Association of Clinical Embryologists 2009 Annual Meeting Abstracts

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Establishing a model of prenatal oogenesis

Geraldine M Hartshorne¹, Emma C Spikings¹ & Emeka Oloto²

¹Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, and ²British Pregnancy Advisory Service, Blackdown, Leamington Spa

E-mail: [email protected]

We are developing a model to study the stages of human oocyte development that occur before birth. Our previous work used fresh tissue, however, we now wish to use cryopreservation, to improve the efficiency of tissue handling for research. We are also establishing methods of viewing oocytes in three dimensions, impossible using our previous cytogenetic methods.

The project was approved by Warwickshire REC. Ovaries were removed from female fetuses after second trimester termination of pregnancy and placed into hepes-buffered medium for transport to the research laboratory. Small pieces were frozen by either (1) slow freezing using DMSO/sucrose as cryoprotectant or (2) vitrification using Medicult kits and cryoleaf. Some tissue was cultured on transwell membranes.

Tissue fragments, fresh or after thaw, were stained with either (a) calcein and ethidium (Invitrogen) for nuclear and cytoplasmic components, or (b) JC1, for mitochondria. Tissues were viewed using a Zeiss LSM510 laser-scanning confocal microscope with UV-argon and HeNe lasers.

Ovaries were obtained from eight fetuses 12-23 weeks gestation. Samples frozen and thawed by either method showed similar cellular viability from fluorescent staining. The tissue remained viable after thawing/warming, as shown by preliminary tissue culture results. Confocal microscopy revealed clusters of oocytes within fresh tissue. In some cases, the oocytes appeared to persist and possibly develop further in tissue culture.

We have successfully vitrified human fetal ovarian tissue. Its application for ovary tissue has not previously been described in humans.

Tissue culture supports the continued development of oocytes in vitro, as demonstrated by confocal microscopy. This confirms our previous observations using cytogenetic spreading methods, and will allow improved molecular analyses in future.

We thank University of Warwick and bpas for support.

Converting Poor IVF Responders to IUI in an NHS IVF Clinic – A Retrospective Audit

Helen Hunter, Yvonne Wilson, Annette Lloyd, Diane Critchlow, Michael Hooper, Ruth Arnesen, Daniel Brison, Cheryl Fitzgerald, & Greg Horne

Regional IVF Unit, Department of Reproductive Medicine, St Mary's Hospital, Manchester M13 OJH

E-mail: [email protected]

Due to NHS funding restrictions, patients developing only one or 2 good size follicles during an IVF cycle are occasionally converted to IUI as an alternative to cancelling the cycle outright. This is possible where one or both oviducts are patent and semen analysis results at clinic were within IVF criteria. This study is a retrospective audit of our results for these patients between November 1998 and April 2007.

53 IUI cycles resulting in insemination from 43 patients were included in the audit. The mean female age was 36.3 (range 28-40), mean duration of infertility 7.3yrs (1-21yrs). One clinical pregnancy resulting in a live birth was achieved (clinical pregnancy rate [CPR]/live birth rate [LBR]1.9% per insemination, 2.2% per patient).

This compares unfavourably with our stimulated husband IUI results from the same period: 355 IUI cycles, 350 inseminations, 153 patients, mean female age 33.1 (range 23-40), mean duration of infertility 6.5yrs (1-17yrs) (CPR 9.9% per cycle, 10% per insemination, 22.9% per patient).

This audit demonstrates that this group of poorly responding IVF patients have a very low chance of success with IUI; although it is relatively inexpensive and may therefore be considered a cost-effective way of concluding a suboptimal IVF cycle. Patients should, however, be counselled accordingly.

A critical role for the PI3K/AKT pathway in self renewal and cell survival in human embryonic stem cells

Robbie Kerr^1,2, Daniel R Brison^1,2, & Susan J Kimber²

North West Embryonic Stem Cell Centre, ¹Department of Reproductive Medicine, Saint Mary's Hospital, Manchester M13 OJH, and ²Faculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton St, Manchester M13 9NT

E-mail: [email protected]

Human embryonic stem (hES) & Embryonal carcinoma (EC) cells form an ideal model system to study early human embryonic development as they are analogous to the transient pluripotent stem cell population in the pre-implantation blastocyst inner cell mass (ICM). Regulation of proliferation and differentiation of the ICM is critical to our understanding of early embryonic development and formation of the primary germ layers after implantation. The phosphoinositide 3-kinase (PI3K) signal transduction pathway is well documented as a mediator of cell growth, proliferation and survival in other systems. We now demonstrate a role for PI3K in the regulation of self renewal in Human ES & EC cells. PI3K signaling is active in human blastocysts, the NTERA-2 EC cell line & the hES cell line HuES-7. Treatment of the NTERA-2 cell line with the reversible PI3K inhibitor LY294002 results in differentiation, as indicated by reduced expression of the cell surface markers of ES cell pluripotency SSEA-3 & TRA1-60 and the pluripotency associated transcription factors, Oct4 & Nanog. PI3K signaling in EC and ES cells regulates cell proliferation and cell survival. Cells treated with LY294002 accumulated in the G₁ phase of the cell cycle and showed a significant increase in apoptosis. The PI3K/Akt pathway may facilitate this role in cell survival via its effects on glucose uptake and metabolism, processes vital in early embryonic development. Our results indicate that treatment with LY294002 results in a decrease in the expression of the major glucose transporter GLUT1 in EC cells. These data clearly demonstrate a role for PI3K/Akt signaling in cell proliferation, survival and self renewal in human EC cells and facilitate the proposal of a model to explain this regulation via interactions with other signaling pathways.

