Duration of progesterone-in-oil support after in vitro fertilization and embryo transfer: a randomized, controlled trial

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1 Duration of progesterone-in-oil support after in vitro fertilization and embryo transfer: a randomized, controlled trial Christine S. Goudge, M.D., Theodore C. Nagel, M.D., and Mark A. Damario, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Women s Health, University of Minnesota, Minneapolis, Minnesota Objective: To determine the efficacy of a short -in-oil protocol (11 days) in comparison with a traditional -in-oil protocol (6 weeks) after IVF and embryo transfer. Design: rospective randomized, controlled trial. Setting: Tertiary referral reproductive medicine unit. atient(s): The first IVF-embryo transfer cycle of women %37 years old. Intervention(s): Either the standard protocol, 50 mg of IM -in-oil daily for 6 weeks (group 1) or the study protocol, 50 mg of IM -in-oil daily for 11 days (group 2). Main Outcome Measure(s): Clinical pregnancy, ongoing pregnancy, and live birth rates. Result(s): One hundred one patients were enrolled (48 in group 1, 53 in group 2). Mean age (31.8 years vs years) and reasons for infertility were not different between the groups. Despite higher embryo quality in group 1 than in group 2 (60.3 vs. 51.6), clinical (63.0% vs. 62.7%) and ongoing (58.7% vs. 51.0%) pregnancy rates (R) were not significantly different. Live birth rates (52.2% vs. 49.0%) were not significantly different. Conclusion(s): regnancy and live birth rates were not different between the two groups, suggesting that the common practice of lengthy (R6 weeks) support of IVF pregnancies may be unnecessary. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Luteal phase support, progesterone-in-oil, in vitro fertilization, pregnancy outcome There is substantial evidence that luteal support with improves clinical outcomes following IVF embryo transfer cycles (1). rogesterone, the natural hormone produced by the corpus luteum (CL), is predominantly responsible for the timely transformation of the uterine endometrium that is necessary for embryonic implantation and the maintenance of pregnancy. It is hypothesized that luteal deficiency after IVF may occur because of impaired CL from residual GnRH agonist/antagonist effects or the loss of granulosa cells (GC) during the process of oocyte retrieval. To make up for this hypothesized deficit, most IVF protocols include supplementation, which is initiated by the time of embryo transfer and continued through the first 6 12 weeks of pregnancy. Recent reports, however, have challenged whether it is necessary to continue supplementation for significant portions of the first trimester (2, 3). Received February 4, 2009; revised April 22, 2009; accepted May 4, 2009; published online June 12, C.S.G. has nothing to disclose. T.C.N. has nothing to disclose. M.A.D. has nothing to disclose. Reprint requests: Theodore C. Nagel, M.D., Reproductive Medicine Center, th Avenue South, Suite 500, Minneapolis, MN (FAX: ; nagel007@umn.edu). A common form of supplementation used during assisted reproductive techniques (ART) in the United States has been IM -in-oil. rogesterone-in-oil is associated with higher implantation rates compared with administered orally or vaginally in some but not all studies (4, 5). A recent metaanalysis has suggested that IM conferred the most benefit compared with oral or vaginal routes (6). rolonged, repeated IM injections of -in-oil, however, may lead to severe inflammatory reactions, sterile abscesses, and significant patient discomfort. rolonged, repeated IM injections of -in-oil have also been reported to cause delayed forms of hypersensitivity reactions, even of a serious nature with marked eosinophilia and pulmonary compromise (7, 8). MATERIALS AND METHODS atient opulation atients were eligible for inclusion if they were 37 years old or less and were undergoing their first IVF-embryo transfer cycle for any indication. Every patient meeting these inclusion criteria was offered participation in the study. Institutional Review Board (IRB) approval was obtained from the University of Minnesota Research Subjects rotection rogram. Once enrolled, subjects were randomly assigned to either receive the standard protocol (group 1): 50 mg of -in-oil IM daily, beginning on the day of oocyte retrieval and continuing until the pregnancy confirmation ultrasound (5 6 weeks) if pregnant, or the study protocol (group 2): 50 mg of -in-oil IM daily, beginning on the day of embryo transfer and continuing for 11 days, until the day of the pregnancy test. Randomization was accomplished using sequentially numbered, opaque, sealed envelopes. 946 Fertility and Sterility â Vol. 94, No. 3, August /$36.00 Copyright ª2010 American Society for Reproductive Medicine, ublished by Elsevier Inc. doi: /j.fertnstert

