Hum. Reprod. Advance Access published March 9, 2010

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1 Human Reproduction, Vol.00, No.0 pp. 1 6, 2010 doi: /humrep/deq059 Hum. Reprod. Advance Access published March 9, 2010 ORIGINAL ARTICLE Infertility Avoidance of weekend oocyte retrievals during GnRH antagonist treatment by simple advancement or delay of hcg administration does not adversely affect IVF live birth outcomes K.P. Tremellen 1,2,* and M. Lane 1,2 1 Repromed, 180 Fullarton Road, Dulwich, SA 5065, Australia 2 Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, SA, Australia *Correspondence address. Fax: ; kelton.tremellen@adelaide.edu.au Submitted on November 5, 2009; resubmitted on February 5, 2010; accepted on February 12, 2010 background: The use of GnRH antagonists in IVF treatment has many advantages over agonist long down-regulation, yet its uptake has been hampered by an inability to program the start date for gonadotrophin stimulation so as to minimize weekend oocyte retrievals (ORs). In this study, we retrospectively analyzed whether conducting a strict Monday to Friday OR program impacts on IVF outcomes. methods: A total of 1642 non-programmed IVF antagonist cycles were analyzed to determine if advancing or delaying the OR by 1 day from ideal to avoid Saturday or Sunday OR, respectively, had any impact on IVF outcomes. The IVF outcomes of Tuesday to Thursday served as a control as no modification in OR timing was required on these days. results: Advancing the OR by 1 day from the ideal resulted in a small but significant decrease in the number of oocytes collected and embryos created. Delaying the OR by 1 day from ideal resulted in a small increase in the number of oocytes collected and embryos created. However, deviation from the ideal day of OR had no significant effect on live birth rates. conclusions: It is possible to safely avoid weekend ORs during GnRH antagonist cycles by simply advancing an ideal Saturday OR to Friday, and delaying an ideal Sunday OR to Monday, without adversely impacting on IVF live birth outcomes. Key words: GnRH antagonist / IVF programming / weekend oocyte retrieval Introduction The evolving view among leading fertility physicians is that GnRH antagonist therapy provides a superior patient friendly IVF experience compared with the traditional long down-regulation agonist protocol, whereas still maintaining excellent pregnancy rates (de Klerk et al., 2006; Devroey et al., 2009). Despite this, many IVF units have been slow to make the transit from GnRH agonist to antagonist treatment protocols, primarily because GnRH agonist therapy has the advantage of allowing effective programming of the gonadotrophin start date, thereby allowing clinics to smooth work demand over the week whereas minimizing the risk of weekend oocyte retrievals (ORs). In 2006, our own IVF program began to move away from the traditional long down-regulation GnRH agonist protocol, with the transition to almost universal GnRH antagonist therapy being complete by late Long down-regulation GnRH agonist therapy is now only used for a few select indications such as oocyte donation, preimplantation genetic diagnosis cycles, and the medical management of severe endometriosis. However, the avoidance of weekend ORs did provide a challenge during this period of transition as our unit does not have the capacity for weekend theatre access to conduct ORs. Although we contemplated programming IVF treatment using the Combined Oral Contraceptive Pill (COCP) to minimize the risk of weekend ORs, we decided not to take this approach as it significantly delays IVF treatment and may increase gonadotrophin dose requirements, whereas decreasing viable pregnancy rates (Kolibianakis et al., 2006a, b; Griesinger et al., 2008). Furthermore, one study reporting on the use of the COCP to avoid weekend ORs in IVF antagonist cycles found that 22% of women still required a weekend OR despite the use of the COCP (Barmat et al., 2005). As an alternative, we elected to avoid weekend OR s by simply bringing forward the day of hcg administration in ideal Saturday ORs to allow for a Friday & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 2 Tremellen and Lane oocyte collection, while delaying ideal Sunday ORs to the Monday. It has already been established that delaying hcg administration by 1 day has no significant negative effect on GnRH agonist IVF outcomes (Dimitry et al., 1991), although a 2-day delay has been reported to be detrimental in GnRH antagonist IVF cycles (Kolibianakis et al., 2004). However, it is presently unclear if advancement or