What makes a viable human embryo?

S.J. Kimber¹, S.F. Sneddon^2,3, D.J. Bloor¹, A.M. El-Bareg¹, B.A. Lieberman^2,3, & D.R. Brison^1,2,3

¹Faculty of Life Sciences, ²Faculty of Medical and Human Sciences, University of Manchester, Manchester, M13 9PT, UK, and ³Department of Reproductive Medicine, St Mary's Hospital, Manchester M13 OJH, UK

Thirty years after the birth of Louise Brown there is still an urgent need for better understanding of the different factors which influence development of the pre- and peri-implantation human embryo in order to further optimise assisted reproduction technologies. Little is understood about the regulation of gene expression in human preimplantation embryos, yet such knowledge will greatly enhance our understanding of what makes for a healthy implantation-competent blastocyst. Over the last 10 years we have been addressing the expression in developmentally significant genes in early human embryos and recently the way in which growth-enhancing proteins (growth factors or cytokines) stimulate the development of human embryos in culture, with a view to improving the effectiveness of in vitro fertilisation (IVF) treatment. We have analysed the expression profile of a number of genes throughout preimplantation development, including those involved in cell adhesion, communication, survival and fate, and responses to metabolic stress. The expression of these genes has also been analysed in response to growth factor stimulation. Patterns of gene expression were successfully analysed in a total of 43 human embryos, cultured either with or without growth factors, using a method of single cell analysis to amplify the transcripts present in each individual embryo. Developmental expression of a number of genes was similar to that seen in murine embryos (e.g. Oct-4, Cdx-2, Nanog). However Gata-6 is expressed throughout preimplantation development in the human. Embryos cultured in the absence of growth factors were compared to those that were cultured in either Insulin like growth factor (IGF) 1, Leukaemia inhibitory factor (LIF) or heparin-binding epidermal growth factor (HB-EGF). These factors are known to stimulate the development of human embryos, but nothing is known of the mechanisms involved. Our data show that culture in HB-EGF and LIF appear to facilitate human embryo expression of a number of genes: Erb-B4 (LIF) and LIF-R and DSC-2 (HB-EGF) while in the presence of HB-EGF no blastocysts expressed Eomes and when cultured with LIF only rare blastocysts expressed Taube Nuss. These data are important in addressing the molecular mechanisms by which growth factors stimulate development of human embryos. Results from this study will improve the understanding of cell fate decisions in early embryos, which has important implications for both IVF treatment and hES cell derivation.

Validation of manual and automated cryopreservation procedures of human spermatozoa

Kyamidou E, Campbell B, Newton T, Hopkisson J, & Tomlinson MJ

School of Human Development, Fertility Unit, Nottingham University Hospital, Queen's Campus, Derby Rd, Nottingham NG7 2UH

E-mail: [email protected]

Introduction. Although the cryopreservation of human embryos and oocytes is relatively complex and requires carefully programmed cooling, the vast majority of Assisted Conception Units still cryopreserve sperm using simple nitrogen vapour cooling (VC). Vapour cooling protocols vary enormously, have little support scientifically and are rarely validated against controlled rate freezing (CRF). This study therefore aims to scientifically validate a simple vapour cooling method against established computer controlled cooling.

Materials & Methods. Preparatory measurements recorded the temperature fluctuations at different heights from the coolant source as a function of time, which enabled the development of a cooling protocol and mimicked that of an established CRF method (2°/min to −5° and 10°/min to −80°C and freefall beyond −135°C). 50 samples of either semen or washed sperm preparation were divided 50:50 and cooled using either the new VC protocol or CRF. Sperm concentration and motion parameters were analysed pre-freeze and post thaw using CASA and supported by manual methods.

Results. There are no overall significant differences between the two methods when post-thaw recovery (motile concentration) was analyzed. As many individual samples showed improved post thaw recovery results using VC as did with CRF. There were no significant differences in any of the sperm motion parameters provided by the CASA analysis between methods.

Conclusions. Manipulation of a simple VC protocol to give a cooling rate equivalent to CRF provides comparable post thaw results. By documentation of this cooling rate and adhering to the procedure rigidly, we believe that simple VC can be considered a fully validated method for the routine cryopreservation of sperm.

Maximising Frozen-Thawed Sperm Survival: Three Cryoprotective Media and Two Freezing Techniques

Carleen A. Gbeho

The Assisted Conception Unit, University College Hospital, London, UK

E-mail: [email protected]

The storage of semen in a frozen state is an invaluable tool, enabling the storage of sperm for future use but this process can lead to large losses of motile sperm because of the damaging effects of extreme temperature change (Stanic et al., 2000). Samples are mixed with cryoprotective media to attempt to preserve the motility and progression of sperm during the freezing process. Key cryoprotective components, often glycerol and sucrose, protect the cells from ice and osmotic cell damage (Lovelock & Polge, 1954). No definite protocol has been proposed for semen freezing although many groups have researched methods giving increased post-thaw results. Protocols which make use of a faster cooling stage often produce the better results (Hammadeh et al., 2001).