2 Treatment rotocols Both groups of patients were treated in an identical manner with the exception of the study variable (-in-oil administration). Most patients underwent down-regulation with the GnRH agonist leuprolide acetate (LA; Lupron; TA harmaceuticals, Deerfield, IL) in a long protocol with 5 days overlap with an oral contraceptive (OC; Ortho-Novum 1/35 or Ortho-Novum 1/50; Ortho harmaceuticals, Raritan, NJ). Leuprolide acetate was administered SC at a dosage of 0.5 mg daily. After withdrawal bleeding and confirmation of adequate ovarian suppression (no ovarian cysts >18 mm and E 2 levels of <50 pg/ml), gonadotropin stimulation was begun. A minority of patients (<5%) received the GnRH antagonist ganirelix acetate (Ganirelix Acetate Injection; Organon Inc., West Orange, NJ) at a dosage of 250 mg SC daily starting during the gonadotropin stimulation (when E 2 levels were R250 pg/ml or lead follicle(s) were R14 mm). Either the LA or ganirelix acetate were discontinued on the day of hcg administration. Because the number of patients that received the GnRH antagonist protocol was so small, no statistical comparison was made between the outcomes of patients who did or did not receive ganirelix acetate. Gonadotropin therapy was begun using either highly purified FSH (Bravelle; Ferring harmaceuticals, arsippany, NJ) or recombinant FSH (Gonal-F; EMD Serono, Inc., Rockland, MA, or Follistim; Organon). In a limited number of cases, purified menotropins (Menopur; Ferring harmaceuticals) were also used for gonadotropin stimulation as part of a mixed protocol. In general, starting total daily dosages of gonadotropins were IU by SC daily or twice daily injections depending on clinical circumstances. On occasion, a patient with a history of previous poor response to gonadotropins was started on a higher initial dosage of gonadotropins ( IU daily). Gonadotropin dosage was increased or decreased as follicular growth was initiated as clinically indicated. Monitoring of serum E 2 concentrations and follicular growth were initiated on the fourth and sixth days of stimulation, respectively. Follicular monitoring was undertaken with a GE Voluson 730 real-time ultrasound device (GE Healthcare, London, United Kingdom) equipped with a 3.7- to 9.3-MHz transvaginal probe. The timing of hcg administration was based on several factors, including the mean lead follicular diameter(s), serum E 2 concentration, and the rate of increase of serum E 2. In general, hcg was administered at a dosage of 10,000 units IM when the lead follicle(s) were R20 mm in mean diameter. Oocyte retrievals were scheduled 36 hours after hcg administration. Transcervical embryo transfers were performed on the third day after oocyte retrieval in all patients. All subjects had hcg and E 2 levels measured on the day of embryo transfer. Also, levels were measured on the day of the pregnancy test for all subjects. For patients in group 2, the level was used to determine whether the study protocol (discontinuation of ) could be safely carried out. If their pregnancy test was positive and their level was R15 ng/ml, the -in-oil injections were discontinued and level rechecked in 3 5 days. If the repeat serum level remained R15 ng/ml, no further testing or -in-oil injections were administered. If the initial or repeat serum level was <15 ng/ml, the patient was instructed to resume daily -in-oil IM injections until the day of confirmation of pregnancy ultrasound at 7 8 weeks estimated gestational age. Those patients in group 1 who were pregnant took daily IM -in-oil injections until the day of confirmation of pregnancy ultrasound (at 7 8 weeks estimated gestational age), regardless of level. Hormonal Assays Estradiol,, and hcg were