hospital, Brussels 1090, Belgium

Introduction: In mice, the first indication of inner cell mass (ICM) and trophectoderm (TE) segregation oc-curs three days post fertilisation (dpf3). After compaction, asymmetric divisions give rise to inner and outer blastomeres that ubiquitously express transcription factor TEAD4 and sequester polarity markers (p-ERM), and TE lineage specifiers (YAP1, GATA3) to outer blastomeres. This is considered the onset of TE lineage segregation in the mammalian embryo. Studies regarding this event in the human embryo are absent and extrapolations from the mouse model cannot be a surrogate for studies in the human embryo.Objectives: Here we aim to identify the time point of when inner cells arise in the human embryo and to investigate the distribution of polarity marker p-ERM, transcription factor TEAD4 and TE lineage specifiers YAP1, and GATA3.Design and Methods: Cryopreserved human 8-cell stage embryos, donated to research, or zygotes created for research after informed consent were warmed (Vit Kit -Thaw, Irvine Scientific, USA) and cultured until dpf5. Immunofluorescence was performed on fixed embryos at predefined stages for TEAD4, YAP1, GATA3, p-ERM as well as F-actin and Hoechst for imaging (LSM800, ZEISS) and the manual estimation of individual blastomere counts per embryo (ImageJ).Results: The first inner blastomeres are observed at dpf4 after compaction at the 16-cell stage. These inner blastomeres are mostly negative for YAP1, GATA3 and p-ERM, whereas the nuclei of the outer blastomeres appear positive for YAP1 (69±22%, n=8) and begin to co-express GATA3 (15±22%, n=8). At this stage the apical membranes of the outer blastomeres appear polarised by p-ERM staining (100%, n=8) and TEAD4 is expressed ubiquitously (100%, n=8).Conclusion: A timeline for blastomere polarisation and TE lineage specifier localisation was created. TEAD4, YAP1 and GATA3 protein expression was demonstrated in some blastomeres of the human embryo during the compaction process. At the fully compacted stage, the embryo consists of polar/outer cells, positive for YAP1 and GATA3 and few apolar/inner cells, negative for either of the proteins. This pattern is maintained in the blastocyst TE and ICM. While the same molecular determinants are involved in the first lineage segrega-tion of mice and humans, their expression pattern differs according to the morphological embryo develop-ment of the respective species.

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FC-07Towards clinical use of germline nuclear transfer to overcome mitochondrial diseases and female infertilityM. Tang1; A. Boel1; G. Guggilla1; D. Bonte1; M. Popovic1; B. Bekaert1; F. Vanden Meerschaut1; P. De Sutter1; P. Coucke2; B. Heindryckx1

1Ghent-Fertility And Stem cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospi-tal, Ghent, Belgium, 2Center for Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University Hospital, Corneel Heymanslaan 10, Ghent 9000, Belgium

Background and objective: To prevent mother-to-child transmission of mitochondrial (mt)DNA mutations, germline nuclear transfer (NT), such as maternal spindle (ST) or pronuclear (PNT) transfer, is currently be-ing proposed. This technique involves transfer of nuclear genome from an oocyte or zygote carrying mtD-NA mutation to an enucleated donor counterpart with unaffected mtDNA. Additionally, NT technology has also been considered for certain infertility indications, such as infertile women experiencing poor embryo development, with the expectation of improving IVF outcomes. However, until now, there is only limited data available on the application of NT in human, either for circumventing mitochondrial diseases or for overcoming female infertility. Here, we assessed the efficiency of NT to reduce transmission of mutated mtDNA, and investigated NT as a novel strategy to restore embryonic development. Furthermore, we de-termined whether NT significantly alters gene expression patterns to verify the safety of this technique.Study design and methods: A 30-year-old woman carrying a homoplasmic mtDNA mutation m.11778G>A, which is known to cause Leber’s hereditary optic neuropathy (LHON) syndrome presented. When undergo-ing PGD to select possible mutation safe embryos, ST and early (e)PNT were performed on the patient’s in vivo matured MII (n=5) and in vitro matured (IVM) MI (n=1) oocytes respectively, for research purposes. Enucleated healthy frozen-thawed in vivo matured MII oocytes with smooth endoplasmic reticulum aggre-gates (SERa) and an enucleated healthy fresh IVM oocyte served as cytoplast recipients, respectively. Fol-lowing NT, embryo development was assessed and mtDNA carry-over was examined using next-generation sequencing (NGS) in reconstructed embryos.In addition, we included a second patient in this study. A 26-year-old woman who experienced two failed IVF cycles characterized by almost all her oocytes having fertilization failure after following ICSI. The pa-tient’s spindle-chromosome was transferred from fresh IVM oocytes (n=5) into enucleated frozen-thawed SERa oocytes donated by fertile women. Fertilization outcome and embryonic development after ICSI were evaluated.Finally, using B6D2F1 mouse, we examined whether NT induces alterations in global gene expression pat-terns. We carried out RNA sequencing (RNA-seq) on individual blastocysts (n=3) obtained after conducting PNT on mouse oocytes (fertilization by ICSI). Individual blastocysts (n=3) obtained from intact MII oocytes undergoing ICSI and pools of 10 unmanipulated oocytes (n=2) served as controls.Main results: For patient 1, harboring the m.11778G>A mtDNA mutation, PGD confirmed close to 100% ho-moplasmic mutation load in all embryos analysed after trophectoderm biopsy (n=4). After ICSI, 100% (5/5) of ST-oocytes were normally fertilized, 100% (5/5) cleaved, and one (20%, 1/5) progressed to a blastocyst (scored as 4AA); while 100% (1/1) of reconstructed ePNT-zygote formed a compacted morula, but failed to reach blastocyst stage. Data from targeted NGS revealed that mtDNA carry-over in two trophectoderm bi-opsy samples from the ST-blastocyst and the ePNT-morula was 3.2%, 2.9% and 3.1%, respectively, while all the patient’s non-manipulated oocytes/zygotes, arrest-embryos and blastocysts showed mutation loads close to 100%. MtDNA carry-over in the remaining arrested ST-embryos (n=4) was 2.9% ± 1.1% (mean ± SD).For patient 2, displaying fertilization failure, we first applied the mouse oocyte activation test (MOAT) to evaluate the sperm activation potential of the patient’s partner. The results revealed that more than 85% of the mouse oocytes were activated after ICSI (MOAT 3 classification, suspected oocyte-related problem). The application of clinical assisted oocyte activation resulted in only one oocyte fertilized (1/12), showing vague 2PN formation and no division until day 3. We further injected the partner’s sperm into fresh non-patient’s IVM and 0PN oocytes, and found most oocytes (83%, 5/6) were activated and cleaved after injec-tion without the application of AOA, indicating that a sperm deficiency can be excluded. Following ST and ICSI, 50% (2/4) of reconstituted oocytes were normally fertilized with 2PN, 25% (1/4) with 1PN, 25% (1/4) with immediate cleavage, and 75% (3/4) subsequently cleaved to four-cell stage, but did not progress to blastocysts.