Semen samples donated to research were prepared for freezing with the commercial cryoprotective media MediCult CryoSperm™ (CS), MediCult Sperm Freezing Medium™ (SFM - both MediCult, Jyllinge, Denmark) and SAGE Assisted Reproduction Products™: Quinn's Advantage™ Sperm Freeze (Rochford Medical Ltd, Oxford, UK - SAGE) and the vials either cryopreserved by a slow-rate, machine-controlled protocol or rapidly by suspension in liquid nitrogen vapour. All were thawed by 5 minute incubations at 37°C and the motility (%) and progression (scored out of 4) analysed. A subset was prepared for flow cytometry via the Sperm Chromatin Structure Assay method (Evenson & Jost, 1994) and the percentage of DNA fragmentation within each group obtained.

Before cryopreservation the mean percentage of motile sperm±standard deviation was 56.57±8.17%, and post-thaw this became 22.50±8.12% for machine frozen samples and 15.22±6.76% for vapour frozen samples. SFM provided significantly better post-thaw motilities (17.12±7.84%) compared to CS (15.04±6.94%, p = 0.046) and SAGE (13.50±5.60%, p = 0.011). Chromatin appeared to be protected most by the use of slow-rate freezing (24.36% fragmented compared to 26.46% fragmented with vapour-freezing).

Differing concentrations of cryoprotectants could be the reason for variations in results obtained between media, and the slower cooling method was shown to have a positive effect on post thaw sperm parameters. These results may aid in optimising post-thaw survival of cryopreserved sperm samples.

Donor IUI in the post-anonymous world: The effect of age, ovarian stimulation and sexual orientation

Linara E, Smith V.J.V, Venkat G, Nair S, Simons E, & Ahuja K K

The London Women's Clinic, 113-115 Harley Street, London, W1G 6AP

E-mail: [email protected]

The consequences of the removal of anonymity for sperm donors in terms of quality and number have been a cause of concern to patients, registered clinics and policy makers. We have studied the impact of these changes on clinical practice and donor compliance in our clinic. A further aim was to determine the role of age, ovarian stimulation and sexual orientation on the live birth rate (LBR) following intrauterine insemination (IUI).

A retrospective analysis of 503 consecutive patients (1036 IUI cycles) who attended for treatment during 2005-6 was performed. Single women and same sex couples contributed to 81% of the treatment cycles. 95% of patients used donor sperm and 5% of patients used partner sperm. The age range of the women was 21-49 (mean 36.3 ±4.5). 437 cycles (183 patients) received no stimulation whilst 599 (320 patients) received mild ovarian stimulation.

Analysis by x²-test confirmed an inverse graded association between age and LBR (p<0.001) (table).

During this period, natural cycles achieved higher LBR per cycle (19.9%) compared to stimulated cycles (14.3%) (p>0.05).

Table

Lesbian women (mean age = 35.1±4.2) achieved significantly higher LBR (21.4%) when compared to single women (mean age = 38.3±3.9), (12.5% LBR) (p<0.05). This is probably due to the age factor though the possible implication of the psychosocial status should not be overlooked.

We conclude that in terms of volume and quality the changes in donor anonymity had minimal effect on the clinical practice.

Transport IVF – an examination of its efficacy

R Lunt, C Kingsland, & S Troup.

Hewitt Centre for Reproductive Medicine, Liverpool Women's Hospital, Crown Street, Liverpool L8 7SS, UK

Transport IVF is often used by assisted conception units primarily for patient convenience, but also to obviate the need for expensive laboratory facilities and reduce regulatory burden. The Hewitt Centre (HC) has transport IVF arrangements with four centres within a 45 mile radius. Patients have the significant part of their treatment managed at the transport centres (ovarian stimulation, egg collection, aftercare). Generally, eggs were identified by an embryologist at the Transport Centres then transported to the HC by the male partner where they undergo routine IVF/ICSI attending two days later for embryo transfer (ET). In transport frozen embryo transfer (FET) cycles patients only attended the HC for ET. The aim of this study was to evaluate the efficacy of our large transport IVF/ICSI/FET service.

Data were analysed for the period 01/01/03–31/07/07, a total of 1435 transport IVF/ICSI/FET cycles were included. When compared to the HC no significant differences were noted in terms of patient age or number of eggs collected. Higher fertilisation rates were observed at some transport centres compared to the HC (eg for IVF, Chester:1852/2902[63.8%] vs HC:9443/15680[60.2%], p = 0.0003). Similarly, the incidence of clinical pregnancy/ET for IVF was comparable (HC:434/1557[28%]; Chester: 90/272[27%]; Arrowe Park: 10/28[36%]; Whiston: 1/8) with Leighton being significantly higher 28/64[44%](p = 0.0058). Results for ICSI and FET were comparable with the HC throughout.

