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What is the preferred method for timing natural cycle frozen–thawed embryo transfer?
RBMOnline - Vol 19. No 1. 2009 66-71 Reproductive BioMedicine Online; www.rbmonline.com/Article/3721 on web 8 May 2009
Article What is the preferred method for timing natural cycle frozen–thawed embryo transfer? Dr Ariel Weissman graduated from the Hadassah-Hebrew University Medical School in Jerusalem in 1988. In 1994 he completed his residency in Obstetrics and Gynaecology at the Kaplan Medical Center, Rehovot, where he spent another 2 years working as a senior physician at the IVF unit. A 2-year research and clinical fellowship with Professor Bob Casper at the University of Toronto followed, where his research focused on transplantation of human ovarian tissue in immunodeficient mice. Returning to Israel in 1998, he joined the IVF unit at the Wolfson Medical Center, Holon, Sackler Tel Aviv University School of Medicine, where he currently holds the position of Senior Lecturer.
Dr Ariel Weissman Ariel Weissman1, Dan Levin, Amir Ravhon, Horowitz Eran, Avraham Golan, David Levran IVF Unit, Department of Obstetrics and Gynecology, Wolfson Medical Center, Holon and Sackler Faculty of Medicine, Tel Aviv University, Israel 1 Correspondence: e-mail: [email protected]
Abstract Spontaneous ovulation during a natural menstrual cycle represents a simple and efficient method for synchronization between frozen embryos and the endometrium. The objective was to compare serial monitoring until documentation of ovulation, with human chorionic gonadotrophin (HCG) triggering, for timing frozen embryo transfer (FET) in natural cycles (NC). In a retrospective study, 112 women with regular menstrual cycles undergoing 132 NC−FET cycles were divided into two groups: group A (n = 61) patients had FET in an NC after ovulation triggering with HCG; group B (n = 71) patients had FET in an NC after spontaneous ovulation was detected. The main outcome measure was the number of monitoring visits at the clinic. Patients in both groups were similar in terms of demographic characteristics and reproductive history. Clinical and laboratory characteristics of fresh and frozen cycles were also found comparable for both groups, as were pregnancy and delivery rates. The number of monitoring visits in group A (3.46 ± 1.8) was significantly lower than in group B (4.35 ± 1.4) (P < 0.0001). In patients undergoing NC−FET, triggering ovulation by HCG can significantly reduce the number of visits necessary for cycle monitoring without an adverse effect on cycle outcome. Ovulation triggering can increase both patient convenience and cycle cost-effectiveness. Keywords: IVF, frozen−thawed embryo transfer, monitoring, natural cycle, ovulation
Introduction Cryopreservation of supernumerary embryos following IVF has been widely practiced as a safe and cost-effective method to increase cumulative pregnancy rates per oocyte retrieval. The current trend towards decreasing the number of embryos being transferred and the increased implementation of single embryo transfer policy in many IVF programmes emphasize the relevance and importance of frozen embryo transfer.
frozen embryo transfer (NC−FET) attractive to many women and clinicians. For the purpose of synchronization between the endometrium and the frozen embryos, the day of spontaneous ovulation in the natural cycle corresponds to the day of egg retrieval in the ‘fresh’ IVF cycle. Thawing and transferring of embryos is scheduled according to the stage at which embryos were frozen.
In ovulatory patients, frozen embryo transfer is commonly performed during a natural cycle (Byrd, 2002). The natural cycle offers the advantages of utilizing the natural physiological process of endometrial preparation for implantation, and decreases medical intervention as compared with hormone replacement cycles. These advantages make natural cycle
Several problems, however, may arise when the NC−FET protocol is being used. Detection and documentation of ovulation may require numerous monitoring visits even in women with regular menstrual cycles. Furthermore, the date of embryo transfer cannot be planned in advance, a difficulty for centres that do not operate 7 days a week.
66 © 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
Patient monitoring prior to frozen embryo transfer in an natural cycle consists of serial blood and ultrasound testing until the detection of ovulation (al-Shawaf et al., 1993). Alternatively, human chorionic gonadotrophin (HCG) may be utilized to trigger ovulation in the presence of a mature follicle and satisfactory endometrial development. This latter approach, which may simplify the monitoring process by saving the patients and the clinic the time and expense involved in extra visits necessary for documentation of ovulation, has not been thoroughly explored. The aim of the present study was to compare the number of clinic visits and cycle outcome between ovulation triggering by HCG and serial monitoring until documentation of ovulation in patient preparation for NC−FET.
Materials and methods Patients All patients who underwent NC−FET in the IVF unit between January 2004 and March 2006 were included in the study. All women had regular ovulatory cycles, and had previously undergone either conventional IVF or intracytoplasmic sperm injection (ICSI) as indicated, with cryopreservation of supernumerary embryos. A total of 132 cycles in 112 patients were retrospectively analysed. Patients were divided into two groups: group A (HCG group) included 61 cycles, in which women were administered HCG to trigger ovulation upon visualization of a dominant follicle on transvaginal sonography (TVS), and group B (no HCG group) included 71 cycles in which HCG was not administered and monitoring was continued until ovulation was detected and documented. Assignment for expectant management or ovulation triggering was done according to the attending physician’s preference. The study was approved by the local institutional review board (permit 1025–08).
Fresh cycle characteristics The stimulation protocols of ‘fresh’ cycles leading to egg retrieval and embryo cryopreservation were standard protocols utilized in the unit as described previously (Levran et al., 2002; Weissman et al., 2003). Previous research has shown no difference in implantation rates of thawed embryos in relation to gonadotrophin-releasing hormone (GnRH) analogue and gonadotrophin type utilized in the initial retrieval cycle (Seelig et al., 2002). Briefly, cycle monitoring in ‘fresh’ cycles consisted of ovarian ultrasonography and serum oestradiol measurements from stimulation day 6 onward, with dose adjustments and monitoring frequency based on patient response. HCG was administered when at least two follicles were ≥18 mm in diameter with serum eoestradiol concentrations within the acceptable range for the number of mature follicles present. Oocyte retrieval was performed 36 h after HCG administration by transvaginal ultrasound-guided needle aspiration under general anaesthesia. After gamete retrieval, oocytes were inseminated using either standard IVF or ICSI, as indicated and described in detail elsewhere (Levran et al., 2002; Weissman et al., 2003).