measured by separate electrochemiluminescence immunoassays. The tests were done using a Roche Elecsys 2010 immunoassay analyzer (Roche Diagnostics, Indianapolis, IN). Statistical Analysis Statistical analysis was performed by the department of biostatistics at the University of Minnesota and included analysis using the Student s t-test, c 2 test, and Fisher s exact test. Continuous data were reported as means and SD. Student s t-tests and c 2 tests were used for comparisons of continuous and noncontinuous data, respectively. Fisher s exact test was carried out when appropriate. Statistical significance was defined as <.05. RESULTS Demographics One hundred one patients were enrolled in the study (48 in group 1, 53 in group 2) between January 2005 and August Four patients withdrew from the study during their treatment (two from group 1, two from group 2). Groups 1 and 2 did not differ significantly in age (mean, 31.8 vs years; ¼.143), although there was a slight trend toward younger women in group 1. Subjects reasons for pursuing IVF were varied (Table 1); no significant differences between groups were identified. Embryo Quality A similar number of embryos were transferred to patients in both groups (mean, 2.12 vs. 2.13; ¼.944). Embryo quality was evaluated both qualitatively and quantitatively. Embryos that were eight cells and grade 1 or 2 were considered high quality. Likewise, embryos with 7, 9, or 10 cells that were grade 1 were also considered high quality. Considering this qualitative measure, there was no significant difference between the number of subjects that had at least one (60.4% vs. 43.4%; ¼.087), or two (37.5% vs. 24.5%; ¼.158) high-quality embryos transferred in group 1 versus group 2. A quantitative measure of embryo quality was derived by calculating the embryo implantation index. The number of cells in an individual embryo was multiplied by a cofactor relating to its grade. The cofactor for a grade 1 embryo was five; for a grade 2 embryo, the cofactor was four; for a grade 3 Fertility and Sterility â 947

3 TABLE 1 Demographics. Age, y (SD) 31.8 (3.2) 32.7 (3.1).143 a Reason for IVF.373 b Male factor only Ovulatory dysfunction 5 7 Endometriosis only 2 1 Tubal factor only 0 2 Elevated FSH only 0 2 Unexplained 6 8 Multiple reasons a Student s t-test. b Fisher s exact test. embryo, the cofactor was three; for a grade 4 embryo, the cofactor was two. For example, an 8-cell, grade 2 embryo was assigned a score of 32. The majority (97%) of subjects had two embryos transferred. The scores for the two embryos transferred were added together to achieve the embryo implantation index for each individual patient. The mean embryo implantation index for patients in group 1 was For group 2, the mean index was This difference was significantly different (¼.022). This significant difference in embryo quality favored higher quality embryos in the control group (Table 2). Hormone Assays Hormone assays on the day of embryo transfer included measurements of hcg and E 2 (Table 3). The mean hcg levels did not differ significantly between groups 1 and 2 (50.6 vs miu/ml; ¼.703). The mean E 2 levels also did not differ significantly between groups 1 and 2 (1,179.7 vs. 969 pg/ml; ¼.133). On the day of the pregnancy test, 11 days after embryo transfer, levels were evaluated before discontinuing -in-oil in group 2 subjects (Table 3). Some subjects had returned to the care of their primary doctor in out-state Minnesota for this portion of care. Because of the inability to access all out-of-network records, levels from this period were only recovered for 82 of the 98 subjects (83.4%) who remained in the study. There were two patients in group 1 and one patient in group 2 whose level was <15 ng/ml on that day. The one patient with a level of <15 ng/ml in group 2 was asked to continue supplementation until the day of her pregnancy confirmation TABLE 2 Embryo Quality. No. of embryos transferred, mean (SD) At least 1 high-quality embryo transferred, n (%) 2 high-quality embryos transferred, n (%) Embryo implantation index, mean (SD), range a Student s t-test. b c 2 test (0.32) 2.13 (0.49).944 a 29 (60.4) 23 (43.4).087 b 18 (37.5) 13 (24.5).158 b 60.3 (17.2), (18.3), a 948 Goudge et al. Duration of support in IVF Vol. 94, No. 3, August 2010