These data demonstrate that performing transport IVF/ICSI/FET causes no adverse effect on fertilisation or chance of pregnancy. These data support the continued use of transport IVF as a way of maximising patient convenience although consideration now needs to be given to the regulatory burden recently placed on the transport centres as a result of the EUTD.

First live birth of an unaffected child in the UK following PGD for Type 2 Gaucher Disease

Colleen Lynch¹, Matt Studt², John Kitchen², Susan Brown², Simon Fishel¹, & Mark Hughes²

¹CARE Fertility, John Webster House, 6 Lawrence Drive, Nottingham Business Park, Nottingham NG8 6PZ, and ²Genesis Genetics Institute, Genomics Centre at Samaritan, Suite A2064, Detroit, MI 48213

E-mail: [email protected]

Type 2 Gaucher Disease is a rare inherited metabolic disorder. Mutations in the gene encoding acid beta glucosidase result in an inability to efficiently break down the lipid glucocerebrosidase. This leads to its accumulation in cells and clinical manifestations including hepatosplenomegaly and extensive and progressive brain damage. Two cycles of PGD were performed for a couple who had already lost a child at 7mths to the disease. The female partner carried a common point mutation in exon 10, leading to the substitution of an amino acid. The male partner carried a rare point mutation in exon 6, leading to the introduction of a premature stop codon. The deceased child was shown to be heterozygous for the maternal mutation and homozygous for the paternal mutation. However, this was inconsistent with the clinical phenotype and was found to be due to the failure of the normal maternal exon 6 to amplify due to a SNP in the primer binding region. In the second treatment cycle, 5 embryos were biopsied on day 3, 2 of which were transferred on day 5. One was homozygous normal and the other a maternal carrier. A singleton pregnancy ensued with prenatal diagnosis confirming the foetus to be unaffected by type 2 Gaucher Disease. A normal birth via caesarean followed. To the best of our knowledge this is the first case of its kind in the UK. NHS funding had been awarded for a maximum of 3 cycles of IVF with PGD.

Current trends in funding provision for patients requesting Preimplantation Genetic Diagnosis

Colleen Lynch & Simon Fishel

Care Fertility, John Webster House, 6 Lawrence Drive, Nottingham Business Park, Nottingham NG8 6PZ

E-mail: [email protected]

Demand for preimplantation genetic diagnosis has increased as people's awareness of the hereditary nature of some conditions, and their reproductive options, grow. This seems to have coincided with, or lead to, a greater willingness on the part of health authorities and primary care trusts to provide funding. However, as with IVF funding, provision remains a “postcode lottery”. Whilst some authorities recognise PGD as an alternative healthcare option, there are still other PCTs and NHS providers considering funding for PGD employing their IVF criteria, which generally includes not having any existing children. Where this criteria is applied to couples wishing PGD it rarely takes in to account the health of the existing child or prognosis. The overall situation, though, is encouraging, as primary care trusts and health authorities begin to weigh up the cost of preimplantation genetic diagnosis with the life-time cost of an ill child. We have analysed the percentage of our patients securing NHS funding on a yearly basis, how many cycles were provided for, and also the situation of couples who had to pay privately for treatment including variation in criteria for funding provision.

Lifestyle and semen quality

Allan A. Pacey

Academic unit of Reproductive and Developmental Medicine, University of Sheffield Medical School, Level 4, The Jessop Wing, Tree Root Walk, Sheffield, S10 2SF

E-mail: [email protected]

The relationship between lifestyle and semen quality is of popular interest to patients and the general public. Newspapers and television programmes seize on new research claims and often present the results to the public uncritically without reference to the biological complexities of the male reproductive system. Many factors influence semen quality, including: (i) the inherent sperm production capacity of the testicles; (ii) the period of time since the last ejaculation; (iii) general health; (iv) the duration and nature of pre-ejaculatory sexual stimulation; and (v) the structural and functional integrity of the male reproductive tract, ejaculatory ducts and accessory glands. Each of these factors will have its own bearing on ejaculate quality and any proposed lifestyle factors need to be interpreted with them in mind. This lecture will discuss two important checkpoints where lifestyle factors may have an important effect on adult semen quality. First is the pre-natal exposure of the male foetus to risk during its time in utero through maternal behaviours such as diet, the use of cosmetics and smoking that may have a detrimental effect on testicular development that in turn impact on adult testicular function. Second is the exposure of adult men to factors that affect the function of the post-pubertal testicle and risks are thought to include the effect of temperature (e.g. hot baths, tight underwear), poor diet and a number of chemical exposures either in the workplace or at home. Whilst lifestyle is an important risk factor in male infertility, its potential effects should be taken in context with other biological variables and known medical conditions.