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Fertilization was assessed 16–18 h following insemination, and was considered normal only when two clearly distinct pronuclei containing nuclei were present. The embryos were cultured in modified P1 medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific) at 37°C in 5% CO2. Embryo transfer was performed using Cook catheter (Soft Pass, J-SPPE; Cook Ob/Gyn, Spencer, IN, USA) by a standard technique under ultrasound guidance. The number of embryos transferred was individualized based on patient age, number of previous failed attempts and embryo quality. Only high-quality surplus embryos were cryopreserved.
Freezing and thawing of embryos Embryos were prepared for cryopreservation in a 1.5 mol/l propanediol solution in human tubal fluid medium (mHTF) supplemented with 12 mg/ml human serum albumin (Embryo Freeze Medium, F-1; Irvine Scientific) for 10 min at room temperature and transferred to a similar solution with the addition of 0.1 mol/l sucrose (Embryo Freeze Medium, F-2; Irvine Scientific) for an additional 30 s at room temperature. Cryopreservation of embryos was performed using a programmable controlled rate-freezing machine (Planer Series III Kryo 10, T.S.; Scientific, Perkasie, PA, USA) precooled to 20°C. Embryos were transferred into 1.8 ml sterile vials (Cryotube Vials; NUNC, Denmark), which were loaded into the freezer. The embryos were cooled from 20 to −8°C at a rate of 2°C\min and manual seeding with liquid nitrogen was performed. The embryos were then cooled to −30°C at a rate of 0.3°C/min, and then at a rate of 50°C/min. At −150°C the straws were transferred to the embryo tank. Thawing of the embryos commenced with rewarming the straw in an incubator at room temperature for 30 s before immersing them in a water bath at 30°C for 40–50 s. The cryoprotectant was subsequently removed by immersing the embryos sequentially in thawing solutions. Beginning with a 1.0 mol/l propanediol solution containing 0.2 mol/l sucrose in mHTF supplemented with 12 mg/ml human serum albumin for 5 min at room temperature (Embryo Thaw Medium T1; Irvine Scientific). Then, embryos were transferred into a medium containing 0.5 mol/l propanediol (Embryo Thaw Medium-T2; Irvine Scientific) for an additional 5 min and then to a similar medium without propanediol (Embryo Thaw Mediu-T3; Irvine Scientific) for an additional 10 min. Embryos were then transferred to mHTF solution for 10 min at room temperature and for an additional 10 min at 37°C. The embryos were then transferred to P1 culture medium at 37°C and 5% CO2 and kept in an incubator until uterine transfer.
NC−FET protocols In group A, patients were monitored from cycle day 11 until the criteria for ovulation triggering by HCG were met. Criteria for HCG administration included: (i) visualization of a leading follicle >17 mm in diameter by TVS; (ii) serum oestradiol concentration >150 pg/ml; and (iii) serum progesterone concentration <1 ng/ml.
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Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
In group B, 71 cycles were monitored from cycle day 11 until documentation of ovulation. Criteria for ovulation detection included: (i) fall in serum oestradiol concentration compared with the previous test; (ii) rise in serum progesterone concentration >1.5 ng/ml; and (iii) disappearance or typical change in the shape of the lead follicle. In both groups, endometrial thickness ≥7 mm was considered mandatory for proceeding with embryo thawing. Embryos were transferred according to the day of development when cryopreserved.
Mannheim, Germany). Intra-assay and inter-assay coefficients of variation were <5% and <10% for oestradiol and <3 and <5% for progesterone respectively. The statistical package SigmaStat (Jandel Corporation, San Raphael, CA, USA) was used for data analysis. Comparisons were made using the unpaired Student’s t-test, the Mann−Whitney rank sum test, chi-squared analysis and Fisher’s Exact Test, where appropriate. P < 0.05 was considered statistically significant. Results are expressed as means with SD.
Results
Data analysis Data recorded for analysis included age, gravidity and parity, infertility duration, day 3 FSH concentrations and the number of previous failed IVF attempts (Table 1). Fresh cycle characteristics included the number of oocytes retrieved and fertilized, the use of ICSI, fertilization rate, cycle outcome and the number of embryos cryopreserved (Table 1). For the frozen−thawed embryo transfer cycles, the following factors were compared: number of monitoring visits at the clinic (primary outcome measure), number of thawed embryos, number of cycles with embryo transfer, number of embryos transferred, endometrial thickness, pregnancy and live birth rate per started cycle and per embryo transfer and implantation rate (secondary outcome measure). A clinical pregnancy was defined as ultrasonographic visualization of an intrauterine gestational sac with fetal heart beat. A spontaneous abortion was defined as a clinical pregnancy lost before 20 weeks’ gestation. Implantation rate was defined as the number of observed intrauterine gestational sacs divided by the number of embryos replaced. Serum oestradiol and progesterone were measured by means of the automated Elecsys Immunoanalyser (Roche Diagnostics,
Demographic characteristics and reproductive history of patients in groups A and B were similar (Table 1). The fresh cycles leading to embryo freezing were also found comparable with regard to clinical and embryology data such as age, duration of infertility, basal FSH concentrations, number of previous IVF cycles, number of oocytes retrieved, utilization rates of ICSI, fertilization rate and number of embryos frozen (Table 1). The pregnancy rate in the original fresh cycle differed between the two groups. Group A achieved 21 pregnancies (21/61; pregnancy rate 34%) compared with nine pregnancies in group B (9/71; pregnancy rate 13%) (P = 0.0057). Characteristics of the cycles in which frozen−thawed embryos were transferred are summarized in Table 2. Clinical and embryology characteristics are similar in both groups, with no statistically significant difference in terms of endometrial thickness, number of thawed embryos, number of cycles without embryo transfer, number of embryos transferred, clinical pregnancy rate per cycle and per transfer. The only parameter found different was the number of monitoring visits until FET. The mean number of visits was 3.5 ± 1.8 versus 4.4 ± 1.4 in groups A and B respectively (P < 0.0001).