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Principal component analysis based on RNA-seq data generated from mouse blastocysts showed that PNT-blastocysts clustered closely with ICSI controls, and both were clearly distinguishable from oocyte con-trols. These findings indicate that NT did not significantly change global gene expression.Conclusions: NT techniques may have potential to overcome mitochondrial disease transmission and cer-tain forms of female infertility. FC-08Mitochondrial DNA mosaicism in early human development J. Mertens1, M. Regin1, N. De Munck2, H. Van de Velde3, K. Sermon1, C. Blockeel3, C. Spits1

1Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels, Belgium, 2IVI Middle East Fer-tility Clinic, Abu Dhabi, United Arab Emirates, 3Center for Reproductive Medicine, UZ Brussel, Brussels, Belgium

Objectives: Historically, the mitochondrial genome has been known to be heterogeneous in terms of the load of an inherited variant across family members, and also between tissues within one individual. By now, this diversity has been refined to the single-cell level, and individuals consist of different cell popu-lations that differ substantially in their variants and mutational burden. The timing of the appearance of these cell lineages has not yet been identified. We hypothesized that mitochondrial DNA mosaicism begins during preimplantation development.Design and Methods: We studied 102 oocytes of 20 donors, 158 individual blastomeres of 25 day-3 embryos and 17 samples of ICM and TE of 7 day-5 blastocysts. Long-range PCR was used to enrich for the mito-chondrial DNA. Deep massive parallel sequencing was carried out on an Illumina NovaSeq platform and the reads were aligned to the reference genome (NC_012920.1). Variants >2% were further analyzed with mtDNA server and MuTect.Results and Conclusion: In all samples, we found two types of variants. Recurrent variants appeared in ei-ther multiple oocytes of the same donor, multiple cells of the same embryo and across TE and ICM samples of the same blastocyst. Unique variants were only found in one oocyte of a cohort, or one cell or TE/ICM sample of an embryo. Recurrent and unique variants differed in their location and heteroplasmic loads in a consistent manner across the sample types. In case of the oocytes, 67% of the recurrent variants were located in the non-coding region. This distribution was similar in early human development, with day-3 embryos having 58% of recurrent variants in non-coding regions and day-5 blastocysts 78%. In contrast, unique variants are predominantly found in the coding-region (50% in oocytes, 89% in day-3 embryos and 87.5% in day-5 blastocysts) of which 25.5% was located in the RNA-coding regions in oocytes, 50% in day-3 embryos and 33.3% in blastocysts. Remarkably, recurrent RNA-coding variants were only found in one day-5 blastocyst. The heteroplasmic load of the unique variants was low, ranging between 2-10%, while the load of recurrent variants was higher, ranging 2-100%. This difference in load likely reflects the higher pathogenic potential variants in rRNA and tRNA genes, which are potentially only tolerated at low loads. Finally, 92% of cleavage-stage embryos had cells with unique variants, proving that mosaicism is already occurring as early as day 3 of human development. FC-09Does the Freeze-all strategy improves the cumulative live birth rate and the time to become pregnant in IVF cycles?S. Johnsona, J. Vandrommeb, A. Delvignea

a CHC Saint-Vincent Rocourt, Liège, Belgique, b CHU Saint-Pierre, Bruxelles, Belgique

Objectives: Elective freezing of all good quality embryos and transfer in subsequent cycles, named as the freeze-all strategy (FAS) is widely used for ovarian hyperstimulation syndrome (OHSS) prevention. The ben-efits of the FAS on live birth rates among high responders have been shown by many studies. Similarly, the benefits of frozen embryo transfer (FRET) compared to fresh embryo transfer (FET) has been demonstrated to prevent preterm birth and small for gestational age. Consequently, why should we limit the FAS to high responders rather than extend it to all?Design and methods: A retrospective and monocentric study was conducted between January 2008 and January 2018 comparing the cumulative live birth rates (CLBR) of patients having undergone FAS to those using FET and having at least one frozen embryo during the same period. Logistic regression (LR) was used to identify confounding variables.