delay of hcg administration by 1 day has any negative consequences for GnRH antagonist IVF outcomes (Devroey et al., 2009). Therefore, the purpose of this retrospective study was to determine if advancing or delaying hcg administration by 1 day from ideal in order to avoid weekend oocyte collections has any adverse impact on IVF treatment outcomes in non-programmed GnRH antagonist cycles. Materials and Methods Study design Couples undergoing IVF or ICSI treatment at our Adelaide site using GnRH antagonist pituitary suppression during the 2008 calendar year were included in this retrospective study. Patients using GnRH agonist therapy (long downregulation or flare protocol) or those receiving COCP programming of their GnRH antagonist cycle (irregular cycling or anovular women) were excluded from the study. Furthermore, women aged 43 years or older were also excluded because of their poor IVF treatment prognosis. All patients involved in the study provided prospective written consent for their medical records to be accessed for the purposes of quality assurance audits such as this study. Furthermore, local scientific advisory committee approval of the study protocol was undertaken. Full Institutional Ethics Review Board approval was not required for this type of retrospective quality assurance audit, as per the Australian National Health and Medical Research Guidelines (NHMRC) guidelines. IVF clinical protocol Gonadotrophin stimulation using recombinant FSH (Puregon, Schering- Plough, Sydney, Australia; Gonal F, Serono, Sydney, Australia) commenced on Day 2 of the menstrual cycle, with a small fraction of patients starting on Day 3. Dosage of rfsh was determined by patient age, prior IVF response and ovarian reserve status assessed by antral follicle count and serum Anti- Mullerian Hormone measurement. Women aged 35 years were normally commenced on 150 IU of rfsh per day, with and 300 IU/day of rfsh being used in the and year age groups, respectively. The GnRH antagonist (250 mg Orgalutran, Schering-Plough, Sydney, Australia) was commenced on the seventh day of the menstrual cycle (sixth stimulation day) and continued until the day of hcg administration. A pelvic ultrasound and blood collection was performed on Day 8 or 9 of the menstrual cycle to assess follicular and endometrial development. Patients at high risk of OHSS or previous rapid follicle development during IVF treatment also had a scan on Day 7 of the cycle (sixth day rfsh stimulation). Repeat scans and blood tests were only performed if the lead follicle development was under 14 mm on the Day 8/9 scan, consistent with the unit s treating philosophy of avoiding unnecessary medical investigations in order to enhance the patient friendliness of IVF treatment. The dosage of rfsh stimulation was able to be modified following the Day 8/9 scan if the clinician suspected a poor or excessive response to gonadotrophin stimulation. The criterion used for ideal timing of the hcg trigger administration for collection of mature oocytes was the presence of two or more follicles 17 mm in diameter, with the majority of follicles being 14 mm. An ideal IVF cycle was defined as one in which it was possible to administer the hcg trigger on the exact day that follicle growth reached these maturity criteria. Conversely, trigger injections were routinely delayed for 1 day past ideal to avoid Sunday oocyte collections or advanced 1 day from ideal to avoid Saturday oocyte collections ( non-ideal IVF cycle). Two senior clinicians made all hcg timing decisions, allowing for a high degree of consistency in scheduling decisions. Oocyte collections were performed transvaginally under sedation 36 h following the administration of the hcg trigger (5000 IU Pregnyl, Schering-Plough, Sydney, Australia; 250 mg Ovidrel, Serono, Sydney, Australia). Luteal support was provided by a combination of nightly vaginal progesterone (Crinone 8%, Serono, Sydney, Australia) commencing on the second day after oocyte collection and a single injection of 500 IU hcg (Pregnyl, Schering-Plough, Sydney, Australia) on the sixth day of the luteal phase. Luteal Pregnyl support was omitted in patients considered at high risk of OHSS. Peripheral blood samples were taken for bhcg, estradiol and progesterone measurements 16 days post-oocyte collection, with progesterone luteal support ceasing at this point provided hormone levels were adequate. First trimester pregnancy scans were conducted at 8 weeks gestation using a 7.5 MHz transvaginal scanner (Toshiba, Japan). Embryology protocol Routine insemination of oocytes using density gradient