Table 1. Demographic characteristics and fresh cycle outcome of the two study groups.
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Characteristic
Ovulation with HCG (Group A)
Ovulation without HCG (Group B)
No. of cycles Age (years) Gravidity Parity Day 3 FSH (IU/l) Infertility duration (years) Previous IVF cycles Oocytes retrieved No. of cycles with ICSI Oocytes fertilized Fertilization rate (fresh cycles) No of clinical pregnancies (fresh cycles) Number of embryos frozen
61 31.4 ± 4.9 1.4 ± 1.3 0.6 ± 0.7 6.6 ± 2.2 4.3 ± 3.6 2.6 ± 3.1 13.3 ± 5.2 40 10.1 ± 4.9 0.75 ± 0.16 21a (34%) 6.5 ± 4.2
71 30.5 ± 4.4 1.5 ± 1.7 0.6 ± 0.9 6.1 ± 2.5 4.2 ± 3.0 2.8 ± 3.1 13.5 ± 5.1 55 9.6 ± 3.8 0.72 ± 0.14 9b (13%) 6.5 ± 4.0
Values are mean ± SD unless otherwise stated. a,b P = 0.0057. HCG = human chorionic gonadotrophin; ICSI = intracytoplasmic sperm injection.
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Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
Table 2. Characteristics and outcome of frozen−thawed embryo transfer cycles. Characteristic/outcome
Ovulation with HCG (Group A)
Ovulation without HCG (Group B)
No. of cycles No. of monitoring visits Cycles without transfer Endometrial thickness (mm) No. of embryos thawed No. of embryos transferred Clinical pregnancy rate per cycle (%) Clinical pregnancy rate per transfer (%) Live birth rate per cycle (%) Live birth rate per transfer (%) Implantation rate (%)
61 3.5 ± 1.8a 7 9.9 ± 2.3 4.2 ± 2.1 2.7 ± 0.9 20/61 (32.8) 20/54 (37.0) 17/61 (27.9) 17/54 (31.5) 23/145 (15.9)
71 4.4 ± 1.4b 9 9.8 ± 2.9 4.0 ± 1.9 2.6 ± 0.8 21/71 (29.6) 21/62 (33.9) 17/71 (23.9) 17/62 (27.4) 26/160 (16.3)
Values are mean ± SD or n (%) unless otherwise stated. a,b P < 0.0001. HCG = human chorionic gonadotrophin; NS = not statistically significant.
Discussion The present study suggests that triggering of ovulation with HCG is as efficient as serial monitoring until ovulation detection in patient preparation for NC−FET in terms of implantation, pregnancy and live birth rates. As ovulation triggering by HCG significantly reduces the number of monitoring visits that are necessary to schedule the day of frozen embryo transfer, this approach may be superior in terms of patient convenience and cost–effectiveness of the cycle. So far as is known, this is the first study that directly evaluates the implementation of ovulation triggering by HCG, and compares it with the common practice of expectant monitoring for scheduling frozen embryo transfer. Cryopreservation of supernumerary embryos has become one of the most common procedures in advanced reproductive technologies (Van Voorhis et al., 1995; Andersen et al., 2006; Wright et al., 2006). Because of the iatrogenic epidemic of multiple gestations (Ozturk et al., 2001), common practice and legislation in many countries has now limited the number of fresh embryos being transferred (Skovmand, 1997; Diniz, 2002; Gordts et al., 2005; Saldeen and Sundstrom, 2005). These trends, along with improvements in laboratory conditions and techniques, have increased the availability of good quality surplus embryos for cryopreservation and have propelled the utilization of cryopreservation dramatically in the last decade (Le Lannou et al., 2006). Many centres have established strong cryopreservation abilities (Kolibianakis et al., 2003). Employment of this technology has been shown to increase the pregnancy rate per follicle aspiration cycle while offering a less demanding and more convenient treatment. Consequently, it has been advocated to use new criteria for reporting assisted reproduction results, i.e. cumulative pregnancy rates per egg retrieval cycle including both fresh and frozen embryo transfers (Van Voorhis et al., 1995; Katsoff et al., 2005). Frozen embryo transfer has been successfully performed in natural cycles following spontaneous ovulation (Sathanandan RBMOnline®
et al., 1991; al-Shawaf et al., 1993) and in cycles in which the endometrium is artificially prepared with exogenous steroids, with or without pituitary suppression by GnRH agonists (al-Shawaf et al., 1993; Dal Prato et al., 2002; Gelbaya et al., 2006). In a recent Cochrane review comparing the above treatment regimens, it was concluded that at the present time there is insufficient evidence to support the use of one intervention in preference to another (Ghobara and Vandekerckhove, 2008). Thawed embryo transfer during a natural cycle utilizes endogenous hormone production and no supplements are usually needed. As the natural cycle protocol is relatively simple and obviates the need for prolonged hormonal supplementation in early pregnancy, it is preferred by many patients and clinics (Kolibianakis et al., 2003). However, problems often occur when this protocol is used. The timing of transfer in a natural cycle requires accurate determination of ovulation. Daily hormone determinations and ultrasonographic monitoring are required, which can become inconvenient for patients, particularly for those with irregular cycles. Natural cycle transfers have a 6% cancellation rate (Sathanandan et al., 1991) because of the inability to determine the exact time of ovulation. Finally, unlike hormone replacement cycles, the date of embryo thaw and transfer cannot be chosen, a particularly vexing problem in centres that do not operate 7 days a week. Because of the above limitations, only patients with regular ovulatory cycles can be included in NC−FET programmes. Among the ovulatory patients that were included in the study, the number of monitoring visits before frozen embryo transfer was found to be significantly lower in the induced ovulation group compared with the expectant monitoring group (3.5 ± 1.8 versus 4.4 ± 1.4 respectively, P < 0.0001). Each patient who received HCG has saved approximately one visit at the clinic in preparation for frozen embryo transfer. This means simplification of the treatment process, which also reduces direct cost for the patient, the need for additional time consuming tests (sonograms and hormonal assays) and the work load for the clinic.