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Analysis were made for the entire cohort and also for different subgroups according to the BELRAP (Belgian Register for Assisted Procreation) criteria and to confounding factors selected by the LR.Results: 2216 patients were studied in all. 233 patients constituted the freeze all (FA) group and 1983 the control (C) group (population 1). Patients selected according to BELRAP criteria (less than 36 years old and first IVF trial) were divided as follows: 124 patients in the FA group with 1241 in the C group (population 2). For these two groups, the CLBR was respectively 50.2% vs 58.1% P=0.021 for population 1 and 53.2% vs 63.3% P=0.023 for population 2. LR revealed the following confounding variables: age, rank of the attempt, tobacco, number of oocytes retrieved, number of embryos obtained, and the date of oocytes retrieval before 2011 and after 2011. Once these confounding variables were excluded, the FA and the C group were restricted to respectively 109 and 770 patients. The CLBR stays in favour of the control group: 70.1% vs 55.9% P=0.03. The time to become pregnant is equally in favour of the C group with a median of 5 days against 61 days for the FA group.Conclusions: The CLBR is significantly lower in the FA group. Nevertheless, the CLBR in the FA group re-mains excellent and superior to that observed in previous studies. The period chosen by the LR underlines the advantages of blastocytes vitrification. These results confirm the high efficiency of FAS but underline the necessity to restrict this strategy to selected cases.

FC-10Pre-implantation development is compromised in Pou5f1-null mouse embryosP. Stamatiadisa,, A. Boelb, M. Van der Jeughta, R. Reddy Guggillaa, M. Tanga, P. De Suttera, P. Couckeb, B. Hein-dryckxa

aGhent-Fertility And Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospi-tal, Corneel Heymanslaan 10, 9000 Ghent, Belgium, bCenter for Medical Genetics, Department of Biomolecular Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium.

Introduction: Early mammalian embryogenesis is controlled by mechanisms that govern the balance be-tween pluripotency and differentiation. Transcription factor OCT4, encoded by Pou5f1, is a key component of the pluripotency regulatory network. Embryonic Pou5f1 expression is initiated at the 4-8 cell stage of the mouse embryo and as the outer cells differentiate into the trophectoderm, Pou5f1 expression becomes downregulated and restricted to cells of the inner cell mass (ICM) of the blastocyst. The aim of our study was to identify the most efficient method of CRISPR-Cas9 introduction and examine the effects of Pou5f1 knock-out during pre-implantation development. Methods: A crRNA:tracrRNA duplex targeting exon 2 of Pou5f1, was microinjected along with Cas9-nuclease in mouse zygotes (S-phase) or was co-injected with mouse sperm during ICSI in M-phase oocytes. We hy-pothesized that earlier injection during M-phase will allow more time for successful targeting prior the first cell division, thereby eliminating mosaicism. S-phase or M-phase injected oocytes, without the CRISPR-Cas9 components served as media controls. The second control group consisted of sham injected S-phase and M-phase oocytes. Reconstructed embryos were cultured for 4.5 days until blastocyst formation. De-tailed embryo scoring was performed daily, and DNA was extracted at day 4.5. The targeted region was PCR amplified and analyzed by fragment analysis and targeted next-generation sequencing (NGS). Immuno-fluorescence analysis was used to confirm the loss of OCT4 expression. Results: A total of 39 blastocysts were analysed, revealing that the editing efficiency (no embryos edited/total no of Embryos) in both groups was over 95% and the mutagenesis efficiency (% edited alleles) was close to 100% pointing to successful editing of both alleles in each blastomere. Comparison of the muta-tional spectrums identified a higher percentage of edited embryos in the M-Phase group exhibiting a sin-gle variant (31% M-phase vs 20% in the S-phase). While embryonic development of S-phase and M-phase Pou5f1-targeted embryos was unaffected up to the morula stage, subsequent development was severely compromised, with the majority of the embryos arresting prior to the blastocyst stage (53% S-Phase, 84% M-Phase). In contrast, blastocyst formation rate in the control groups was 94%. Immunofluorescence anal-ysis confirmed the concordant loss of OCT4 protein. OCT4 depletion resulted to absence of Sox17 expres-sion in agreement with previous studies, suggesting that OCT4 promotes primitive endoderm genes.Conclusion: Both methods resulted in similar editing/mutagenesis efficiencies but created distinct muta-tional patterns. Overall, we confirmed that embryonic Pou5f1 expression is required for the development of the mouse embryo to the blastocyst stage supporting the important role of OCT4 in pre-implantation development.

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FC-11Time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome?L.T.M. Vandenberghe1, S. Santos-Ribeiro2, N. De Munck3, B. Desmet1, W. Meul1, H. Van de Velde1, G. Verheyen1

1Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel, Centre for Reproductive Medicine, Brussels, Bel-gium, 2IVI-RMA Lisboa, Lisbon, Portugal, 3IVI-RMA Middle East Fertility Clinic, Abu Dhabi, UAE