Efficient derivation of embryonic stem cells from human blastocysts using animal product-free conditions for inner cell mass isolation

Priddle H¹, Chamberlain S¹, Devlin L¹, Barbadillo Muňoz M¹, Smith N², Morris T³, Parkin T², Watson S³, Hopkisson J¹, Sjoblom C¹, Campbell B¹, & Young LE¹

¹School of Human Development, Queen's Medical Centre, Nottingham, NG7 2UH, ²Department of Cytogenetics, Nottingham City Hospital, NG5 1PB, and ³Academic Unit of Cancer Studies, Queen's Medical Centre, Nottingham, NG7 2UH

E-mail: [email protected]

Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of the blastocyst may provide a promising resource for transplant medicine. For safe clinical use, animal products employed in hESC derivation must be circumvented. The ICM is commonly isolated by immunosurgery (using animal antibodies and complement) as trophectoderm is believed to inhibit ICM expansion. Here we report efficient (40%) derivation of hESCs by whole embryo culture and mechanical dissection to isolate the ICM.

Donated embryos were cultured in GIII media (Vitrolife) resulting in 5 blastocysts. Zona pellucidae were removed using acid Tyrodes, and embryos were cultured with mitotically inactivated mouse embryonic fibroblasts (MEFs). After 5 days, the ICM was distinct, enabling isolation by dissection and plating onto fresh MEFs. For two blastocysts, further ICM expansion occurred resulting in a morphology characteristic of hESCs. Cells were successfully vitrified at early passage and continuously cultured to demonstrate self-renewal. The cell lines were characterised by karyotyping, immunocytochemistry, differentiation in vitro and in vivo.

NOTT1 and NOTT2 are karyotypically normal hESC lines (46,XX and 46,XY respectively) and have been cultured continuously for more than six months. Both lines express Oct4, Nanog, TRA-1-60, TRA-1-81 and SSEA-4, but not SSEA-1; and differentiate to three germ layers in vitro or in vivo. These lines are available from the UK Stem Cell Bank (R-07-001 & R-07-002).

Supported by the University of Nottingham.

A Prospective Randomised blind study to compare two flushing media on oocyte collection and fertilisation rates during in vitro fertilisation.

Jane Pritchard, Louise Reeve, Helen S Clarke, Karen L Martin, William L Ledger, & Rachel C Cutting.

Assisted Conception Unit, Centre for Reproductive Medicine and Fertility, Sheffield Teaching Hospitals (STH) NHS Foundation Trust, Level 1, Jessop Wing, Tree Root Walk, Sheffield

E-mail: [email protected]

Introduction. One of the main factors affecting the outcome of an IVF (in vitro fertilisation) cycle is the implantation potential of the embryos transferred back into the patient's uterus. The viability of the oocytes used to create these embryos therefore has a major impact on success rates. We wanted to assess whether using a complex heparinised culture medium, Sydney IVF Follicle Flush Buffer, is more effective than simple Hartmann's solution when used during follicular flushing during oocyte recovery.

Material and Methods. A Prospective randomised blind study was performed on 102 patients having IVF or ICSI treatment. Patients were randomised to have the follicles on the left ovary flushed with either heparinised saline or Sydney IVF follicle flush buffer at the time of oocyte recovery. The contralateral ovary was flushed with the other solution. Oocytes were cultured and assessed for fertilisation and embryo development.

Results. A total of 1066 follicles were aspirated yielding 807 oocytes. There was no significantly difference observed between Hartmanns compared to Sydney IVF follicle flush buffer for fertilisation rate of mature oocytes (risk ratio (RR) = 0.97; 95% confidence interval (CI) of 0.88–1.07), collection rate (RR = 0.98; CI 0.91 to 1.05), zygote quality (RR = 1.04; CI 0.89–1.23), embryo cleavage rate (RR of 1.02; CI of 0.92–1.14) and clinical pregnancy rate (RR = 1.08 CI 0.49–2.31). There was a slight increase in the number of good quality embryos (RR = 1.09; CI of 1.01 to 1.18) and the number of embryos frozen (RR = 1.54; 95% CI of 1.08 to 2.19) when Follicle Flush Buffer was used. Conclusions: Hartmann's solution would be continued to be used in the Assisted Conception Unit during oocyte retrievals as it provides a cheap alternative but is as effective as specialised culture media in supporting the oocytes during initial retrieval.

Factors affecting Percutaneous Epidydimal Sperm Aspiration (PESA) &Testicular Sperm Extraction (TESE) treatment

Shantal Rajah¹ & Jayanta Barua²

¹Brentwood Fertility, Essex Nuffield Hospital, and ²London South Bank University, Essex

E-mail: [email protected]

PESA and TESE technique are widely used to retrieve sperm for ICSI treatment in males who have Azoozospermia.

A total of 42 cycles of PESA/TESE cycles were performed for men with obstructive & non obstructive azoozospermia. 32 cases of PESA were successful in both non and obstructive males with normal FSH (<10.0). TESE was carried out in 10 cases who failed PESA. 5 cases (2male) TESE had high FSH.

Sperm count in PESA with obstructive (Ob) and non obstructive (NO) were 11.4 & 4.4 million/ml and motility 20&22%. In TESE 0.5million and <5%. Men ages range from 42 to 65yrs for Ob/PESA&TESE and 32 to 41 yrs for NO/PESA.