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Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
Simplification of IVF has been a longstanding goal in assisted reproduction practice (Tan, 1994). Frozen embryo transfer by itself is both simple and cost effective. Further simplifications in the evolution of assisted reproduction include the ability to reduce monitoring to a necessary minimum and the self-administration of subcutaneous (s.c.) gonadotrophin preparations. Ovulation triggering by HCG in NC−FET cycles meets both requirements, as it reduces the number of visits at the clinic, and HCG is selfadministered by an s.c. injection. Furthermore, the possibility to time ovulation by HCG trigger allows some flexibility in scheduling the day of frozen embryo transfer. For example, in a patient who was found to have an 18 mm follicle and met all other requirements for HCG administration, embryo thawing and transfer were scheduled to take place on a Friday. Awaiting spontaneous ovulation could cause a delay of 1 or 2 days in timing frozen embryo transfer, as follicles often rupture at diameters greater than 18 mm. In such case, frozen embryo transfer would be scheduled on a Saturday or a Sunday, which may be inconvenient for both patients and the staff. The cost−benefit evaluation for this approach differs by country and even by clinic, and thus should be individually assessed for every clinical set-up. Factors that should be taken into consideration include the cost of hormonal assays and ultrasound scans, the cost of time (work hours), travel and the HCG preparation for the patients, and the reduction of work load by the clinic. Experience suggests that patients were happy to trade extra monitoring visits for a single s.c. injection of HCG. The implantation, clinical pregnancy and live birth rates reported in this study are comparable with those of previous larger studies (Mandelbaum et al., 1998; Wang et al., 2001; Salumets et al., 2006). It has been previously shown that establishment of pregnancy in a fresh cycle positively affects the chance of pregnancy achievement in a subsequent frozen embryo transfer cycle when sibling embryos are being used (Toner et al., 1991; Lin et al., 1995; Karlstrom et al., 1997; Wang et al., 2001). In the present study, the pregnancy rate in the fresh cycle was significantly higher in group A (HCG group), compared with group B. This difference, which is most likely related to the retrospective nature of this study, and perhaps also to its limited size, did not translate into a difference in implantation, pregnancy and live birth rates in frozen embryo transfer cycles. Further randomized prospective studies will clarify this issue.
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The major drawback of the present study is its retrospective design. The administration of HCG was based on physicians’ preference and only two physicians (AW and AR) actually prescribed HCG, which could be a source of bias. Twenty patients were included twice in the study, 10 were treated twice in the same group (A or B), and 10 had changed groups (A and B). The number of visits, which was the primary outcome measure in this study, was counted per treatment cycle, not per patient. Lack of true randomization also affected the homogeneity of the study groups, as reflected in higher pregnancy rates in the fresh cycles in group A, as discussed above. Nevertheless, the results of the study indicate that triggering ovulation with HCG can be successfully used for timing NC−FET. The present study should be therefore regarded as a ‘feasibility’ or ‘proof of concept’ study, which should be further investigated in a prospective manner. Indeed, a randomized prospective study exploring further this approach is currently under way.
In summary, in patients undergoing NC−FET, triggering ovulation by HCG can significantly reduce the number of visits necessary for scheduling embryo transfer without an adverse effect on cycle outcome. Ovulation triggering can increase both patient convenience and cycle cost effectiveness. Utilizing this new treatment approach can therefore be attractive to both patients and clinics.
References al-Shawaf T, Yang D, al-Magid Y et al. 1993 Ultrasonic monitoring during replacement of frozen/thawed embryos in natural and hormone replacement cycles. Human Reproduction 8, 2068–2074. Andersen AN, Gianaroli L, Felberbaum R et al. 2006 Assisted reproductive technology in Europe, 2002. Results generated from European registers by ESHRE. Human Reproduction 21, 1680–1697. Byrd W 2002 Cryopreservation, thawing, and transfer of human embryos. Seminars in Reproductive Medicine 20, 37–43. Dal Prato L, Borini A, Cattoli M et al. 2002 Endometrial preparation for frozen–thawed embryo transfer with or without pretreatment with gonadotropin-releasing hormone agonist. Fertility and Sterility 77, 956–960. Diniz D 2002 New reproductive technologies, ethics and gender: the legislative process in Brazil. Developing World Bioethics 2, 144–158. Gelbaya TA, Nardo LG, Hunter HR et al. 2006 Cryopreserved−thawed embryo transfer in natural or down-regulated hormonally controlled cycles: a retrospective study. Fertility and Sterility 85, 603–609. Ghobara T, Vandekerckhove P 2008 Cycle regimens for frozen−thawed embryo transfer. Cochrane Database of Systematic Reviews, CD003414. Gordts S, Campo R, Puttemans P et al. 2005 Belgian legislation and the effect of elective single embryo transfer on IVF outcome. Reproductive BioMedicine Online 10, 436–441. Karlstrom PO, Bergh T, Forsberg AS et al. 1997 Prognostic factors for the success rate of embryo freezing. Human Reproduction 12, 1263–1266. Katsoff B, Check JH, Choe JK, Wilson C 2005 A novel method to evaluate pregnancy rates following in vitro fertilization to enable a better understanding of the true efficacy of the procedure. Clinical and Experimental Obstetrics and Gynecology 32, 213–216. Kolibianakis EM, Zikopoulos K, Devroey P 2003 Implantation potential and clinical impact of cryopreservation − a review. Placenta 24 (Suppl. B), S27–S33. Le Lannou D, Griveau JF, Laurent MC et al. 2006 Contribution of embryo cryopreservation to elective single embryo transfer in IVF−ICSI. Reproductive BioMedicine Online 13, 368–375. Levran D, Farhi J, Nahum H et al. 2002 Prospective evaluation of blastocyst stage transfer vs. zygote intrafallopian tube transfer in patients with repeated implantation failure. Fertility and Sterility 77, 971–977. Lin YP, Cassidenti DL, Chacon RR et al. 1995 Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they were derived. Fertility and Sterility 63, 262–267. Mandelbaum J, Belaisch-Allart J, Junca AM et al. 1998 Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Human Reproduction 13 (Suppl. 3), 161–174; discussion 175–177. Ozturk O, Bhattacharya S, Templeton A 2001 Avoiding multiple pregnancies in ART: evaluation and implementation of new strategies. Human Reproduction 16, 1319–1321. Saldeen P, Sundstrom P 2005 Would legislation imposing single embryo transfer be a feasible way to reduce the rate of multiple pregnancies after IVF treatment? Human Reproduction 20, 4–8. Salumets A, Suikkari AM, Makinen S et al. 2006 Frozen embryo transfers: implications of clinical and embryological factors on the