Introduction: In assisted reproduction, ovarian stimulation (OS) is used to induce the simultaneous growth of multiple follicles, followed by final maturation and ovulation triggering with exogenous hCG. Generally, oocyte retrieval (OR) is performed 35-36h later. In addition, a 1-3 hour culture period of the cumulus-oocyte complexes prior to oocyte injection is believed beneficial for fertilization and embryo quality, probably due to improved oocyte cytoplasmic maturation. However, in large ART centers with heavy workload, re-specting these exact time intervals is frequently challenging. Therefore, we questioned whether the time interval between ovulation triggering and oocyte injection affects embryological and clinical outcome in ICSI cycles.Methods: A single-center retrospective cohort analysis was performed including 8811 ICSI cycles from 2010 until 2015. Regarding the time interval between ovulation triggering and oocyte injection, seven categories were considered: <36h, 36h, 37h, 38h, 39h, 40h and ≥41h. The interval of <36h and 36h occurred only if OR was carried out before the planned 36h trigger interval and followed by immediate injection. The main outcome measures were oocyte maturation, fertilization and embryo utilization (embryos adequate for transfer or cryopreservation) rate per injected MII. Clinical pregnancy (CPR) and live birth rates (LBR) were considered as secondary outcomes. During the study period, oocyte retrieval was routinely performed 36h post-triggering except in the <36h and 36h groups. Only cycles with fresh autologous gametes were includ-ed. Exclusion criteria were: injection with testicular/epididymal sperm, managed natural cycles, conven-tional IVF, combined conventional IVF/ICSI, PGT and IVM cycles. Statistical analysis was performed by mul-tivariable multilevel mixed modeling regression analysis. Female age, number of oocytes, pre-preparation sperm concentration, post-preparation sperm motility, day of transfer, number of embryos transferred and embryo quality were identified as potential confounders.Findings: Among the 7 interval groups, maturation rates ranged from 76.4%-83.2% and differed significant-ly (p<0.001). Similarly, there was a significant difference in fertilization rates (range 69.2%-79.3%; p<0.001). Pairwise comparisons between the time intervals showed that the adjusted maturation and fertilization rates were higher in the >41h interval category compared to the other time interval categories (P<0.010 and P<0.001, respectively). However, the adjusted embryo utilization rates of this group did not differ signifi-cantly. Oocyte injection <36h post-triggering had no significant effect on maturation (P=0.462), fertilization (P=0.824) or embryo utilization rates (P=0.696) compared to injection at 38h.No effect of the time interval was observed on clinical pregnancy rates (range 23.1%-34.7%; p=0.595) and live birth rates (range 16.2%-25.8%; p=0.486), after adjusting for potential confounders. The adjusted anal-ysis showed that the chance of having a live birth tends to be lower (OR 0.533, 95%CI: 0.252-1.126; P=0.099) when oocyte injection was performed before 36h compared to 38h after ovulation triggering. However, no significant difference was reached. Injection ≥41h post-triggering did not affect live birth rate (OR 0.971, 95%CI: 0.802-1.176; P=0.765) compared to injection at 38h post-ovulation.Conclusion: Our results indicate that the optimal insemination time window is less stringent than previ-ously thought as both embryological and clinical outcome parameters were not significantly affected in our analysis. This is reassuring for busy centers that might not always be able to follow strict time intervals.

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FC-12Rnasequencing of human trophectoderm cells reveal major pathways predicting implantation using a new three-dimensional in vitro model W. Essahib1, E. Couvreu De Deckersberg2, A. Aberkane1, P. Verdyck3, Y. Guns5, C. Spits2, I. Van Riet4, S. Mackens1,5, M. Debrucker5, G. Verheyen5, H. Tournaye5, H. Van de Velde1,5

1Reproduction and Immunology (REIM), Laarbeeklaan 103, 1090 Brussels; 2Reproduction and Genetics (REGE), Laarbeeklaan 103, 1090 Brussels; 3Centre for Medical Genetics, UZ Brussel, Laarbeeklaan 101, 1090 Brussels; 4Hematology, UZ Brussel, Laarbeeklaan 101, 1090 Brussels; 5Centre for Reproductive Medecine, UZ Brussel, Laarbeeklaan 101, 1090 Brussels

Objective: Successful implantation requires a receptive endometrium and a competent embryo but little is known about their crosstalk. A number of human in vitro models have been set up but so far only limited advancements have been achieved. Therefore, we aimed to setup a 3D in vitro model to study implanta-tion. Additionally, we aimed to highlight pathways decisive for implantation by RNA-sequencing of tro-phectoderm (TE) cells.Design and methods: The research was approved by the Local Ethical Committee and the Federal Com-mittee for Embryo; patients donated embryos for research after informed consent (IC). The 3D model was composed of an epithelium monolayer of Ishikawa cells and stromal cells (donated after IC by oocyte do-nors the day of oocyte retrieval) which were decidualized and embedded in Matrigel. Day 5 good quality vitrified human blastocysts were warmed. TE biopsies taken on day 6 were divided into two parts for (1) RNA sequencing and (2) DNA sequencing using Next Generation Sequencing (NGS). Embryos were sequenced using Illumina Hiseq. Biopsied embryos were put in co-culture and after 48 hours allocated to a group: “implantation” (I) or “no implantation” (NI) in the 3D model. The implanted embryos were fixed and stained for epiblast (OCT4) and trophoblast (CK7 and hCG).Main results and the role of chance: Twenty-four biopsied embryos were co-cultured in the 3D model until day 12. Attachment and invasion in the model were observed and confirmed with secreted hCG levels. An invasion rate of 60% was achieved after 48 hours of co-culture. Immunohistochemistry of invaded em-bryos showed trophoblast formation with CK7 and hCG. OCT4 confirmed further development of the inner cell mass. TE biopsies obtained before co-culture were further allocated according the fate (I or NI) of the embryos. DNA sequencing outcome (euploidy vs aneuploidy) could not be linked to the invasion capacity. However RNA sequencing revealed differentially expressed (DE) genes. 141 transcripts were upregulated and 17 downregulated in the NI group. Further analysis of DE genes revealed pathways involved in cell polarity (Planar Cell Polarity) and inflammation (p38MAPK). Finally, regulatory effects were found playing a role in cell movement (INHIBIN and VEGF) and protrusion formation.Conclusion: The 3D model sustains human embryo development and allows the study of early embryogen-esis. Our findings support the importance of TE cells during human implantation. The identified pathways may lead to specific markers that could be used in the clinic to select the best embryo for transfer and as a result reduce the time to pregnancy.

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POSTERSP01Morphokinetic parameters of embryos with numerical or structural anomalies are delayed in com-parison to euploid embryos. K. Declerck1, S. De Gheselle1, M. Baetens2, K. Tilleman1

1Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospi-tal, Ghent, Belgium.2Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University and Ghent University Hospital, Ghent, Belgium.

