- Email: [email protected]
Does endometrial integrin expression in endometriosis patients predict enhanced in vitro fertilization cycle outcomes after prolonged GnRH agonist therapy?
Does endometrial integrin expression in endometriosis patients predict enhanced in vitro fertilization cycle outcomes after prolonged GnRH agonist therapy? Eric S. Surrey, M.D., Annette K. Lietz, L.V.N., Robert L. Gustofson, M.D., Debra A. Minjarez, M.D., and William B. Schoolcraft, M.D. Colorado Center for Reproductive Medicine, Lone Tree, Colorado
Objective: To determine whether endometrial expression of the integrin avb3 vitronectin can predict which endometriosis patient subgroup will benefit from pre-IVF cycle prolonged GnRH agonist (GnRHa) therapy. Design: Prospective randomized institutional review board approved pilot trial. Setting: Private assisted reproductive technology program. Patient(s): IVF candidates with regular menses, surgically confirmed endometriosis, and normal ovarian reserve. Intervention(s): All patients underwent endometrial biopsy 9 to 11 days post-LH surge to evaluate avb3 integrin expression. Patients were randomized either to receive depot leuprolide acetate 3.75 mg every 28 days for three doses before controlled ovarian hyperstimulation (COH) or to proceed directly to COH and IVF. Group 1: integrin-positive controls (N ¼ 12); group 2: integrin-positive administered prolonged GnRHa (N ¼ 8). Group A: integrin-negative controls (N ¼ 7); group B: integrin-negative administered prolonged GnRHa (N ¼ 9). Main Outcome Measure(s): COH responses, ongoing pregnancy and implantation rates. Results: There were no significant effects of GnRH agonist treatment in either of the integrin expression strata regarding ongoing pregnancy or implantation rates, although these outcomes were more frequent in group 2 vs. 1 (62.5% vs. 41.6% and 35% vs. 20.6%, respectively). This effect may have because of limited sample size. The value of a negative integrin biopsy in predicting an ongoing pregnancy after prolonged GnRH agonist therapy was only 44.4%. Conclusion(s): Endometrial avb3 integrin expression did not predict which endometriosis patients would benefit from prolonged GnRHa therapy before IVF. (Fertil Steril 2010;93:646–51. 2010 by American Society for Reproductive Medicine.) Key Words: Implantation, integrin, endometriosis, GnRH agonist, infertility, in vitro fertilization
It has been previously demonstrated that endometrial expression of the integrin subtype avb3 vitronectin and its receptor appears to occur 6 to 10 days after ovulation, the presumed window of implantation (1, 2). This cell adhesion molecule has also been demonstrated on the surface of embryos (3). It has been proposed that this integrin and its receptor may act not only as a site of interaction between the embryo and the endometrium, but may also play a role in initiating trophoblast invasion of the endometrium (4, 5). Lessey et al. (6) reported that aberrant endometrial avb3 integrin expression in 39% of women with unexplained infertility and ‘‘in-phase’’ endometrial biopsies. Most of the patients in this trial were noted to have stage I and II endometriosis. These same investigators demonstrated aberrant expression in 44% of endometriosis patients (7). These findings, however, have not been consistently reported (8, 9). Received April 14, 2008; revised December 5, 2008; accepted December 11, 2008; published online January 27, 2009. E.S.S. has received grant support from TAP Pharmaceuticals. A.K.L. has nothing to disclose. R.L. Gustofson has nothing to disclose. D.A.M. has nothing to disclose. W.B.S. has nothing to disclose. Presented in part at the 63rd Annual Meeting of the American Society for Reproductive Medicine, Washington, DC, October 13–17, 2007. Supported in part by a grant from TAP Pharmaceuticals, Waukegan, IL. NIH Clinical Trials Registry Identifier: NCT00621179. Reprint requests: Eric S. Surrey, M.D., Colorado Center for Reproductive Medicine, 10290 RidgeGate Circle, Lone Tree, CO 80124 (FAX: 303-788-8310; E-mail: [email protected]).