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pregnancy outcome. Human Reproduction 21, 2368–2374. Sathanandan M, Macnamee MC, Rainsbury P et al. 1991 Replacement of frozen-thawed embryos in artificial and natural cycles: a prospective semi-randomized study. Human Reproduction 6, 685–687. Seelig AS, Al-Hasani S, Katalinic A et al. 2002 Comparison of cryopreservation outcome with gonadotropin-releasing hormone agonists or antagonists in the collecting cycle. Fertility and Sterility 77, 472–475. Skovmand K 1997 First-ever fertilisation bill passed in Denmark. Lancet 349, 1678. Tan SL 1994 Simplifying in-vitro fertilization therapy. Current Opinion in Obstetrics and Gynecology 6, 111–114. Toner JP, Veeck LL, Acosta AA, Muasher SJ 1991 Predictive value of pregnancy during original in vitro fertilization cycle on implantation and pregnancy in subsequent cryothaw cycles. Fertility and Sterility 56, 505–508. Van Voorhis BJ, Syrop CH, Allen BD et al. 1995 The efficacy and cost effectiveness of embryo cryopreservation compared with other assisted reproductive techniques. Fertility and Sterility 64, 647–650.
Wang JX, Yap YY, Matthews CD 2001 Frozen–thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Human Reproduction 16, 2316–2319. Weissman A, Farhi J, Royburt M et al. 2003 Prospective evaluation of two stimulation protocols for low responders who were undergoing in vitro fertilization−embryo transfer. Fertility and Sterility 79, 886–892. Wright VC, Chang J, Jeng G, Macaluso M 2006 Assisted reproductive technology surveillance − United States, 2003. Morbidity and Mortality Weekly Report. Surveillance Summaries 55, 1–22.
Declaration: The authors report no financial or commercial conflicts of interest. Received 12 June 2008; refereed 3 September 2008; accepted 11 February 2009.
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Article What is the preferred method for timing natural cycle frozen–thawed embryo transfer? Dr Ariel Weissman graduated from the Hadassah-Hebrew University Medical School in Jerusalem in 1988. In 1994 he completed his residency in Obstetrics and Gynaecology at the Kaplan Medical Center, Rehovot, where he spent another 2 years working as a senior physician at the IVF unit. A 2-year research and clinical fellowship with Professor Bob Casper at the University of Toronto followed, where his research focused on transplantation of human ovarian tissue in immunodeficient mice. Returning to Israel in 1998, he joined the IVF unit at the Wolfson Medical Center, Holon, Sackler Tel Aviv University School of Medicine, where he currently holds the position of Senior Lecturer.
Dr Ariel Weissman Ariel Weissman1, Dan Levin, Amir Ravhon, Horowitz Eran, Avraham Golan, David Levran IVF Unit, Department of Obstetrics and Gynecology, Wolfson Medical Center, Holon and Sackler Faculty of Medicine, Tel Aviv University, Israel 1 Correspondence: e-mail: [email protected]
Abstract Spontaneous ovulation during a natural menstrual cycle represents a simple and efficient method for synchronization between frozen embryos and the endometrium. The objective was to compare serial monitoring until documentation of ovulation, with human chorionic gonadotrophin (HCG) triggering, for timing frozen embryo transfer (FET) in natural cycles (NC). In a retrospective study, 112 women with regular menstrual cycles undergoing 132 NC−FET cycles were divided into two groups: group A (n = 61) patients had FET in an NC after ovulation triggering with HCG; group B (n = 71) patients had FET in an NC after spontaneous ovulation was detected. The main outcome measure was the number of monitoring visits at the clinic. Patients in both groups were similar in terms of demographic characteristics and reproductive history. Clinical and laboratory characteristics of fresh and frozen cycles were also found comparable for both groups, as were pregnancy and delivery rates. The number of monitoring visits in group A (3.46 ± 1.8) was significantly lower than in group B (4.35 ± 1.4) (P < 0.0001). In patients undergoing NC−FET, triggering ovulation by HCG can significantly reduce the number of visits necessary for cycle monitoring without an adverse effect on cycle outcome. Ovulation triggering can increase both patient convenience and cycle cost-effectiveness. Keywords: IVF, frozen−thawed embryo transfer, monitoring, natural cycle, ovulation
Introduction Cryopreservation of supernumerary embryos following IVF has been widely practiced as a safe and cost-effective method to increase cumulative pregnancy rates per oocyte retrieval. The current trend towards decreasing the number of embryos being transferred and the increased implementation of single embryo transfer policy in many IVF programmes emphasize the relevance and importance of frozen embryo transfer.
frozen embryo transfer (NC−FET) attractive to many women and clinicians. For the purpose of synchronization between the endometrium and the frozen embryos, the day of spontaneous ovulation in the natural cycle corresponds to the day of egg retrieval in the ‘fresh’ IVF cycle. Thawing and transferring of embryos is scheduled according to the stage at which embryos were frozen.
In ovulatory patients, frozen embryo transfer is commonly performed during a natural cycle (Byrd, 2002). The natural cycle offers the advantages of utilizing the natural physiological process of endometrial preparation for implantation, and decreases medical intervention as compared with hormone replacement cycles. These advantages make natural cycle
Several problems, however, may arise when the NC−FET protocol is being used. Detection and documentation of ovulation may require numerous monitoring visits even in women with regular menstrual cycles. Furthermore, the date of embryo transfer cannot be planned in advance, a difficulty for centres that do not operate 7 days a week.