4 TABLE 3 Hormone Assays. Day of embryo transfer hcg, mean (SD), range 50.6 (71.4), (22.5), a E 2, mean (SD), range 1,179.7 (603.5), 355 3, (557.5), 251 3, a Day of pregnancy test level >15 ng/ml, % (n) 94.4 (34/36) 98.0 (50/51).567 b 3 5 days after discontinuation of level >15 ng/ml, % (n) 100 (34/34) a Student s t-test. b Fisher s exact test. ultrasound, as per the study protocol. There were three other patients in group 2 who continued taking for the full 6 weeks despite the study protocol. The outcomes of these three patients were retained in group 2 during the subsequent intention-to-treat analysis of our study results. All group 2 patients who discontinued their use after 11 days with levels >15 ng/ml had their levels rechecked 3 5 days later. In all cases, the level of >15 ng/ml was maintained after discontinuation of supplementation. Outcomes The main outcome measures for this study were clinical and ongoing pregnancy rates (R) and live birth rate (Table 4). The clinical Rs (63% vs. 62.7%; ¼.976) and ongoing Rs (58.7% vs. 51%; ¼.446) were not significantly different between groups 1 and 2. The live birth rates were also not significantly different between the two groups (52.5% in group 1 vs. 49.0% in group 2; ¼.839). There were 4 twin births in group 1 and 12 twin births in group 2. There were no other multiple births. DISCUSSION Although it remains common practice, there is surprisingly little data to support prolonged administration during the first trimester after IVF-embryo transfer. A single prospective, randomized trial has evaluated the effect of supplementation (rogestan, 200 mg vaginally three times a day) either withdrawn on the day of positive hcg test or taken for another 3 weeks (2). Deliveries occurred in 78.7% and 82.4% of patients with positive hcg tests in the study and control groups, respectively, showing no statistically significant difference. One retrospective, uncontrolled study compared 200 pregnant women receiving vaginal beginning the day of embryo transfer and continuing for 3 additional weeks after a positive hcg test with 200 pregnant women receiving vaginal beginning the day of embryo transfer and discontinuing on the day of the positive hcg test. Of the TABLE 4 Outcome Data. Biochemical pregnancies, n (%) 31 (67.4) 35 (68.8).677 a Clinical pregnancies, n (%) 29 (63.0) 32 (62.7).976 a Ongoing pregnancies, n (%) 27 (58.7) 26 (51.0).446 a Live births, n (%) 24 (52.2) 25 (49.0).839 a Multiple pregnancies, n (%) 4 (16.7) 12 (48).032 b a c 2 test. b Fisher s exact test. Fertility and Sterility â 949

5 200 pregnancies in which vaginal was discontinued early, 63% ended in live birth compared with 64% of controls (3). One retrospective study evaluated outcomes in patients receiving 50 mg of daily IM -in-oil beginning on the day of oocyte retrieval (9). In this report, supplementation was discontinued on the day of the positive hcg test only in those pregnant patients in whom serum levels were >60 ng/ml. rovided subsequent levels were maintained at >30 ng/ ml, supplementation remained discontinued with comparable live birth rates. Another retrospective cohort study evaluated mg of daily IM -in-oil beginning on the day of oocyte retrieval and either discontinuing on the day of the positive hcg test or continuing until 12 gestational weeks (10). Similar Rs occurred between the luteal protocol (46.3%) and the first trimester protocol (49.0%). At present, however, there have been no prior prospective, randomized controlled trials comparing a luteal phase IM protocol with standard first trimester IM supplementation. The study we present here was done in an effort to evaluate the optimal length of luteal phase IM support in a prospective, randomized fashion. There were two major differences between the regimens in the control and study groups. The first difference was the timing of initiation. The study group started taking IM -in-oil injections on the day of the embryo transfer, which was 3 days later than the control group, who began taking -in-oil injections on the day of oocyte retrieval. The IM injection of hcg that was given to all women in this study 36 hours before oocyte retrieval is likely to be adequate for luteal support for IVF-embryo transfer patients during the first 3 days after oocyte retrieval. Nationally, IVF-embryo