University Hospital Dr. Peset, Valencia, Spain

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1 A prospective, randomized, controlled trial comparing three different gonadotropin regimens in oocyte donors: ovarian response, in vitro fertilization outcome, and analysis of cost minimization Marco Melo, M.D., a Jose Bellver, M.D., a Nicolas Garrido, Ph.D., a,b Marcos Meseguer, Ph.D., a,b Antonio Pellicer, M.D., a,b,c and Jose Remohı, M.D. a a Instituto Valenciano de Infertilidad, Universidad de Valencia, b Fundacion IVI, and c Department of Obstetrics and Gynaecology, University Hospital Dr. Peset, Valencia, Spain Objective: To compare the efficacy of three different gonadotropin regimens in an oocyte donation program. The analysis of cost minimization also was evaluated. Design: Prospective, randomized, controlled study. Setting: Instituto Universitario IVI, Valencia, Spain. Patient(s): One thousand twenty-eight donors undergoing a GnRH agonist protocol were assigned randomly to one of three groups: group 1 (n ¼ 346), only recombinant FSH (rfsh); group 2 (n ¼ 333), only highly purified menotropin (HP-hMG); and group 3 (n ¼ 349), rfsh plus HP-hMG. One thousand seventy-nine oocyte recipients. Intervention(s): Controlled ovarian stimulation. Main Outcome Measure(s): Controlled ovarian stimulation parameters, IVF outcome, and cost analysis. Result(s): No differences were found among the groups with respect to days of stimulation, gonadotropin dose, final E 2 and P levels, number of oocytes retrieved, and cancellation rate. Similarly, there were no differences among the groups in terms of embryo development parameters. Moreover, implantation, pregnancy, and miscarriage rates with the three regimens were similar. However, the cost of rfsh was greater than that of the other protocols. Conclusion(s): This study suggests that in the GnRH agonist protocol the three different gonadotropin regimens evaluated herein are equally effective. Protocols using HP-hMG would appear to be the best in terms of costeffectiveness in an oocyte donation program. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Cost minimization, embryo quality, IVF outcome, menotropin, recombinant FSH, oocyte donation, oocyte quality, ovarian stimulation The impact on IVF outcome of the different gonadotropin preparations, such as urinary hmg, highly purified menotropin (HP-hMG), and recombinant FSH (rfsh), has been widely debated (1 12). A PubMed search of the field yields approximately 690 publications, of which 56 are reviews, 11 are meta-analyses, and 7 are randomized controlled trials. In an oocyte donation program, the primary objective is to obtain oocytes that can generate good embryos with a high potential for implanting and developing into a pregnancy. Discrepancies in the literature make it difficult to decide what kind of gonadotropin to administer to women undergoing the long protocol. In addition, the cost-effectiveness of the chosen regimen must be taken into account, as ovarian stimulation represents approximately 28% of the total cost of the treatment. Received March 26, 2009; revised April 25, 2009; accepted May 4, 2009; published online November 19, M.M. has nothing to disclose. J.B. has nothing to disclose. N.G. has nothing to disclose. M.M. has nothing to disclose. A.P. has nothing to disclose. J.R. has nothing to disclose. Reprint requests: Marco Melo, M.D., Instituto Valenciano de Infertilidad, Plaza de la Policıa Local, 3, Valencia, 46015, Spain (FAX: ; marcoabm@hotmail.com or mmelo@ivi.es). Bearing in mind the controversial results, we decided to carry out a trial that would clarify the matter. The primary objective was to evaluate the implantation, pregnancy, and miscarriage rates associated with three different protocols of ovarian stimulation. The secondary objective was to compare the parameters of controlled ovarian stimulation, embryo development, and the cost observed with these regimens. MATERIALS AND METHODS The project was approved by the Institutional Review Board on the Use of Human Subjects in Research of the Instituto Universitario IVI, Valencia, Spain, and complied with the Spanish Law of Assisted Reproductive Technologies (14/ 2006). The ClinicalTrials.gov identifier is NCT This was a prospective, randomized, controlled, parallelgroups study conducted in a private infertility clinic (Instituto Valenciano de Infertilidad, Valencia, Spain) between January 2005 and December Inclusion and Exclusion