66 © 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
Patient monitoring prior to frozen embryo transfer in an natural cycle consists of serial blood and ultrasound testing until the detection of ovulation (al-Shawaf et al., 1993). Alternatively, human chorionic gonadotrophin (HCG) may be utilized to trigger ovulation in the presence of a mature follicle and satisfactory endometrial development. This latter approach, which may simplify the monitoring process by saving the patients and the clinic the time and expense involved in extra visits necessary for documentation of ovulation, has not been thoroughly explored. The aim of the present study was to compare the number of clinic visits and cycle outcome between ovulation triggering by HCG and serial monitoring until documentation of ovulation in patient preparation for NC−FET.
Materials and methods Patients All patients who underwent NC−FET in the IVF unit between January 2004 and March 2006 were included in the study. All women had regular ovulatory cycles, and had previously undergone either conventional IVF or intracytoplasmic sperm injection (ICSI) as indicated, with cryopreservation of supernumerary embryos. A total of 132 cycles in 112 patients were retrospectively analysed. Patients were divided into two groups: group A (HCG group) included 61 cycles, in which women were administered HCG to trigger ovulation upon visualization of a dominant follicle on transvaginal sonography (TVS), and group B (no HCG group) included 71 cycles in which HCG was not administered and monitoring was continued until ovulation was detected and documented. Assignment for expectant management or ovulation triggering was done according to the attending physician’s preference. The study was approved by the local institutional review board (permit 1025–08).
Fresh cycle characteristics The stimulation protocols of ‘fresh’ cycles leading to egg retrieval and embryo cryopreservation were standard protocols utilized in the unit as described previously (Levran et al., 2002; Weissman et al., 2003). Previous research has shown no difference in implantation rates of thawed embryos in relation to gonadotrophin-releasing hormone (GnRH) analogue and gonadotrophin type utilized in the initial retrieval cycle (Seelig et al., 2002). Briefly, cycle monitoring in ‘fresh’ cycles consisted of ovarian ultrasonography and serum oestradiol measurements from stimulation day 6 onward, with dose adjustments and monitoring frequency based on patient response. HCG was administered when at least two follicles were ≥18 mm in diameter with serum eoestradiol concentrations within the acceptable range for the number of mature follicles present. Oocyte retrieval was performed 36 h after HCG administration by transvaginal ultrasound-guided needle aspiration under general anaesthesia. After gamete retrieval, oocytes were inseminated using either standard IVF or ICSI, as indicated and described in detail elsewhere (Levran et al., 2002; Weissman et al., 2003).
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Fertilization was assessed 16–18 h following insemination, and was considered normal only when two clearly distinct pronuclei containing nuclei were present. The embryos were cultured in modified P1 medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific) at 37°C in 5% CO2. Embryo transfer was performed using Cook catheter (Soft Pass, J-SPPE; Cook Ob/Gyn, Spencer, IN, USA) by a standard technique under ultrasound guidance. The number of embryos transferred was individualized based on patient age, number of previous failed attempts and embryo quality. Only high-quality surplus embryos were cryopreserved.
Freezing and thawing of embryos Embryos were prepared for cryopreservation in a 1.5 mol/l propanediol solution in human tubal fluid medium (mHTF) supplemented with 12 mg/ml human serum albumin (Embryo Freeze Medium, F-1; Irvine Scientific) for 10 min at room temperature and transferred to a similar solution with the addition of 0.1 mol/l sucrose (Embryo Freeze Medium, F-2; Irvine Scientific) for an additional 30 s at room temperature. Cryopreservation of embryos was performed using a programmable controlled rate-freezing machine (Planer Series III Kryo 10, T.S.; Scientific, Perkasie, PA, USA) precooled to 20°C. Embryos were transferred into 1.8 ml sterile vials (Cryotube Vials; NUNC, Denmark), which were loaded into the freezer. The embryos were cooled from 20 to −8°C at a rate of 2°C\min and manual seeding with liquid nitrogen was performed. The embryos were then cooled to −30°C at a rate of 0.3°C/min, and then at a rate of 50°C/min. At −150°C the straws were transferred to the embryo tank. Thawing of the embryos commenced with rewarming the straw in an incubator at room temperature for 30 s before immersing them in a water bath at 30°C for 40–50 s. The cryoprotectant was subsequently removed by immersing the embryos sequentially in thawing solutions. Beginning with a 1.0 mol/l propanediol solution containing 0.2 mol/l sucrose in mHTF supplemented with 12 mg/ml human serum albumin for 5 min at room temperature (Embryo Thaw Medium T1; Irvine Scientific). Then, embryos were transferred into a medium containing 0.5 mol/l propanediol (Embryo Thaw Medium-T2; Irvine Scientific) for an additional 5 min and then to a similar medium without propanediol (Embryo Thaw Mediu-T3; Irvine Scientific) for an additional 10 min. Embryos were then transferred to mHTF solution for 10 min at room temperature and for an additional 10 min at 37°C. The embryos were then transferred to P1 culture medium at 37°C and 5% CO2 and kept in an incubator until uterine transfer.
NC−FET protocols In group A, patients were monitored from cycle day 11 until the criteria for ovulation triggering by HCG were met. Criteria for HCG administration included: (i) visualization of a leading follicle >17 mm in diameter by TVS; (ii) serum oestradiol concentration >150 pg/ml; and (iii) serum progesterone concentration <1 ng/ml.
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Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
In group B, 71 cycles were monitored from cycle day 11 until documentation of ovulation. Criteria for ovulation detection included: (i) fall in serum oestradiol concentration compared with the previous test; (ii) rise in serum progesterone concentration >1.5 ng/ml; and (iii) disappearance or typical change in the shape of the lead follicle. In both groups, endometrial thickness ≥7 mm was considered mandatory for proceeding with embryo thawing. Embryos were transferred according to the day of development when cryopreserved.