In PESA group18 cycles were women <35 and 6 were >35yrs in Ob/PESA group and 6 & 2 were in NO/PESA. In TESE 2&8 women <35 & >35 age. Fertility rate (FR) for <35 & >35 in Ob & NOPESA were 64, 67 & 50, 65%. FR for TESE was 43 & 36%. Cleavage rate(CLR) for <35 & >35 women were 76 & 50% for Ob and 75 & 55% for NOb/PESA & 50, 55% in TESE. Clinical pregnancy rate (PR) in <35 & >35yrs women in Ob & NO/PESA were 50, 20% & 60, 40% respectively. In TESE 40% & 0.

In our study, PESA treatment FR and PR is not affected by male age. There is no difference in FR between Ob and NOb/PESA in all male ages. FR & CLR&PR lower in TESE. High FSH had cleavage arrest. Cleavage rate is high in <35 women age gp than >35 in PESA but no difference in TESE. There is a significant higher PR with women age <35 in Ob, NOPESA & TESE than women >35 age gp.

Non-invasive metabolomic profiling of human embryo culture media correlates with pregnancy outcome

Emre Seli, M.D., Denny Sakkas, Ph.D., & David H. Burns, Ph.D. 300 George Street Suite 770 New Haven, CT 06511 USA

Objective. More than 100,000 assisted reproductive technology (ART) cycles are started yearly in the U.S. Partly due to our inability to select the best embryo(s) to be transferred, a mean number of 3.1 embryos are transferred in ART cycles using fresh nondonor oocytes. This leads to a 34.3% overall pregnancy rate, 29.0% of which result in multiple-infant live births. Similarly, a mean number of 2.5 embryos are transferred in ART cycles using fresh donor oocytes, achieving a 50.4% pregnancy rate, 44.7% of which result in multiple-infant live births (SART 2002). These statistics have remained essentially unchanged for several years and reflect a need for improvement over our current embryo selection methodology that is based on cleavage rates and morphology. We hypothesized that, embryos that result in pregnancy may be different in their metabolomic profile compared to embryos that do not, and that the difference may be detected by the rapid, non-invasive evaluation of the embryo culture media using targeted spectroscopic analysis and bioinformatics.

Materials and Methods. Prospective studies were conducted in one academic, and three private ART centers. Embryo culture medium of transferred embryos from ART cycles using fresh donor or nondonor oocytes were evaluated. Media (n = 619) were individually collected after embryo transfer, and analyzed using Raman, and/or Near Infrared Spectroscopy (NIR). Initially 69 samples were tested. In order to develop a model, the spectra obtained from each instrument were separately analyzed using a wavelength selective genetic algorithm (GA) to determine regions predictive of pregnancy outcome as determined by a logistic regression of the light attenuation from the wavelength included. To avoid random correlations, a leave-one out cross-validation was used. Sensitivity and specificity of predicting pregnancy (defined as presence of fetal cardiac activity) were calculated. Once spectral models developed were tested using embryo culture medium samples from different centers that perform multiple (n = 41) or single (n = 509) embryo transfers.

Results. Spectral profile describing differences in -CH, -NH and -OH concentrations showed distinct differences between culture media of embryos that resulted in pregnancy and those that did not. The ratio of the -CH to ROH content in the media that is reflective of oxidative stress was also different between the two groups. Using GA with Raman, three spectral regions associated with these molecular species were identified as predictive of pregnancy outcome. Compiled outcomes from the leave-one-out cross-validation of the logistic regression using the Raman measurements resulted in a specificity of 86% and a sensitivity of 76.5%. Analysis of the NIR required four wavelength regions, and provided a specificity of 75% and a sensitivity of 83.3%. These findings were confirmed in studies using embryo culture media samples collected from centers that use multiple embryo transfer and single embryo transfer.

Conclusion. Our findings suggest that a detectable difference exists in the metabolomic profiles found in culture media obtained from embryos that cause pregnancy compared to those that do not. The reported metabolomic parameters were established using two different forms of spectroscopic analysis, with samples from different centers, and achieved high sensitivity and specificity. Metabolomic profiling may serve as a useful methodology for rapid, non-invasive embryo assessment and selection. Consequently, metabolomic evaluation may make it possible to decrease the number of embryos transferred, lead to a decrease in multiple-infant live birth rates, and possibly also improve the pregnancy rates.

Potential for embryo mediated immuno-protection from uterine NK cells through co-expression of HLA-E and HLA-G at the cell surface of blastocyst trophectoderm

Valerie Shaikly^1,2, Anna Withey², Ian Morrison¹, Ayesha Shakhawat¹, James Cooper², Vladimir Ovsyankin², Mohamed Taranissi², Clare Noble², Richard Cherry¹, & Nelson Fernández¹

¹Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SQ, and ²Assisted Reproduction and Gynecology Centre London

E-mail:[email protected]

HLA-G and HLA-E are designated as ‘non-classical class I molecules'. Their expression has been observed in the placenta at the foetal maternal interface. HLA-G is thought to confer immune tolerance of placental tissues through binding with inhibitory receptors of T-lymphocytes and uterine NK cells. HLA-G protein has also previously been observed in human embryos but its function has yet to be elucidated. The aim of this study was to determine whether HLA-G and/or HLA-E were expressed on the trophectoderm of blastocysts; these cells of the embryo are involved in the formation of placental tissue and are exposed to the uterine environment during implantation.