Criteria Donors were healthy women of 18 to 34 years of age, with regular menstrual cycles, no family history of hereditary or 958 Fertility and Sterility â Vol. 94, No. 3, August /$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 chromosomal diseases, normal karyotype, and body mass index (BMI) 18 to 29 kg/m 2 and showing negative screening for sexually transmitted diseases. Written informed consent was obtained. Women with polycystic ovary syndrome were excluded from this study (13, 14). Recipients were healthy women of 18 to 49 years of age, with BMI 18 to 29 kg/m 2, and whose male partner did not present severe male factor (fresh spermatozoa < / mm 3, <5% normal forms, and/or nonobstructive azoospermia). The recipient couple gave their written informed consent. Cases with uterine pathology (submucous or more than 2 cm intramural fibroids, polyps, adhesions, adenomyosis, or m ullerian defects), implantation failure, and recurrent miscarriage were excluded (13). Randomization Method and Sample Size Calculation One thousand twenty-eight donors were recruited and randomly assigned to groups fulfilling our inclusion criteria. The randomization visit took place on the first day of controlled ovarian stimulation (after pituitary down-regulation). Donors were assigned randomly to three groups by a study nurse, using computer-generated random numbers: group 1 (n ¼ 284), 225 IU of rfsh (Gonal; Serono, Madrid, Spain); group 2 (n ¼ 280), 225 IU of HP-hMG (Menopur; Ferring Pharmaceuticals, Madrid, Spain); group 3 (n ¼ 290), 150 IU of rfsh (Gonal; Serono) plus 75 IU HP-hMG (Menopur; Ferring). The study nurse coordinated the randomization process and distribution of medication throughout the controlled ovarian stimulation cycles. All embryologists, laboratory personnel, and sponsor staff, including the statistician responsible for the statistical analysis, were blinded to the treatment allocation. The sample size had been calculated previously assuming a pregnancy rate of 55% per cycle, alpha risk of 0.05, and beta risk of 0.20, thereby making it possible to detect differences >10% in a bilateral test. Oocyte Donors For controlled ovarian stimulation, only GnRH agonist protocols were used as previously described (15). In short, administration of 0.5 mg of leuprolide acetate (Procrin; Abbott, Madrid, Spain) began in the midluteal phase of the previous cycle and continued until negative vaginal ultrasound defined ovarian quiescence. The dose of GnRH agonist then was decreased to 0.25 mg until the day of hcg administration. The fixed starting dose of 225 IU gonadotropins per day was initiated on day 3 of menstruation and sustained for the first 5 days of controlled ovarian stimulation, during which serum E 2 was assessed. The gonadotropin dose was adjusted if necessary. Serial transvaginal ultrasound examinations were initiated on day 5 of controlled ovarian stimulation and were performed every 48 hours to monitor the follicular growth. Human chorionic gonadotropin (Ovitrelle, 250 mg; Serono) was administered when three or more follicles reached 18 mm in diameter, and oocyte retrieval was scheduled 36 hours later. Serum E 2 and P levels were measured on the morning of hcg administration. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifico S.A., Madrid, Spain). The serum E 2 kit had a sensitivity of 28 pg/ml and intraobserver and interobserver variation coefficients of 6.6% and 7.7%, respectively. The serum P kit had a sensitivity of 0.2 ng/ml, with intraobserver and interobserver variation coefficients of 9.6% and 3.9%, respectively. Groups were compared regarding controlled ovarian stimulation parameters, number of oocytes and proportion of mature oocytes retrieved (number of metaphase II oocytes/total oocytes retrieved calculated for patients having intracytoplasmic sperm injection only), ovarian hyperstimulation syndrome (OHSS) incidence (16), and cost of treatment. Oocyte Recipients The protocol for hormonal replacement therapy has been described previously (17). In brief, down-regulation was achieved with use of an IM dose of 3.75 mg of triptorelin (Decapeptyl; Ipsen Pharma, Barcelona, Spain) beginning in the midluteal phase. Hormonal replacement therapy was initiated on day 1 to 3 of the following cycle, and doses of E 2 valerate (Progynova; Schering, Madrid, Spain) were increased as follows: 2 mg/d for the first 8 days of treatment, 4 mg/d for the following 3 days, and at least 6 mg/d until a