Mannheim, Germany). Intra-assay and inter-assay coefficients of variation were <5% and <10% for oestradiol and <3 and <5% for progesterone respectively. The statistical package SigmaStat (Jandel Corporation, San Raphael, CA, USA) was used for data analysis. Comparisons were made using the unpaired Student’s t-test, the Mann−Whitney rank sum test, chi-squared analysis and Fisher’s Exact Test, where appropriate. P < 0.05 was considered statistically significant. Results are expressed as means with SD.
Results
Data analysis Data recorded for analysis included age, gravidity and parity, infertility duration, day 3 FSH concentrations and the number of previous failed IVF attempts (Table 1). Fresh cycle characteristics included the number of oocytes retrieved and fertilized, the use of ICSI, fertilization rate, cycle outcome and the number of embryos cryopreserved (Table 1). For the frozen−thawed embryo transfer cycles, the following factors were compared: number of monitoring visits at the clinic (primary outcome measure), number of thawed embryos, number of cycles with embryo transfer, number of embryos transferred, endometrial thickness, pregnancy and live birth rate per started cycle and per embryo transfer and implantation rate (secondary outcome measure). A clinical pregnancy was defined as ultrasonographic visualization of an intrauterine gestational sac with fetal heart beat. A spontaneous abortion was defined as a clinical pregnancy lost before 20 weeks’ gestation. Implantation rate was defined as the number of observed intrauterine gestational sacs divided by the number of embryos replaced. Serum oestradiol and progesterone were measured by means of the automated Elecsys Immunoanalyser (Roche Diagnostics,
Demographic characteristics and reproductive history of patients in groups A and B were similar (Table 1). The fresh cycles leading to embryo freezing were also found comparable with regard to clinical and embryology data such as age, duration of infertility, basal FSH concentrations, number of previous IVF cycles, number of oocytes retrieved, utilization rates of ICSI, fertilization rate and number of embryos frozen (Table 1). The pregnancy rate in the original fresh cycle differed between the two groups. Group A achieved 21 pregnancies (21/61; pregnancy rate 34%) compared with nine pregnancies in group B (9/71; pregnancy rate 13%) (P = 0.0057). Characteristics of the cycles in which frozen−thawed embryos were transferred are summarized in Table 2. Clinical and embryology characteristics are similar in both groups, with no statistically significant difference in terms of endometrial thickness, number of thawed embryos, number of cycles without embryo transfer, number of embryos transferred, clinical pregnancy rate per cycle and per transfer. The only parameter found different was the number of monitoring visits until FET. The mean number of visits was 3.5 ± 1.8 versus 4.4 ± 1.4 in groups A and B respectively (P < 0.0001).
Table 1. Demographic characteristics and fresh cycle outcome of the two study groups.
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Characteristic
Ovulation with HCG (Group A)
Ovulation without HCG (Group B)
No. of cycles Age (years) Gravidity Parity Day 3 FSH (IU/l) Infertility duration (years) Previous IVF cycles Oocytes retrieved No. of cycles with ICSI Oocytes fertilized Fertilization rate (fresh cycles) No of clinical pregnancies (fresh cycles) Number of embryos frozen
61 31.4 ± 4.9 1.4 ± 1.3 0.6 ± 0.7 6.6 ± 2.2 4.3 ± 3.6 2.6 ± 3.1 13.3 ± 5.2 40 10.1 ± 4.9 0.75 ± 0.16 21a (34%) 6.5 ± 4.2
71 30.5 ± 4.4 1.5 ± 1.7 0.6 ± 0.9 6.1 ± 2.5 4.2 ± 3.0 2.8 ± 3.1 13.5 ± 5.1 55 9.6 ± 3.8 0.72 ± 0.14 9b (13%) 6.5 ± 4.0
Values are mean ± SD unless otherwise stated. a,b P = 0.0057. HCG = human chorionic gonadotrophin; ICSI = intracytoplasmic sperm injection.
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Article - Timing natural cycle frozen–thawed embryo transfer - A Weissman et al.
Table 2. Characteristics and outcome of frozen−thawed embryo transfer cycles. Characteristic/outcome
Ovulation with HCG (Group A)
Ovulation without HCG (Group B)
No. of cycles No. of monitoring visits Cycles without transfer Endometrial thickness (mm) No. of embryos thawed No. of embryos transferred Clinical pregnancy rate per cycle (%) Clinical pregnancy rate per transfer (%) Live birth rate per cycle (%) Live birth rate per transfer (%) Implantation rate (%)
61 3.5 ± 1.8a 7 9.9 ± 2.3 4.2 ± 2.1 2.7 ± 0.9 20/61 (32.8) 20/54 (37.0) 17/61 (27.9) 17/54 (31.5) 23/145 (15.9)
71 4.4 ± 1.4b 9 9.8 ± 2.9 4.0 ± 1.9 2.6 ± 0.8 21/71 (29.6) 21/62 (33.9) 17/71 (23.9) 17/62 (27.4) 26/160 (16.3)
Values are mean ± SD or n (%) unless otherwise stated. a,b P < 0.0001. HCG = human chorionic gonadotrophin; NS = not statistically significant.