Blastocysts were donated to this study with informed consent by patients at the Assisted Reproduction Centre. After zona pellucida removal embryos were double stained with the HLA-G specific monoclonal antibody MEMG9/goat anti-mouse IgG FITC conjugate followed by HLA-E specific antibody MEME07/goat anti-mouse APC conjugate. Embryos were fixed, whole mounted and analysed by high resolution confocal microscopy. Co-localisation analysis of the two fluorescent probes was performed using computer software that discriminates the intensity and the physical localisation of fluorescent spots.

HLA-G and HLA-E molecules were found to be co-expressed on the surface of trophoblast cells in all 12 blastocysts studied. Analysis of confocal images showed that HLA-E was almost always co-expressed with HLA-G. HLA-G was also detected alone, in the absence of a physical association with HLA-E.

Surface expression of HLA-E and its binding with NK cell inhibitory receptors is known to depend upon the expression of a nonamer peptide derived from HLA-class I molecules. Our results show that in trophoblast cells of human blastocysts expression of HLA-E requires co-expression of HLA-G. The resultant HLA-E and HLA-G complex may be important in the immuno-protection of the implanting embryo via inhibition of NK cell activity.

Clinically unusable oocytes as a source of embryos for derivation of human embryonic stem cells and for studies of gene expression

Sharon Sneddon^1,2, Paul DeSousa³, Susan Kimber¹, & Daniel Brison^1,2

¹Faculty of Medical and Human Sciences and Faculty of Life Sciences, University of Manchester, ²Department of Reproductive Medicine, St Mary's Hospital Manchester, and ³Roslin Cells, Edinburgh

E-mail: [email protected]

Progress in the study of early human development and in the derivation of human embryonic stem cell (hESC) lines has been limited by the scarcity of embryos available for research. Up to 30% of oocytes in any IVF cycle are unsuitable for use because they are immature or fail to fertilise. These oocytes retain some developmental potential; however the normality of these has yet to be established.

Patients donated immature or failed to fertilise oocytes after informed consent according to standard guidelines developed by the UK Human Embryonic Stem Cell Co-ordinators group. Oocytes were subjected to several treatments, including in vitro maturation, parthenogenetic activation and intracytoplasmic sperm injection. Resulting embryos were cultured for up to 7 days. A cohort of embryos underwent gene expression analysis. The majority of blastocysts were used for stem cell derivation.

50% of immature oocytes emitted a polar body after in vitro maturation, suggestive of progression to metaphase II. Fertilisation occurred in 58% of these with a blastocyst formation rate of 12%. Fertilisation occurred in 65% of activated or re-inseminated oocytes, with a blastocyst formation rate of 8%. One of these resulted in the hESC line RCMAN-1. Embryos expressed a range of transcripts and proteins at comparable levels to control embryos.

Embryos created from clinically unusable oocytes represent a valuable research resource. Derivation of hESCs from these has the potential to yield a supply of tissues for regenerative medicine as well as for use in the study of early human development.

Preliminary implementation of an elective single embryo transfer: a clinics and patients perspective

Nickolas Vringas, Jane Pritchard, Anne Mowforth, & Rachel Cutting

Assisted Conception Unit, Jessop Wing, Sheffield Teaching Hospitals, Sheffield, UK

E-mail: [email protected]

Background. Around 1 in 4 IVF births are twins, ten times the natural conception twin rate. The increased rates of morbidity, neonatal mortality and maternal complications associated with twin pregnancies have made this unacceptable and it is essential for clinics to move to elective single embryo transfer (eSET) policy in high risk patients. In advance of the HFEA issuing guidance for single embryo transfers our clinic has undertaken a gentle shift in offering eSET to good prognosis patients under the age of 35. In this study we wanted to acertain whether or not an increase in the number of eSETs had the desired effect of reducing the twin rate without damaging patients' chances to conceive. We also wished to assess patients' views on eSET with regards to the type of funding they receive and whether this has any influence on their decision making.

Methods. Questionnaires were collected from patients regarding opinions on eSET. In addition statistical analysis was performed on retrospective data regarding eSET, two embryo transfers and pregnancy outcomes.

Results. Preliminary results show that although 82.5% had received information about eSET only 32.5% of patients considered eSETs. 70% of patients would consider having eSET if further cycles were NHS funded. 48% of patients felt that at least three cycles should be funded. Analysis of the retrospective data showed that in ‘good prognosis' group pregnancy rate was not significantly affected (eSETs 39.2% and two embryo transfers 42.8%) by eSET and that the twin pregnancy rate was greatly reduced. (0% in patients having eSET compared to 15.8% in patients having 2 embryos replaced).

Conclusions. A barrier for patients to except eSET is clearly the lack of NHS funded cycles, although increased education regarding the chances of success with one embryo is achieving the desired affect of increasing the proportion of patients accepting this option. An increase in the number of single embryo transfers has had the desired effect of reducing the twin rate without a significant reduction of the clinical pregnancy rate. This data has given our clinic confidence to offer eSET more widely to selected patients.