Objective: To determine differences in morphokinetic development of embryos with a normal chromo-somal profile and embryos with numerical and/or structural anomalies.Design and methods: In a retrospective single centre study (January 2016-September 2019) 322 biopsied embryos from 84 different cycles were analysed for morphokinetic development using time-lapse imag-ing (EmbryoScopeTM). Laser assisted hatching (LAH) was performed to assist trophectoderm (TE) hernia-tion and to benefit the TE biopsy on day 5 or 6. PGT for structural rearrangements (PGT-SR) or aneuploidy screening (PGT-A) was performed by Next Generation Sequencing (NGS). The embryos were allocated into two groups based on the genetic result of the TE biopsy. Embryos having an euploid profile (n=154; 123D5/31D6) and embryos displaying a chromosomal anomaly (n=149; 115D5/34D6). Nineteen embryos were inconclusive after NGS. Seventeen morphokinetic time-points [second polar body extrusion (tPB2), appearance of two pronuclei (tPNa), pronuclear fading (tPNf), times to reach two- to nine-cell divisions (t2–t9), start of compaction (tSC), end of compaction (tM), initiation of blastulation (tSB), blastocyst stage (tB), expanded blastocyst stage (tEB) , hatched blastocyst stage (tHB)] were analyzed and 7 time-intervals [dura-tion of the PN stage (PN), duration of the first cell cycle (CC1), duration of the second cell cycle (CC2), syn-chronization of cell divisions (S2); synchronization of cleavage pattern (SS3), duration of compaction and the blastulation] were calculated. For statistical analysis, morphokinetic parameters were log-transformed and linear-regression analysis (mixed models) was applied (SPSSv24). Fixed effects were determined by comparing euploid embryos and embryos with a numerical and/or structural anomaly (p≤0.05).Results: Three time-points and 1 calculated time-interval were significantly delayed in embryos display-ing a chromosomal numerical and/or structural anomaly compared to embryos with an euploid pro-file. Respectively, tPB2 [Geometric mean(GM) 2.90h versus GM 2.61h;p=0.011], t3 [GM 37.7h versus GM 35.98h;p=0.029]; tHB [GM 116.41h versus GM 114.29h;p=0.026] and the time-interval for the duration of compaction [GM 8.91h versus GM 7.08h;p=0.004]. In the remaining morphokinetic parameters no statisti-cal significant differences where seen between the chromosomal normal embryos and abnormal ones. Limitations of the study: data were not corrected for the moment of LAH and for semen origin. Time-lapse analysis was not subdivided between numerical and structural anomalies due to the small sample size.Conclusions: tPB2, t3, tHB and the duration of compaction time are faster in embryos without numerical and/or structural anomalies. These morphokinetic parameters could be used as a predictive tool for ane-uploidy in relation to preimplantation genetic testing.

P02The role of macrophages and mast cells in testicular fibrosis seen in Klinefelter menM. Willems, D. Van Saen, E. Goossens Research Group Biology of the Testis (BITE), Vrije Universiteit Brussel, Basic Research

Introduction and objectives: Klinefelter syndrome (KS; 47,XXY) is a genetic disorder which affects 1-2 in 1000 newborn males. Diagnosis mostly occurs at adult age since 95% of KS men suffer from azoospermia due to a loss of spermatogonial stem cells. From puberty onwards, testicular fibrosis is detected. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. The aim of this study was to identify factors involved in the fibrotic remodeling of KS testes. The role of mast cells and macrophages was investigated since previous research reported an increase in these immune cells in non-KS infertile pa-tients. In addition, the presence of decorin was investigated since its expression is regulated by the above mentioned immune cells.

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Material & Methods: Immunohistochemistry (IHC) was used to detect mast cells (anti-tryptase) and mac-rophages (anti-CD68) in the interstitial and tubular area of prepubertal (n=4), peripubertal (n=18) and adult (n=25) KS testicular biopsy samples. In addition, age-related controls and adult Sertoly Cell Only (SCO) samples were included. The presence of the secretory products TNF-alpha, tryptase and decorin was evaluated in adult KS samples in comparison to SCO and control samples through RT-qPCR. Differential gene-expression analysis was performed to detect a difference in up- and downregulated genes between azoospermic patients with and without fibrosis. Results: Increased immune cell numbers in adult KS testis samples in comparison to controls were found in the tubular area. No significant difference was observed between KS and control samples from peri- or prepubertal patients. Adult samples with more fibrosis showed a higher number of immune cells in the tubular area compared to control samples. Decorin was upregulated in KS samples in comparison to con-trols. Through the differential gene expression analysis, the upregulation of several fibrotic-related genes which have been previously described in other tissues was detected in KS compared to control samples. In addition, the gene which encodes lumican, which is, as decorin, a member of the small leucine-rich pro-teoglycan (SLRP) family, has been found to be upregulated in KS samples compared to control samples. Conclusion: Mast cells and macrophages are not directly responsible for the initiation of testicular fibrosis in KS men since a great variability in cell number and the presence of their secretory products was de-tected. Whether decorin, lumican or other SLRP family members play a role in the initiation of testicular fibrosis in KS will need further research. P03Oocyte vitrification : How to go further by reducing the osmotic stress ?Déborah Desmeta Pierre Vanderzwalmena,b