Discussion The present study suggests that triggering of ovulation with HCG is as efficient as serial monitoring until ovulation detection in patient preparation for NC−FET in terms of implantation, pregnancy and live birth rates. As ovulation triggering by HCG significantly reduces the number of monitoring visits that are necessary to schedule the day of frozen embryo transfer, this approach may be superior in terms of patient convenience and cost–effectiveness of the cycle. So far as is known, this is the first study that directly evaluates the implementation of ovulation triggering by HCG, and compares it with the common practice of expectant monitoring for scheduling frozen embryo transfer. Cryopreservation of supernumerary embryos has become one of the most common procedures in advanced reproductive technologies (Van Voorhis et al., 1995; Andersen et al., 2006; Wright et al., 2006). Because of the iatrogenic epidemic of multiple gestations (Ozturk et al., 2001), common practice and legislation in many countries has now limited the number of fresh embryos being transferred (Skovmand, 1997; Diniz, 2002; Gordts et al., 2005; Saldeen and Sundstrom, 2005). These trends, along with improvements in laboratory conditions and techniques, have increased the availability of good quality surplus embryos for cryopreservation and have propelled the utilization of cryopreservation dramatically in the last decade (Le Lannou et al., 2006). Many centres have established strong cryopreservation abilities (Kolibianakis et al., 2003). Employment of this technology has been shown to increase the pregnancy rate per follicle aspiration cycle while offering a less demanding and more convenient treatment. Consequently, it has been advocated to use new criteria for reporting assisted reproduction results, i.e. cumulative pregnancy rates per egg retrieval cycle including both fresh and frozen embryo transfers (Van Voorhis et al., 1995; Katsoff et al., 2005). Frozen embryo transfer has been successfully performed in natural cycles following spontaneous ovulation (Sathanandan RBMOnline®
et al., 1991; al-Shawaf et al., 1993) and in cycles in which the endometrium is artificially prepared with exogenous steroids, with or without pituitary suppression by GnRH agonists (al-Shawaf et al., 1993; Dal Prato et al., 2002; Gelbaya et al., 2006). In a recent Cochrane review comparing the above treatment regimens, it was concluded that at the present time there is insufficient evidence to support the use of one intervention in preference to another (Ghobara and Vandekerckhove, 2008). Thawed embryo transfer during a natural cycle utilizes endogenous hormone production and no supplements are usually needed. As the natural cycle protocol is relatively simple and obviates the need for prolonged hormonal supplementation in early pregnancy, it is preferred by many patients and clinics (Kolibianakis et al., 2003). However, problems often occur when this protocol is used. The timing of transfer in a natural cycle requires accurate determination of ovulation. Daily hormone determinations and ultrasonographic monitoring are required, which can become inconvenient for patients, particularly for those with irregular cycles. Natural cycle transfers have a 6% cancellation rate (Sathanandan et al., 1991) because of the inability to determine the exact time of ovulation. Finally, unlike hormone replacement cycles, the date of embryo thaw and transfer cannot be chosen, a particularly vexing problem in centres that do not operate 7 days a week. Because of the above limitations, only patients with regular ovulatory cycles can be included in NC−FET programmes. Among the ovulatory patients that were included in the study, the number of monitoring visits before frozen embryo transfer was found to be significantly lower in the induced ovulation group compared with the expectant monitoring group (3.5 ± 1.8 versus 4.4 ± 1.4 respectively, P < 0.0001). Each patient who received HCG has saved approximately one visit at the clinic in preparation for frozen embryo transfer. This means simplification of the treatment process, which also reduces direct cost for the patient, the need for additional time consuming tests (sonograms and hormonal assays) and the work load for the clinic.
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Simplification of IVF has been a longstanding goal in assisted reproduction practice (Tan, 1994). Frozen embryo transfer by itself is both simple and cost effective. Further simplifications in the evolution of assisted reproduction include the ability to reduce monitoring to a necessary minimum and the self-administration of subcutaneous (s.c.) gonadotrophin preparations. Ovulation triggering by HCG in NC−FET cycles meets both requirements, as it reduces the number of visits at the clinic, and HCG is selfadministered by an s.c. injection. Furthermore, the possibility to time ovulation by HCG trigger allows some flexibility in scheduling the day of frozen embryo transfer. For example, in a patient who was found to have an 18 mm follicle and met all other requirements for HCG administration, embryo thawing and transfer were scheduled to take place on a Friday. Awaiting spontaneous ovulation could cause a delay of 1 or 2 days in timing frozen embryo transfer, as follicles often rupture at diameters greater than 18 mm. In such case, frozen embryo transfer would be scheduled on a Saturday or a Sunday, which may be inconvenient for both patients and the staff. The cost−benefit evaluation for this approach differs by country and even by clinic, and thus should be individually assessed for every clinical set-up. Factors that should be taken into consideration include the cost of hormonal assays and ultrasound scans, the cost of time (work hours), travel and the HCG preparation for the patients, and the reduction of work load by the clinic. Experience suggests that patients were happy to trade extra monitoring visits for a single s.c. injection of HCG. The implantation, clinical pregnancy and live birth rates reported in this study are comparable with those of previous larger studies (Mandelbaum et al., 1998; Wang et al., 2001; Salumets et al., 2006). It has been previously shown that establishment of pregnancy in a fresh cycle positively affects the chance of pregnancy achievement in a subsequent frozen embryo transfer cycle when sibling embryos are being used (Toner et al., 1991; Lin et al., 1995; Karlstrom et al., 1997; Wang et al., 2001). In the present study, the pregnancy rate in the fresh cycle was significantly higher in group A (HCG group), compared with group B. This difference, which is most likely related to the retrospective nature of this study, and perhaps also to its limited size, did not translate into a difference in implantation, pregnancy and live birth rates in frozen embryo transfer cycles. Further randomized prospective studies will clarify this issue.
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The major drawback of the present study is its retrospective design. The administration of HCG was based on physicians’ preference and only two physicians (AW and AR) actually prescribed HCG, which could be a source of bias. Twenty patients were included twice in the study, 10 were treated twice in the same group (A or B), and 10 had changed groups (A and B). The number of visits, which was the primary outcome measure in this study, was counted per treatment cycle, not per patient. Lack of true randomization also affected the homogeneity of the study groups, as reflected in higher pregnancy rates in the fresh cycles in group A, as discussed above. Nevertheless, the results of the study indicate that triggering ovulation with HCG can be successfully used for timing NC−FET. The present study should be therefore regarded as a ‘feasibility’ or ‘proof of concept’ study, which should be further investigated in a prospective manner. Indeed, a randomized prospective study exploring further this approach is currently under way.
In summary, in patients undergoing NC−FET, triggering ovulation by HCG can significantly reduce the number of visits necessary for scheduling embryo transfer without an adverse effect on cycle outcome. Ovulation triggering can increase both patient convenience and cycle cost effectiveness. Utilizing this new treatment approach can therefore be attractive to both patients and clinics.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 12 June 2008; refereed 3 September 2008; accepted 11 February 2009.
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