purified sperm was performed if good quality sperms were available. If the male partners normal sperm morphology was,10% (normal range in our laboratory for normal morphology 15%) or if there were less than motile sperm available, ICSI was used to achieve fertilization. Light microscopic examination was used to verify fertilization 16 h later. Embryos were graded excellent (Grade 1), good (Grade 2), poor (Grade 3) and very poor (Grade 4) according to the usual morphological criteria for grading embryos (cell number, fragmentation, nuclear appearance for cleavage stage embryos; compaction and blastocyst cavity formation, cell number in the inner cell mass and trophectoderm for Day 4/5 embryo assessment). Grade 1 and 2 embryos were preferentially transferred and only Grade 1 and 2 embryos were cryopreserved. Embryo transfer was normally performed under ultrasound guidance on Day 4 or 5 after OR ( blastocyst transfer). Embryo transfers were only conducted on a Monday to Saturday basis. Avoidance of transfers on Sundays was achieved by transferring Day 4 embryos on the Saturday, rather than transferring these embryos as blastocysts the following day. Previous published work from our unit has shown that Day 4 and 5 embryo transfers have comparable pregnancy rates (Feil et al., 2008). When the number of fertilized oocytes equaled the number of embryos allocated to be transferred, cleavage stage (Day 2/3) embryo transfers were performed. Statistical analysis The primary end-point for this study was live birth rate, which was defined as the proportion of IVF cycles reaching OR that resulted in the birth of at least one live born child. The reporting of pregnancy outcomes (live birth, pregnancy complications and fetal abnormalities) is mandated by law in South Australia, resulting in a 100% complete follow-up of all pregnancies. The studies secondary pregnancy end-points included biochemical pregnancy rate (proportion of ORs that resulted in a day 16 luteal bhcg 20 IU/l) and implantation rate (proportion of transferred embryos that resulted in a gestation sac at 8 weeks gestation). Embryology secondary outcomes included fertilization rate (proportion of collected oocytes that developed two pronuclei) and the number of embryos of sufficient quality to warrant cryo-preservation. Data were analyzed using commercial software (GraphPad Prism Version 5.02, Graphpad Software Inc, La Jolla, CA, USA). Baseline demographic and fertility-related variables between groups were analyzed using either the one-way ANOVA or the Mann Whitney U-test for continuous variables and x 2 for categorical variables. Differences in pregnancy outcomes were

3 Avoiding weekend oocyte retrievals in antagonist cycles 3 analyzed by x 2 analysis and also expressed as an odds ratio (95% confidence interval). A P-value 0.05 was considered statistically significant. Results A total of 1642 IVF cycles are included in this study, with 472 ORs (28.7%) occurring on a Monday (both on the ideal day and 1 day after the ideal), 372 ORs (22.7%) on a Friday (both on the ideal day and 1 day before the ideal) and the remaining 798 ORs (48.6%) being performed from Tuesday to Thursday (Table I). The results in Table I depict the various IVF prognostic variables for each weekday OR group. No significant differences in patient s age, number of previous IVF cycles, etiology of infertility or ovarian reserve status were noted between the groups. This would suggest that the important patient covariants affecting IVF response and pregnancy outcome are equally distributed throughout the week, allowing for meaningful non-biased comparisons between the groups. The outcome of IVF stimulation is recorded in Table II. As expected, a significant difference in the duration of gonadotrophin Table I Baseline characteristics of patients according to the day of OR. stimulation was observed between the three groups. Advancement of Saturday ORs to Friday resulted in a small reduction (0.59 days) in the average duration of rfsh stimulation for the Friday OR group compared with the control group. Conversely, avoidance of Sunday ORs by delaying the OR until Monday resulted in a small but significant increase (0.63 days) in the average duration of rfsh stimulation compared with the control group. Embryology outcomes did not appear to be significantly influenced by the day on which OR occurred (Table II). No significant difference was observed between the groups with regard to the numbers of oocytes collected or embryos generated, nor fertilization rates. Furthermore, embryo quality, as assessed by morphological grading of the