transfer protocols differ with respect to the day that luteal support is initiated. There has been some evidence to suggest that the endometrium at the time of oocyte retrieval in women undergoing IVF is advanced beyond what is otherwise normally expected at the time of ovulation in natural cycles (11). Whether these precocious changes within the endometrium are clinically relevant remains to be determined. One potential benefit of delaying -in-oil administration initiation from the day of oocyte retrieval to the day of embryo transfer, although, is that it causes less exogenous hormone influence on the endometrium and would contribute less to any possible abnormal maturation of the endometrium that may or may not be important in IVF-embryo transfer outcomes. The second major difference between the two treatment protocols was the timing of the discontinuation. Continuing supplementation beyond the time of the positive pregnancy test (11 days after embryo transfer) did not confer an outcome benefit to the control subjects, whose clinical Rs, ongoing Rs, and live birth rates were not significantly different from those subjects in the study group. As there was one patient in the study group whose was continued because of a level <15 ng/ml on the day of her pregnancy test, the conclusions of this study can only be assumed to apply to patients with a level >15 ng/ml at the time of their pregnancy test. Interestingly, there was an observation of higher quality embryos transferred in the control group. This significant difference in embryo quality is attributable to chance as the treatment intervention in this study did not alter any characteristic that would have been expected to influence embryo quality. Our data showing no difference in any outcome data, including live birth rates, between subjects in these two groups exists despite the higher quality embryos transferred in the control group, and support a conclusion that there was no benefit to a longer course of supplementation in the control group. One of the limitations of this study is that the sample size was small enough that chance differences in embryo quality were present, which may have had an impact on outcomes. This weakness can be overcome by repeating a similar trial with a larger sample size. ower analysis suggested that a larger sample size than could be accomplished at one institution would be required to have enough confidence in the outcome to affect treatment protocols. In addition, our study is limited by having made changes to both the day of -in-oil supplementation initiation and the day of discontinuation. We had hypothesized that changes to both the initiation and discontinuation days would permit a maximal reduction in the number of required days of luteal supplementation. Although we believe that initiating -in-oil supplementation on the day of embryo transfer to be largely indistinguishable from initiation on the day of oocyte retrieval, future studies investigating the optimal length of supplementation might facilitate a more straight-forward statistical analysis if the initiation date is the same in all subject groups. Despite a limited sample size and significantly higher quality embryos transferred in the control group, there was no significant difference in clinical Rs, ongoing Rs, or live birth rates between the group of IVF-embryo transfer patients who received 6 weeks of -in-oil supplementation and the group that only received 11 days of supplementation. Thus, in this small randomized, controlled trial of 101 patients undergoing IVF-embryo transfer there was no benefit to a longer (6 weeks) length of supplementation when compared with the study group receiving supplementation for only 11 days. This finding suggests that further study in larger groups of women may reveal that our standard practice of lengthy supplementation in IVF pregnancies is unnecessary. REFERENCES 1. Soliman S, Daya S, Collins J, Hughes EG. The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil Steril 1994;61: Nyboe Andersen A, opovic-todorovic B, Schmidt K, Loft A, Lindhard A, Højgaard A, et al. rogesterone supplementation during early gestations after IVF or ICSI has no effect on the delivery rates: a randomized controlled trial. 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