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We have recently described a 48.6% prevalence of absent integrin expression in a case–control study of 74 consecutive IVF candidates felt to be at high risk for implantation defects because of prior IVF failure but adequate embryo quality and/ or endometriosis (10). Of the patients who had previously undergone laparoscopy, 52.8% had been diagnosed with endometriosis, of which 57.1% was stage III–IV disease. Return of avb3 vitronectin expression with administration of a GnRH agonist or danazol has been described in prospective but nonrandomized trials (11, 12). This may represent an explanation for the improved pregnancy rates reported in endometriosis patients administered a prolonged course of a GnRH agonist before IVF-ET (13, 14). This approach has also been shown to result in IVF cycle outcomes that were no different than in integrin-positive patients who were not administered this agent (10). However, the question of whether endometrial avb3 vitronectin expression can predict which subgroup of endometriosis patients will benefit from prolonged pre-IVF cycle GnRH agonist therapy has not been assessed, and is the objective of the current investigation.
MATERIALS AND METHODS This investigation is a prospective randomized pilot trial approved by the HCA/HealthOne institutional review board. The study population consisted of patients with surgically
Fertility and Sterility Vol. 93, No. 2, January 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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documented endometriosis and no current sonographic evidence of an ovarian endometrioma >2 cm in mean diameter. All patients had evidence of normal ovarian reserve testing (day 3 serum FSH level <11 mIU/mL, E2 level <60 pg/ mL, and bilateral antral follicle count >4). A normal clomiphene challenge test was required for all women >38 years. All patients had a normal uterine cavity documented at precycle office hysteroscopy. Patients had received no depot preparations of a GnRH agonist, danazol, or other hormonal suppressive therapy for endometriosis within 6 months of study entry. Endometrial biopsies were performed on all patients 9 to 11 days after an LH surge was documented by urinary ovulation predictor kits. Tissue was evaluated for the presence or absence of avb3 vitronectin by commercial assay (Adeza, Sunnyvale, CA) using previously described techniques (1). All endometrial biopsy samples were confirmed to be in phase 2 days by standard histologic criteria. After the biopsy results were obtained, patients were randomized by two separate computer generated randomized number tables, one for women expressing the integrin and one for women who did not express the integrin, to either receive an intramuscular preparation of the GnRH agonist leuprolide acetate (TAP Pharmaceuticals, Waukegan, IL) 3.75 mg every 28 days for three injections before initiation of controlled ovarian hyperstimulation or to proceed directly to controlled ovarian hyperstimulation and IVF. This randomization scheme was designed to allow for more equal distribution of treatment regimens for both integrin-positive and -negative patients given the presumed unequal distribution of biopsy results on previously published data (10). Patients were randomized into one of four groups based on avb3 vitronectin expression and treatment protocol. Those integrin-positive patients randomized to the control group were assigned to group 1, and those who were integrin negative were assigned to group A. Patients who were randomly assigned to receive a prolonged course of the GnRH agonist who were integrin positive were assigned to group 2, whereas those who were integrin negative were assigned to group B. All patients subsequently underwent controlled ovarian hyperstimulation employing standard GnRH agonist downregulation or microdose flare protocols. The determination for which protocol was to be employed was based on ovarian reserve testing and response in previous cycles when appropriate. Indications for hCG administration, day 3 vs. 5 embryo transfer and numbers of embryos to transfer were based on previously published protocols and ASRM/SART guidelines (15–17). Remaining viable embryos were subsequently cryopreserved at the blastocyst stage 5 and/or 6 days after oocyte aspiration. Pregnancy tests were performed 14 days after oocyte aspiration. Biochemical pregnancy rate was defined as the number of positive serum pregnancy tests obtained 14 days after oocyte aspiration per embryo transfer procedure. Ongoing pregnancy rate was defined as the presence of an intrauterine gesFertility and Sterility
tational sac with fetal cardiac activity documented by ultrasound evaluation performed 4 to 5 weeks after a positive pregnancy test per embryo transfer procedure. Implantation rate was defined as the number of intrauterine gestational sacs with fetal cardiac activity as documented by ultrasound examination per number of embryos transferred. Data were analyzed by Student’s group t tests and chisquare analyses as appropriate. Values of P<.05 were considered to be statistically significant.