lead embryo or the number of embryos of sufficient quality to cryopreserve was not significantly different between the groups. Finally, the number of embryos transferred, and the proportion of embryos transferred at the blastocyst stage (overall 81.89%, P ¼ 0.77) was not significantly different between the groups. A total of 650 cycles of IVF resulted in biochemical evidence of implantation, giving an overall biochemical pregnancy rate of 39.6% Control OR Monday OR Friday OR P-value (Tuesday Thursday OR)... Number OR s performed Maternal Age (years) Paternal age (years) Number of prior IVF cycles Tubal factor (%) 8.8% 11.4% 11.2% Endometriosis (%) 5.7% 6.0% 5.7% Male factor (%) 47.3% 47.4% 44.8% Other (%) 25.1% 22.5% 25.4% Unexplained (%) 13.1% 12.7% 12.9% Maternal BMI Serum AMH (pmol/l) AFC (2 5 mm) BMI, body mass index; AMH, Anti-Mullerian hormone; AFC, antral follicle count; OR, oocyte retrieval. Table II IVF treatment outcomes according to the day of OR. Control Monday OR Friday OR P-value (Tuesday Thursday OR)... Days of rfsh stimulation , Number oocytes collected Number embryos produced Fertilization rate (%) 58.66% 58.85% 57.40% Number of embryos transferred Number of embryos cryopreserved Biochemical pregnancy rate per OR (%) 39.84% (318/798) 38.98% (184/472) 39.78% (148/372) Implantation rate (%) 31.03% (296/954) 30.72% (173/563) 33.40% (150/449) Live birth rate (%) 32.83% (262/798) 31.77% (150/472) 34.14% (127/372) 0.525

4 4 Tremellen and Lane per OR. No significant difference was observed in the biochemical pregnancy or implantation rates between the various days of the week (Table II). The overall live birth rate per OR conducted was 32.8%, with no significant difference in live birth rates being observed over the various days of the week. A subgroup analysis was then conducted in which ORs performed on a Monday and Friday were classified as having been performed on the ideal or non-ideal (delayed or advanced by 24 h) day. When comparing IVF outcomes between ideal and non-ideal ORs conducted on the same day (Table III), several differences that were not apparent from the original analysis (Table II) were identified. As would be expected, the differences in the numbers of days of gonadotrophin stimulation between ideal and non-ideal ORs became more marked, with approximately a day difference being observed in each group. Bringing forward ideal Saturday ORs to Friday resulted in approximately one less oocyte being collected and as a result one less embryo being created. Conversely, delaying ideal Sunday ORs to Monday resulted in two extra oocytes being collected and one additional embryo being created. In the non-ideal Friday OR group, significantly less oocytes were collected and less embryo embryos were generated, which meant that slightly fewer embryos on average were transferred fresh or were available for cryopreservation. Despite these differences in IVF stimulation outcomes between ideal and non-ideal OR groups, they did not translate into any differences in biochemical pregnancy rates, implantation rates or live birth rates. The odds ratio (95% confidence intervals) for live births when comparing ideal Friday with ideal Saturday ORs was 1.17 ( ) and the corresponding values when comparing ideal Monday with ideal Sunday ORs was 0.90 ( ). Discussion The results of this study suggest that avoidance of weekend ORs in GnRH antagonist IVF cycles can be successfully achieved by simply advancing ideal Saturday OR s to Friday, while delaying ideal Sunday ORs to Monday, without any detrimental effect on IVF pregnancy outcomes. Therefore, in the interests of patient friendly IVF, the use of the COCP for programming the start date of gonadotrophin stimulation in order to minimize the risk of weekend ORs should be abandoned for women with regular menstrual cycles, especially as the COCP may increase gonadotrophin dose requirements and may reduce viable pregnancy rates (Kolibianakis et al., 2006a, b; Griesinger et al., 2008). Furthermore, even with the use of COCP programming, it is impossible to predict an individual patient s response rate to gonadotrophin stimulation with absolute certainty, still placing them at risk of requiring a weekend OR (Barmat et al., 2005). On first principles one could anticipate three potential problems related to our technique of advancement of hcg scheduling. First, advancement of ideal Saturday ORs to Friday could be expected to lead to the collection of fewer oocytes, with an increase in the proportion of oocytes that are immature, and could possibly result in inadequate endometrial development. We did