aCHIREC, Brussels, Belgium; bIVF Centers Prof. Zech, Bregenz, Austria

Objective(s): During vitrification process, oocytes have to tolerate and adapt to non-physiological condi-tions when exposed to hypertonic cryoprotectant (CP) solutions. This can negatively affect fundamental functions of oocytes. Stress induced by non-optimal oocyte exposure to CP solutions may compromise intracellular spatial orientation, function and distribution of the organelles and may disrupt the microtu-bular and microfilament network. Are membrane, cytoskeleton or organelles affected by a too pronounced dehydration – leading to a reduction of cell volume that becomes lethal for the oocyte - or by too faster shrinkage/swelling or too high intracellular CP? The objective of the study was to increase the number of exposure steps of oocytes to CP solutions in order to reduce and evaluate the osmotic stress.Design and methods : or this study, 127 GV-oocytes (GV) unsuitable for IVF-ICSI cycles were randomly dis-tributed into two groups: the "5-steps" group, including 56 GV and the "9-steps" group, including 71 GV.After denuding, GV were vitrified using FertiVit Cooling Kit (FERTIPRO). The manufacturer procedure was applied for the 5-steps group while intermediate concentrations of DMSO/EG were added for the 9-steps group, as following : 1,25/1,25 (1,5 min) ; 2,5/2,5 (1,5 min) ; 3,75/3,75 (1,5 min) ; 5/5 (1,5 min) ; 6,25/6,25 (1,5 min) ; 7,5/7,5 (1,5 min) ; 8,75/8,75 (1,5 min) ; 10/10 (3 min) and 20/20 (60 sec.). GV were vitrified in closed system using Vitrisafe carrier (VitriMed) and CBSTM high security embryo straws (CryoBio System). Warm-ing was performed with FertiVit Warming Kit (FERTIPRO) according to the manufacturer. Following rates were evaluated : survival (SR = nb of survived GV / total number of vitrified/thawed GV x 100) and in-vitro maturation to MII stage (MII).Results: Out of the 56 GV vitrified with the 5-steps protocol, 34 survived after warming (60.7%) and 22 ma-tured to MII stage (64,7%). In the 9-steps group, out of the 71 vitrified/warmed GV, 62 survived (87,3%, P<0.001) and 45 (72,6%, NS) matured to MII stage. Conclusion: This preliminary study indicates that we can obtain a significant increase in the survival rate, permitting to obtain higher number of MII, by multiplying exposition steps to CP from 5 to 9 with a constant addition of 2.5 % of permeable CP. This new 9-steps protocol reduces the osmotic shock and could lead to better outcomes after oocytes vitrification and warming, but also to increase the oocyte pool for patients with high immature oocytes rate such in cases of PCOS or oncofertility. To go further, it would be interesting to evaluate the activation and fertilization potential of such vitrified-warmed GV, and test this new protocol with mature oocytes.

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P04Rapid and cost-effective method to assess semen quality of heat stressed Belgian blue cattle under field conditionsG. Residiwati1, H.S.A. Tuska1, Budiono2, G.K.V. Kawai1, A. Seifi-Jamadi1,3, D. Santoro1, B. Leemans1,4, C. Boccart5, G. Opsomer1, A. Van Soom1

1Department of Reproduction, Obstetrics, and Herd Health, Ghent University, 9820 Merelbeke, Belgium, 2Ga-jayana University, Malang, East Java 65144, Indonesia, 3Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj 31587-77871, Iran, 4Utrecht University, 3512 JE Utrecht, Netherlands, 5Association Wallonne De L'Élevage, 1300 Wavre, Belgium

Objective : Since several Asian developing countries are starting to breed Belgian Blue cattle, a rapid and cost-effective method is necessary to assess their semen quality under field conditions. Bright field mi-croscopy is considered a reliable and inexpensive tool, which can be applied in farm or breeding places. Belgian Blue cattle are notoriously sensitive to heat stress, and with the tropical climate that exists in most Asian countries, it is important to investigate how they react on heat stress. Belgian Blue bulls are double-muscled, have smaller scrota and less cooling properties which is increasing their susceptibility to heat stress, leading to lower fertility. It was the aim of this study to evaluate the quality of cryopreserved semen of Belgian Blue bulls collected under heat stress (August) and non-heat stress conditions (March) using rapid and easy staining methods.Design and methods : We evaluated DNA integrity using toluidine blue staining, acrosome integrity using rose bengal, mitochondrial activity using diaminobenzidine 3’3, and plasma membrane integrity using eo-sin nigrosin. Statistical analysis was done with SPSS by comparing the mean percentages of frozen sperm in each technique by paired t-Test. The correlation between each staining technique was done by Pearson test. The significance level was set up with 5% (statistical differences were considered significant if P < 0.05). Results : We found that the mean percentages of DNA integrity, acrosome integrity, plasma membrane in-tegrity, and mitochondrial activity of non-heat stressed semen (93.03 ± 1.30, 86.47 ± 1.88, 51.55 ± 2.23, and 91.11 ± 2.34 respectively) were higher compared with the heat stressed semen derived from Belgian Blue bulls (5.08 ± 1.04, 59.16 ± 2.56, 34.92 ± 1.15, and 81.22 ± 3.18) (P <0.05)). Conclusion : Using several rapid and cost-effective staining techniques, we found that frozen-thawed Bel-gian Blue semen collected under non-heat stress conditions had a significantly better quality of DNA in-tegrity, acrosome integrity, mitochondrial activity, and plasma membrane integrity rather than semen col-lected under heat stress conditions.Keywords: heat stress, non-heat stress, Belgian Blue sperm, staining technique, field condition

P05Is the use of SpermMobil to activate immotile spermatozoa a valid approach, looking at embryo qual-ity and clinical outcome? L. Van Landuyt, I. Mateizel, P. Drakopoulos, V. Vloeberghs and G. VerheyenCentrum voor Reproductieve Geneeskunde, UZBrussel