The future of embryo freezing

Maureen J Wood

Department of Obstetrics and Gynaecology, University of Aberdeen, Foresterhill, Aberdeen, UK

E-mail: [email protected]

There is no doubt that embryo cryopreservation has a future in fertility management, even though, given recent advances in oocyte cryopreservation, there may be an increase in the number of women preferring oocyte storage. The demand for storage of supernumerary embryos will grow as we learn how to produce and identify high quality embryos that are resistant to cryo-injury, and as single embryo transfer becomes the norm. Wider application of PGD will add to the demand, and present the challenge of storing biopsied embryos that are likely to be hypersensitive to the stresses of cryopreservation. However, it is less certain how embryos will be cryopreserved in future. Vitrification is an attractive alternative to slow controlled rate freezing, but its reliability for routine use in a wide variety of clinics and the safety of the samples during storage remain to be proven. Interrupted rapid cooling, so-called “two-step” freezing, has been neglected for more than a decade, but may combine the advantages of controlled rate freezing and vitrification. The challenge facing the embryologist will be to devise a robust evidence based cryopreservation strategy for each patient. Currently there is a paucity of sound evidence on which to base decisions. Well designed trials are needed with live birth as the primary endpoint rather than intermediates such as implantation and pregnancy.

The effect of cytoplasmic displacement volume during ICSI on fertilisation and embryo quality

Woodward BJ¹, Montgomery SJ², Rai J², Bains H², Campbell KHS¹, Hartshorne GM², & Kennedy CR²

¹School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leics. LE12 5RD, UK, and²Centre for Reproductive Medicine, University Hospital, Clifford Bridge Road, Coventry, CV2 2DX, UK

E-mail: [email protected]

Introduction. ICSI involves aspiration of the oolemma into the injection pipette until it stretches so thinly that it eventually ‘snaps', as indicated by a sudden cytoplasmic rush. This study examined the effects of the volume of cytoplasm displaced during ICSI in terms of fertilisation and early cleavage.

Material & Methods. For each oocyte (n = 232), the length of cytoplasm aspirated into the injection pipette was noted at the points of ‘snap’ (oolemma breach) and ‘return’ (maximum length reached). The respective cytoplasmic volumes within the injection pipette were then calculated. The fertilisation status and Day 2 embryo quality were then compared according to whether the cytoplasmic displacement volume was considered small (<1000μm³), medium (1000–2000μm³) or large (>2000μm³). Statistical differences were analysed by chi-squared test (P<0.05).

Results. The mean cytoplasmic displacement volume observed during ICSI was approximately 1735μm³, ranging from 147μm³ to 6771μm³. There were no significant differences in fertilisation according to small (n = 59), medium (n = 91) or large (n = 82) cytoplasmic displacement. However, there was a trend for better quality embryos to develop from oocytes subjected to less cytoplasmic displacement, with 62.2%, 56.1% and 48.4% good quality embryos developing from oocytes in the small, medium and large cytoplasmic displacement groups respectively.

Conclusions. These results suggest that although fertilisation may be unaffected, the ability of the human embryo to develop optimally during early cleavage may be affected by cytoplasmic disruption at ICSI. Some publications have reported that embryo quality is poorer after ICSI than after IVF. This work may help to explain these observations in terms of differences in ICSI technique which may have consequences for embryo development.

A review of thawing strategy for frozen embryo transfer

D Yell, C Kingsland, & S Troup

Hewitt Centre for Reproductive Medicine, Liverpool Women's Hospital, Crown Street, Liverpool L8 7SS, UK

E-mail: [email protected]

The optimum number of pronucleate embryos that should be thawed for a frozen embryo transfer (FET) is an ongoing topic of discussion, with the aim being to optimise pregnancy rates whilst minimising embryo ‘wastage’. Currently, for two embryo transfers and when possible, embryos are thawed until four are available for culture. However, our unit does not re-freeze embryos resulting in some being discarded. The aim of this study was to determine the cleavage rate of pronucleate embryos following the freeze/thaw procedure, the number and quality of those discarded, and pregnancy rates according to the number of embryos available for culture.

400 FET cycles between January 2006 and June 2007 were examined. 1194/1567(76.2%) embryos survived the freeze thaw process with 1067/1194(89.4%) going on to cleave. 357/1067(33.5%) were discarded of which 202(56.6%) were of ‘high’ quality (grade 1 or 2). The clinical pregnancy rates (per ET) for patients having one, two, three or four embryos in culture were 1/32(3.1%), 11/99(11.1%), 12/73(16.4%) and 28/164(17.1%), respectively. Although the differences in pregnancy rates were not statistically significant there was a clear trend towards increased pregnancy rates where more embryos were cultured (e.g. one vs. three, p = 0.0886; one vs. four, p = 0.0727)

The increased pregnancy rates where more embryos were cultured suggest better quality embryos are available for transfer, perhaps a result of increased choice. However, cleavage rates and hence the number of cleaved embryos discarded are high. Clearly a balance needs to be found between achieving good pregnancy rates and ‘wasting’ embryos. In conclusion, it may be prudent to adopt a policy for thawing until only three embryos are available for culture.