observe a small reduction in the number of oocytes collected and fertilized, equating to just less than one embryo per IVF cycle. The absence of any significant effect of advancing the OR by 1 day on live birth rates, however, suggests that embryo number and inadequate endometrial development is not a significant issue. It is possible that the small non-significant trend toward a reduction in the number of embryos available for cryopreservation observed in the Saturday ideal OR group may translate into a significant reduction in overall pregnancies once frozen embryo transfer pregnancies are analyzed. The outcome for all the frozen embryos generated in this study is unlikely to be known for several years, making such an analysis impossible to conduct at this point. Delaying a Sunday oocyte collection to Monday could theoretically harm IVF outcomes by leading to the post mature loss of oocyte quality in the lead follicles. However, any potential loss of lead follicles is more than likely to be offset by the development of more mature follicles from the previously borderline mature cohort. Our observation of a small increase in the number of oocytes collected and comparable fertilization rate on ideal Sunday ORs compared with ideal Monday ORs suggests that delaying the OR by 1 day has no negative impact on oocyte quality or quantity. Furthermore, pregnancy and live birth rates were not significantly different between the ideal and nonideal Monday ORs, suggesting that there was no clinically significant negative affect on embryo quality or endometrial development brought about by delaying the OR by 1 day. This is consistent with Table III IVF outcomes for ORs conducted on the ideal day of scheduling compared with those advanced or delayed by 1 day from ideal. Monday OR... P-value Friday OR... P-value Ideal Monday Ideal Sunday Ideal Friday Ideal Saturday OR (n 5 221) OR (n 5 251) OR (n 5 207) OR (n 5 165)... Number of days rfsh stimulation , , Number of oocytes , Number of embryos , Fertilization rate (%) 60.0% 58.0% % 55.0% Embryos transferred Embryos cryopreserved Biochemical pregnancy rate (%) 38.91% (86/221) 39.04% (98/251) % (83/207) 35.8% (59/165) Implantation rate (%) 32.15% (82/255) 29.54% (91/308) % (91/263) 31.72% (59/186) Live birth rate (%) 30.76% (68/221) 33.06% (83/251) % (74/207) 32.12% (53/165) 0.463

5 Avoiding weekend oocyte retrievals in antagonist cycles 5 previous reports suggesting that delaying the hcg trigger by 24 h has no detrimental effect on GnRH agonist IVF pregnancy outcomes (Dimitry et al., 1991). The effect of delaying hcg administration on endometrial development is still under debate. As we have shown that delaying the hcg trigger by 24 h does not reduce pregnancy rates in IVF treatment, one can assume that any negative effect on the endometrium is not clinically significant. However, studies have shown that delaying the hcg trigger by 2 days may alter endometrial development and have a detrimental effect on pregnancy rates (Kolibianakis et al., 2004). In one study, endometrial biopsies were taken on the day of OR in women administered hcg either as per normal protocol or with a 48 h delay. Endometrial development was normal in the per protocol group but advanced by up to 3 days in the delayed hcg group (Kolibianakis et al., 2005). In this study, progesterone levels taken 24 h after the hcg trigger were increased by 100% in the delayed hcg group, probably related to the fact that more mature follicles were present. Since elevated early luteal progesterone levels have been associated with accelerated glandular development (Develioglu et al., 1999), the authors proposed that delaying the hcg trigger by 2 days may cause an early closure of the implantation window and therefore decrease pregnancy rates. Our interpretation of these reports in the context of our results is that it would appear to be safer to advance an ideal Saturday OR to Friday, rather than to delay it by 2 days to a Monday. The link between premature luteinization, advanced endometrial development and pregnancy outcome is still not clear. A recent meta-analysis of five studies examining the influence of elevated progesterone concentration on the day of hcg administration on pregnancy outcome failed to identify any significant association (Venetis et al., 2007). Similarly, another study has suggested that while 18.2% of women undergoing an IVF antagonist cycle have evidence of premature luteinization (late follicular phase progesterone.1.5 ng/ml or 5.5 nmol/l), this premature exposure of the endometrium to progesterone only negatively impacts cleavage