Objective: SpermMobil is a ready-to-use theophylline for activation of motility in immotile (but vital) sper-matozoa, and is used in order to distinguish vital and non-vital immotile spermatozoa for ICSI. A faster selection of viable sperm and higher fertilization, embryo quality and pregnancy rates were previously observed in patients with obstructive azoospermia (Ebner et al. 2011). In a small sibling-oocyte in-house validation test, SpermMobil was found to reduce sperm searching time in frozen-thawed testicular biop-sies, with equivalent fertilization and embryo quality. The objective of this retrospective analysis was to evaluate the use of SpermMobil (Gynemed, Germany) when immotile spermatozoa had to be selected from ejaculates or testicular biopsies. The outcome in terms of embryo development and live birth delivery rate was analysed.Study design/methods: The study includes ICSI cycles in which SpermMobil exposed spermatozoa were in-jected in either part of (27 sibling oocyte cycles) or all of the oocytes (120 cycles, 100% SpermMobil cycles) between July 2013 and February 2016. In the 27 sibling cycles, fertilization and embryo quality were compared between the motile (86 oocytes) and SpermMobil-exposed spermatozoa (144 oocytes). In 120 cycles with 100% SpermMobil (1070 oocytes),

25

fertilization, embryo quality and clinical outcome were evaluated. In total, spermatozoa originated from fresh or frozen ejaculates (n=42), fresh epididymal sperm (n=6) and fresh or frozen testicular biopsies (n= 99) of which 82 were from non-obstructive azoospermic patients.Results: In sibling cycles, fertilization was similar between motile and immotile-SpermMobil exposed sper-matozoa (69/144, 47.9% versus 41/86, 47.7%). After the use of SpermMobil, the percentage of good-quality embryos was higher on day 3 (53/69, 76.8% versus 20/41, 48.8% (p=0.005), but not on day 5 (15/35, 39.4% versus 6/21, 28.5% (p=0.579). In the 100% SpermMobil cycles, fertilization rate was 42.5% (455/1070), good embryo quality on day 3 was 41.8% (190/455) and on day 5 was 18.5% (27/146). Transfer rate was 83.5% (81/97, 23 cycles were freeze-all cycles) and pos hCG rate was 40.7% per transfer (33/81). Clinical pregnancy rate and live birth delivery rate were 25.9% (21/81) and 20.5% (16/78, 3 unknown outcomes). Conclusions: Using SpermMobil to select vital immotile sperm is a valid approach, resulting in reassuring embryo development and a live birth delivery rate of 20.5%. This analysis offers the reassurance that Sper-mMobil is a simple and useful method to overcome difficult ICSI cases with immotile spermatozoa, even in non-obstructive azoospermia, and renders acceptable embryo quality and live birth delivery rates.

P06Anonymous sperm donors’ attitude towards donation and the release of identifying informationF. Mahieu, W. Decleer, K. Osmanagaoglu, V. ProvoostAZ Jan Palfijn Gent

Introduction: Belgian legislation allows only strictly anonymous gamete donation and known donation (donation to a recipient known by the donor). Recently, an amendment of the legislation was proposed to grant donor offspring, as of 18 years old, the right to claim identifying information about their donor.Objective: The aim is to explore the attitude of actual spermdonors towards donation and the release of identifying information and to investigate which donors would be willing to donate when anonymity would be prohibited by law.Design and Methods: All men who were accepted as sperm donors (n = 242) by AZ Jan Palfijn Hospital (Ghent, Belgium) were invited to complete an anonymous online survey. The response rate was 65.5%. Results: One in five (20.1%; n = 30) would continue sperm donation upon a legislation change towards identifiable donation. Three in four donors (75.2%) would agree to provide basic non-identifiable informa-tion about themselves and one in three (32.9%) would provide extra non-identifiable information such as a baby photo or a personal letter. Almost half of the donors (45.6%) would agree to donate in a system where the hospital can trace the donor at the child’s request and contact the donor, leaving it to the donor to decide whether or not to have contact with the requesting donor child.Conclusion: These findings show that only one in five current donors would continue to donate when iden-tifiable. The study also demonstrates that current donors think more positive about alternative options and that nearly half of them are willing to be contacted by the hospital at the donor child’s request, provid-ing the donor can decide at that time whether or not to release his identity. P07Factors associated with colostrum quantity and quality in Belgian Blue cowsH.S.A. Tuska1, G. Residiwati1, K. Verdru1, A. Raes1, D. Santoro1, Budiono2, A. Van Soom1, G. Opsomer1

1Department of Reproduction, Obstetrics, and Herd Health, Ghent University, 9820 Merelbeke, Belgium2Gajayana University, Malang, East Java, Indonesia

Objectives : Colostrum is a paramount feed for newborn calves. Bovine colostrum is secreted from the mammary gland during the first 24 h after calving and contains a highly significant source of nutrients, nu-traceuticals and various bioactive compounds promoting the growth and development of newborn calves. Good quality colostrum (i.e., containing high levels of Ig) fed as soon as possible after birth, is a necessity to decrease disease susceptibility and neonatal calf mortality. Identification of the best quality colostrum has always been a major challenge. The present study aimed to evaluate the impact of several maternal factors (including parity, age, month of birth) and the newborn calves (birth weight, gender, and birth month) on the quantity and quality of colostrum of Belgian Blue (BB) cows. Design and methods : The data include 346 records of BB calves born in the Clinic of Reproduction and Obstetrics at the Faculty of Veterinary Medicine, Ghent University (Belgium), collected between 2017 and