pregnancy test was performed. On day 15, a scan was performed to evaluate endometrial growth. On the day after donation, 800 mg/d of micronized intravaginal P (Progeffik; Effik Laboratories, Madrid, Spain) were added. Embryo transfer was performed under ultrasound guidance on day 3 of development. Oocyte and Embryo Evaluation IVF Outcome An oocyte was considered to be normally fertilized when two pronuclei were visible. Fertilization rate was defined as the proportion of oocytes resulting in the formation of two pronuclei. Cleavage rate was calculated as the number of cleaving embryos divided by the total number of normally fertilized oocytes. Embryos were classified according to cell number, symmetry, and degree of fragmentation (18). The number of top-quality embryos was calculated by adding the number of cryopreserved embryos to the number of transferred embryos. Serum b-hcg was measured in recipients 16 days after oocyte donation. Implantation rate was obtained by dividing the number of gestational sacs revealed by the scan by the number of replaced embryos. Clinical pregnancy was confirmed 2 weeks later if a heartbeat was confirmed by transvaginal scan. Ectopic pregnancy was defined as a pregnancy sited out of the endometrium, detected by ultrasound or laparoscopy, or highly suspected because of symptoms and/or the b-hcg serum curve of the patient. Miscarriage rate was defined as the percentage of pregnancies not Fertility and Sterility â 959

3 TABLE 1 Oocyte donor characteristics and controlled ovarian stimulation parameters. Group 1, rfsh Group 2, HP-hMG Group 3, rfsh D HP-hMG P value No Age (y) NS BMI (kg/m 2 ) NS Antral follicle NS count Days of stimulation NS Gonadotropin dose (IU) 2, , , NS E 2 level hcg day (pg/ml) 2, , , NS P level hcg day (ng/ml) NS Oocytes retrieved NS Cancellation rate (%) 62/346 (18) 53/333 (16) 59/349 (17) NS Oocyte pickup (%) 284/346 (82) 280/333 (84) 290/349 (83) NS Mild and moderate OHSS 20/284 (7.04) 19/280 (6.78) 16/290 (5.52) NS rate (%) Severe OHSS rate (%) Melo. Gonadotropin regimens and IVF outcome. Fertil Steril surpassing the 20th week of gestation after previous detection of a heartbeat (13). Cost-minimization Analysis The cost-minimization analysis was calculated considering the cost per vial (75 IU) at the time of the clinical trial, which was V ($38.88 US) and V ($29.45 US) for rfsh and HP-hMG, respectively. The economic impact of the other procedures was the same for all study groups and thus was not taken into consideration. Cost-minimization analysis was preferred to cost-effectiveness analysis as it is more appropriate for comparative studies in which the treatments compared are assumed to be similar in effectiveness, as previously reported (8, 9, 12, 19, 20 23). Statistical Analysis The analysis of variance was used to test the significance of differences among the groups, and the c 2 test was used to assess the significance of categorical parameters and pregnancy and miscarriage rates among groups. A P value <.05 was considered significant. All statistical analyses were performed with use of the Statistical Package for the Social Sciences for Windows (version 11.0; SPSS, Chicago, IL). RESULTS A total of 1,028 oocyte donors were randomized and divided into three groups, according to the gonadotropin regimen, as described previously: group 1 (n ¼ 346), group 2 (n ¼ 333), and group 3 (n ¼ 349). No differences were found among the three groups with respect to age, BMI, and antral follicle count. Regarding controlled ovarian stimulation parameters, the mean number of days of stimulation and mean gonadotropin dose were similar in the three treatment groups. Moreover, the mean concentration of E 2 and P on the day of hcg administration did not differ. In terms of ovarian response, there was no difference among the groups in the mean number of oocytes retrieved. Moreover, there was a similar incidence of mild and moderate OHSS among the groups. No severe OHSS was observed. The overall cancellation rate ranged between 16% and 18%. The most frequent causes for cycle cancellation were the existence of fewer than six follicles that fulfilled the criterion for administering hcg on the last day of controlled ovarian stimulation (follicular diameter >14 mm), >30% drop in the level of E 2 on the day of hcg administration, risk of OHSS, and poor administration of medication. No differences were found between the groups with respect to these parameters. Finally, no differences were found among the groups in percentage of oocyte pickup and mean number of oocytes retrieved (Table 1). Regarding the recipients, a total of 1,079 patients were included in the study: 351 in group 1, 363 in group 2, and 365 in group 3. No differences were found among the three groups 960 Melo et al. Gonadotropin regimens and IVF outcome Vol. 94, No. 3, August 2010