stage embryo implantation but not blastocyst implantation (Papanikolaou et al., 2009). In our study in excess of 80% of transfers were conducted at the blastocyst stage, thus possibly extending the implantation window even if serum progesterone levels were prematurely increased by delaying the hcg trigger. We would therefore advocate a preference for blastocyst over cleavage stage embryo transfer when the oocyte collection is delayed from ideal, or when there is a premature increase in late follicular phase progesterone concentration. The physiologically ideal criteria for administration of hcg in GnRH antagonist IVF cycles has not been conclusively determined. In most studies, hcg is administered when 3 follicles 17 mm are present (Borm and Mannaerts, 2000; Fluker et al., 2001; Kolibianakis et al., 2002), although other studies have successfully used the criteria of 3 follicles with a maximum diameter 18 mm (Garcia-Velasco et al., 2001) or at least 1 follicle of 18 mm and 3 follicles 15 mm (de Jong et al., 2001). It would therefore appear that follicles between 15 and 18 mm in diameter have good reproductive potential. If we assume follicles grow at 2 mm per day, the 3-mm span in good follicle development should allow for a grace period of at least 24 h in scheduling the hcg trigger injection. Our observation of no significance difference in pregnancy outcomes with advancement or delay of the hcg injection by one day supports this conclusion. Furthermore, studies have suggested that considerable observer error exists for ultrasound measurement of follicular diameter (Prins and Vogelzang, 1984, Forman et al., 1991). It is likely that many hcg scheduling decisions based on ideal follicle dimensions are actually only occurring within 1 2 days of ideal size due to the margins of error experienced in follicle measurement. It is our view that human follicular physiology appears to be much more forgiving than many reproductive physicians would believe. This large study is the first of its kind to clearly show that in a typical clinical population, avoidance of weekend oocyte collections during GnRH antagonist treatment can be achieved by simply advancing or delaying ideal weekend ORs by one day without negatively impacting on IVF pregnancy outcomes. Our pragmatic approach to IVF treatment works very well provided the IVF unit has sufficient medical/ scientific manpower and theatre access to cope with the increased work loads generated on Mondays and Fridays. We acknowledge that smaller IVF units with less staff and theater flexibility may not be able to accommodate these increased workloads. For these units, the use of COCP programming may still be required even though it is perceived as being less patient friendly. Simply delaying the start date for gonadotrophin stimulation from day two to five in an attempt to smooth demand is not a viable alternative as this has been shown to result in a very significant increase in cycle cancellation due to insufficient response (Hohmann et al., 2003). In conclusion, the results of this study indicate that follicular and endometrial development during GnRH antagonist IVF cycles is quite robust and can withstand advancement or delay in hcg administration by 1 day without adversely impacting on IVF live birth success. However, we acknowledge that retrospective studies such as ours are prone to bias and require confirmation by randomized controlled trials (RCTs) before any definitive conclusions on the efficacy of week day that only GnRH antagonist IVF treatment can be made. However, based on the very small non-significant differences in pregnancy outcomes observed between ideal and non-ideal ORs in our study, the sample size for such equivalence RCTs would be prohibitively large. It is hoped that by showing that GnRH antagonist IVF cycles can be conducted on a week day-only basis without compromising live birth rates, more IVF units will make the transition to the patient friendly GnRH antagonist approach. Authors roles K.T. was responsible for the study design, data analysis and writing of the manuscript M.L. was involved in data collection and review of the manuscript. Acknowledgements The authors wish to thank Deb Tippins for her assistance in data collection and Dr Christine Kirby for her helpful review of this manuscript. Conflict of interest: K.T. has previously received honoraria from Schering-Plough, manufactureres of the GnRH antagonist Ganarelix. Funding This study was supported by Schering-Plough Australia as part of a quality assurance grant.

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