2018. The quantity and quality of colostrum was measured using a colostrum densimeter as soon as possible after the cows were delivered by C-section. Data were analyzed using SPSS using 0.05 as significance level. Results : We found that there were significant associations between the quantity of colostrum and parity, cow age, as well as calf birth month (with P = 0.00; 0.00; 0.00 respectively). The highest and lowest mean colostrum produc-tion was found in cows that were of third (3.44 liter) and first (1.50 liter) parity respectively. The highest (3.47 liter) respectively lowest (1.42 liter) amounts of colostrum were produced by cows that were 6-6.99 and 1-1.99 years old. Moreover, colostrum production was on average higher in cows that calved in spring (3.08 liter) versus those that calved in autumn (2.50 liter). Further, there were significant associations between calf birth weight and calf gender (P = 0.015) and between calf birth month and calf birth weight (P = 0.022). Male calves (63.13 kg) were significantly heavier than female ones (55.84 kg). Also, calves born in the summer (72.84 kg) were heavier than those born in spring (54.69 kg).Conclusion : Present data show that in the BB breed, neonatal calf weight and colostrum production are associated with multiple environmental factors. The latter is important in order to minimize the loss of neonatal calves. Keywords: colostrum quantity, colostrum quality, Belgian Blue, cow, calf

P08Luteal phase support in IVF: contradiction between evidence-based medicine and real-life practicesF. Di Guardo1,2, H. Midassi3, A. Racca1, H. Tournaye1, M. De Vos1, C. Blockeel1

1Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium, 2 De-partment of General Surgery and Medical Surgical Specialties, Gynecology and Obstetrics Section, University of Cata-nia, Catania, Italy, 3 Polyclinique Ibn annafis, Sfax, Tunisie.

Introduction: luteal phase support in assisted reproduction cycles has been widely investigated during the last 10 years. Although there is strong scientific consensus reporting that progesterone represents the preferential product for luteal phase supplementation, when to start, which is the best route, dosage and duration of progesterone ad-ministration, and whether there is a place for additional agents is still under debate. Nevertheless, fertility special-ists do not always adhere to evidence-based recommendations in their daily clinical practice. In this context, the aim of this worldwide web-based survey is to document the currently used protocols for luteal phase support in clinical practice and appraised tendencies of drug prescription behavior comparing them to the existent evidence-based literature.Material and methods: in the first phase of the survey (April-May 2017), the questionnaire was sent to physicians based in Tunisia. In the second phase (August-October 2018), the survey was internationally expanded including physicians involved in ART worldwide, in order to increase the generalizability of the study data. 1480 physicians received the survey questionnaire which was sent by secure e-mail. 148 gynaecologists/reproductive endocrinolo-gists involved in ART from 32 countries returned completed questionnaires. The survey was conducted in agreement with the GDPR privacy policy.Results: most of the clinicians (71%) started progesterone luteal phase support on the day of egg collection. Vaginal progesterone administration was used alone by 80% of doctors or in combination with intramuscular or oral routes (4% of doctors). As a single agent, intramuscular progesterone was used by 6% of doctors while oral progestin and subcutaneous progesterone were used by 5% of clinicians respectively. HCG use for luteal phase support had been completely abandoned. Progesterone was administered until 8-10 weeks’ gestation in 35% of respondents and until 12 weeks of pregnancy in 52% of respondents. Conclusion: vaginal administration represented the preferred route for luteal phase support. However, this survey highlighted also the increased tendency of using other routes, apart from the vaginal, such as subcutaneous and oral. The increasing use of the oral route reported in the survey, both alone or in combination with the vaginal route confirmed the clinical practice shift expected due to the recent evidences available on oral progestin about its safety and tolerability. Furthermore, in spite of the lack of scientific evidence supporting the continuation of luteal phase support until 12 weeks’ gestation, this practice was used by more than half of the clinicians surveyed highlighting the contradiction between evidence-based medicine and real-life practices. Finally, the intramuscular route seemed to represent still a considerable option by clinicians.

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BSRM and VFS acknowledge the following sponsors

PLATINUM SPONSORS

GOLD SPONSORS

SILVER SPONSORS AlfaSigmaBesins HealthcareCryos InternationalEuropean Sperm BankIM ServicesMedifirstMellowood Medical

Sponsors Abbott rapid diagnostics medicalCryo SolutionsDensmoreLabotect Labor-Technik-Göttingen GmbHLBT Testing and CalibrationMicropticSTB zorg

We thank Gedeon Richter & Merck for the symposia

© COOK 10/2018 RH-D45768-EN-F

MEDICAL

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© COOK 10/2018 RH-D45768-EN-F

MEDICAL

Place embryos with confidence.

The original

Guardia embryo transfer catheters are designed for ease of access:

• The bulb-shaped tip promotes passage

through the cervix and into the uterine cavity.

• The curved guide catheter and soft, flexible

transfer catheter may aid in navigation.

Guardia™ Access Curved Embryo Transfer Catheter

Guardia™ AccessET Curved Embryo Transfer Catheter

Guardia™ Access Nano Curved Embryo Transfer Catheter

With EchoTip®

Smaller diameter

cookmedical.eu

CustomerService

Belgium/Flemish: +32 27001633, nl.orders@cookmedical.comBelgium/French: +32 27001633, be.orders@cookmedical.com

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1. Bontekoe et al. Adherence compounds in embryo transfer media for assisted reproductive technologies (Review). Cochrane Database syst Rev.2014. Published by John Wiley & Sons, Ltd. 2. Urman et al. Fertility and Sterility 90 (3). 2008. 3. Turley and Moore. (1984). Biochem Biophys Res Commun 121. 4. Campbell et al.(1995). Hum Reprod 15 (Suppl 6). 5. Behzad et al. (1994). Biol Reprod 51. 6. Yaegashi et al. (1995). Hum Pathol 26. 7. Stojkovic et al.(2003). Biol Reprod 68.

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