4 TABLE 2 Oocyte recipient characteristics and IVF outcome. Group 1, rfsh Group 2, HP-hMG Group 3, rfsh D HP-hMG P value No. Characteristics Menopause NS Low ovarian response NS Premature ovarian failure NS Age NS Age (y) NS BMI (kg/m 2 ) NS Endometrial thickness NS (mm) Oocytes received NS Fertilization rate (%) NS Cleavage rate (%) NS Top-quality embryo NS Embryos transferred NS Implantation rate (%) NS Clinic pregnancy rate (%) 199/351 (56.7) 207/363 (57) 216/365 (59.2) NS Multiple pregnancy rate (%) 56/199 (28.1) 55/207 (26.6) 59/216 (27.3) NS Miscarriage rate (%) 35/199 (17.6) 37/207 (17.8) 38/216 (17.6) NS Ectopic pregnancy rate (%) 3/315 (0.85) 4/363 (1.1) 4/365 (1.09) NS Melo. Gonadotropin regimens and IVF outcome. Fertil Steril with respect to their age, BMI, and endometrial thickness (Table 2). These patients were included in our oocyte donation program because of menopause, 371 (34.3%); low ovarian response, 350 (32.4%); premature ovarian failure, 264 (24.3%); or advanced female age, 94 (9%). Oocyte recipients received a similar mean number of oocytes. In terms of embryo development, no differences were found among the groups with respect to fertilization and cleavage rates. Moreover, the mean numbers of top-quality embryos and embryos transferred per patient were similar in the three groups (Table 2). We obtained a total of 1,193 (36.5%) top-quality embryos in group 1, 1,270 (38.4%) in group 2, and 1,314 (38.3%) in group 3. Good-quality supernumerary embryos were frozen. Of 3,266 embryos obtained in group 1, 491 (15%) were frozen, compared with 544 (16.4%) of the 3,303 embryos obtained in group 2 and 584 (17%) of the 3,431 embryos obtained in group 3. Both parameters were similar in all three groups. In terms of IVF outcome, no statistically significant difference was detected among the regimens with respect to pregnancy rate, frequency of multiple pregnancies, ectopic pregnancy, and miscarriage rate. Regarding the cost-minimization analysis, oocyte donors given treatment with HP-hMG and rfsh þ HP-hMG required a lower number of vials than donors given treatment with rfsh (Table 3). Considering the 24.2% higher cost per vial of rfsh, the cost per cycle in this group was 25.8% and 13.6% higher than HP-hMG and rfsh þ HP-hMG groups, respectively. Moreover, the total number of vials of gonadotropins required per pregnancy was 9.5% and 12.8% higher for oocyte donors receiving rfsh than for those who received the other two treatments. Finally, the cost per pregnancy was 31.4% and 23.2% higher in the rfsh group than in the HP-hMG and rfsh þ HP-hMG groups, respectively. DISCUSSION The present clinical study represents the first large, prospective, randomized, controlled trial to evaluate the impact of three different gonadotropin protocols on oocyte donation program outcome and their respective cost. We decided to use this model because it is a powerful tool with which to discriminate between the effects of different regimens on the implantation process. Opting for a donor helps to reduce the variability in oocyte quality that can result from infertility. Moreover, the endometrium of the recipient is spared from the potentially harmful side effects of ovarian stimulation. Finally, the treatment is expensive, and a significant component of the cost of this assisted reproductive technology (ART) is the drugs administered for controlled ovarian stimulation. Fertility and Sterility â 961

5 TABLE 3 Cost-minimization analyses and percentage variation of economic parameters in rfsh versus other protocols. Parameter RFSH (n [ 346) HP-hMG (n [ 333) RFSH D HP-hMG (n [ 349) Mean gonadotropin IU/cycle Mean no. of vials/cycle Cost per vial, V (US$) Mean cost/cycle, V (US$) Total no. of vials No. of clinic pregnancies Total no. of vials/pregnancy Cost/pregnancy, V (US$) 2, , , ( 2.1%) RFSH (20.9) þ HP-hMG (10.4) ¼ 31.3 ( 6%) (38.88) (29.45) (38.88) þ (29.45) ( 24.2%) (1,294.85) (960.21) þ ¼ ( 25.8%) (1,119.01) ( 13.6%) 11,522 10,855 ( 5.8%) RFSH (7,294) þ HP-hMG (3,629) ¼ 10,923 ( 5.2%) ( 9.5%) RFSH (33.7) þ HP-hMG (16.8) ¼ 50.5 ( 12.8%) 1, (2,251.40) 1, (1,543.41) þ ¼ 1, ( 31.4%) (1,805.23) ( 23.2%) Melo. Gonadotropin regimens and IVF outcome. Fertil Steril To obtain more than one embryo for replacement in the oocyte donation cycle and thus improve the chances of pregnancy, it is necessary to stimulate the growth and maturation of several follicles. Recently, however, the aim of ovarian stimulation has shifted from obtaining the maximum number of oocytes possible to achieving an adequate cohort of goodquality embryos (8). The ovarian stimulation regimen appears to have an effect on the aneuploidy rate in embryos (24). Recently, administration of exogenous LH activity has been shown to modulate the magnitude of the follicular response (25 29) and to decrease the incidence of aneuploidy (24), perhaps by an effect on the quality of the oocyte (12, 24, 30 33). In studies of primates, a lack of LH activity affected oocyte maturation, embryo development, and implantation (34). This evidence suggests that significant suppression of LH during the midfollicular phase of ovarian stimulation has a detrimental effect on the outcome of ART. However, controversy over the most adequate gonadotropin preparation for induction of follicular maturation in IVF still exists. Several studies have shown that the stimulation profile of women receiving HP-hMG differs from that of those receiving rfsh. It has been observed that, at equal doses, HP-hMG displays a milder stimulation pattern, reflected in a lower mean number of oocytes, lower E 2 serum levels, and a trend toward a lower incidence of OHSS (8, 9, 26 28, 35). However, in the present study, no differences were found with respect to controlled ovarian stimulation parameters. A possible explanation for these contradictory data is that we did not use a strict fixed-dose protocol as used in the previous studies. After 4 days of controlled ovarian stimulation, the gonadotropin dose was adjusted according to the ovarian response. We believe that a fixed-dose protocol has disadvantages and may not be ideal for evaluating controlled ovarian stimulation parameters, as the dose in question may be inadequate for the subject, leading to a low or high ovarian response. In our study, all treatments proved similar with respect to the incidence of OHSS of both moderate and severe forms. In fact, we observed a low incidence in all groups, perhaps also due to the nonfixed dose used for the gonadotropin protocol. Previous studies of rfsh versus HP-hMG suggest that the former produces a more potent ovarian response than the latter, leading to a higher number of retrieved oocytes, but a lower number of top-quality embryos (8, 9, 24). However, our experience with oocyte donors has demonstrated that the two protocols are equipotent. When we analyzed the impact of the three different gonadotropin protocols on embryo development, we did not find any difference with respect to the different parameters. These findings may be explained by our study population, which was composed of young, healthy, and fertile donors. Finally, it is clear that the price of rfsh has an impact on patient budget (or that of the oocyte donation program) and on the final cost of ART procedures. If rfsh is more effective than HP-hMG, its use is obviously justified. However, recent randomized controlled trials have shown that HP-hMG and rfsh are equally effective (8, 9, 12, 19 21, 36). In the present study, the cost-minimization analysis revealed a higher cost 962 Melo et al. Gonadotropin regimens and IVF outcome Vol. 94, No. 3, August 2010

6 per cycle and per pregnancy with rfsh than with the other protocols, principally with respect to the HP-hMG regimen. These observations are in accordance with those of recent reports (23, 37). In summary, we have observed that both HP-hMG and rfsh preparations can be used with success for controlled ovarian stimulation in IVF cycles. It is encouraging and positive that controversy exists in our field, but recent studies have shown similar results and a higher pregnancy rate when HP-hMG was used. We believe that the most important finding of this study is that it highlights the higher cost per cycle and per pregnancy achieved when only rfsh is used in an oocyte donation program. On the basis of our results, we believe that a high success rate can be achieved at a lower cost by using HP-hMG to carry out controlled ovarian stimulation in oocyte donors. 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