RESULTS Forty patients were initially recruited to participate in the trial. The overall incidence of absent endometrial integrin expression was 45%. Thirty-seven underwent randomization after three dropped out: two for personal reasons and one who elected to proceed with oocyte donation because of multiple failed IVF cycles with uniformly compromised embryo quality. A summary of the randomization scheme is displayed in Figure 1. Baseline clinical characteristics are displayed in Table 1. There were no significant differences between group 1 and 2 or groups A and B with regard to age, characteristics of ovarian reserve, extent of endometriosis, or number of prior failed cycles. It is interesting to note that only 18.9% of the patients who underwent randomization had previously undergone an unsuccessful IVF cycle. Of these 7 patients, only one had failed more than one prior cycle. Outcomes of controlled ovarian hyperstimulation are displayed in Table 2.There were no statistically significant differences between group 1 and 2 or groups and B for any of the parameters assessed. However, higher gonadotropin dose requirements that did not reach statistical significance were noted for both groups 2 and B, who received prolonged courses of the GnRH agonist. Similarly, there were no significant differences between group 1 vs. group 2 and group A vs. group B with regard to the incidence of day 3 vs. 5 embryo transfer, use of intracytoplasmic sperm injection, or assisted hatching. One group B cycle was cancelled before oocyte aspiration for poor response. One patient randomized to group A patient conceived spontaneously before initiation of therapy. Cycle outcomes are displayed in Figures 2 and 3. There were no significant differences between groups 1 and 2 or groups A and B with regard to biochemical pregnancy, ongoing pregnancy, or implantation rates. An apparent trend toward higher ongoing pregnancy and implantation rates in group 2 vs. 1 did not achieve statistical significance (odds ratio [OR] 2.33; 95% confidence interval [CI]: 0.37–14.61 and OR 2.08; 95% CI: 0.60–7.14, respectively). Based on the small patient numbers evaluated in this study, the value of a negative integrin biopsy in predicting an ongoing pregnancy after a prolonged course of GnRH agonist was 44.4%. The value of a positive integrin biopsy in predicting 647
FIGURE 1 Randomization summary of patients eligible to participate in the current trial. 40 Patients Eligible
3 Drop-Outs Prior to Randomization1
Randomization
Group 1 12 Patients
Group 2 8 Patients
Group A 7 Patients
Group B 10 Patients
Completed Therapy: 12 Patients
Completed Therapy: 8 Patients
Completed Therapy: 6 Patients2
Completed Therapy: 9 Patients3
1. 2 drop-out for personal reasons, 1 proceeded with oocyte donation 2. 1 patient conceived pre-therapy 3. 1 patient cancelled due to a poor response
Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
an ongoing pregnancy without prolonged GnRH agonist therapy was 41.7%. DISCUSSION In the current investigation, we hypothesized that endometrial avb3 vitronectin expression might predict response to prolonged GnRH agonist therapy before an IVF cycle in endometriosis patients. The randomization scheme in this prospective pilot trial was designed to assign relatively equal numbers of patients with either positive or negative biopsies to each of the two treatment groups given the unequal distribution of aberrant integrin expression. Unfortunately, we noted that there were no significant differences in ongoing pregnancy or implantation rates in those patients who failed to express avb3 vitronectin who were treated with a 3-month course of a GnRH agonist before an IVF cycle in comparison to untreated controls. The predictive values of biopsy results in determining response to prolonged GNRH agonist therapy was uniformly poor, with a 44.4% likelihood that a negative biopsy would predict IVF pregnancy after GnRH agonist therapy and a 41.7% likelihood that a positive biopsy would predict pregnancy if prolonged GnRH agonist therapy was not administered. The use of prolonged courses of GnRH agonists has been shown to enhance assisted reproductive technology cycle outcomes in patients with endometriosis. Three prospective randomized trials demonstrated significantly higher clinical pregnancy rates after a 3- to 6-month course of therapy before 648
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Integrin and GnRH agonist response
IVF or gamete intrafallopian transfer (14, 18, 19). In a recent meta-analysis, Sallam et al. (13) noted that the clinical pregnancy rate in endometriosis patients administered a GnRH agonist for 3 to 6 months before the assisted reproductive technologies was increased fourfold in comparison to controls (OR 4.28; 95% CI 2–9.15). In the current study, a 3month course of agonist administration was selected because this was the shortest duration demonstrated to be efficacious in previously published trials (11, 14). However, it is important to note that ongoing pregnancy and implantation rates in the control group reported more recently by Surrey and coworkers (14) were still rather high (53.80% and 30.38%, respectively). Therefore, it would be advantageous to be able to predict which endometriosis patient groups might better benefit from prolonged GnRH agonist therapy to minimize the additional expense and delay associated with this treatment. Aberrant endometrial avb3 integrin expression was reported by Lessey and coworkers (6) in 39% of women with unexplained infertility and an ‘‘in-phase’’ endometrial biopsy performed between cycle days 20 to 24. Sixty-four percent of these women had a prior diagnosis of minimal to mild endometriosis. These investigators also demonstrated that 44% of infertile women with endometriosis failed to express this integrin subtype (7). It is interesting to note that in this trial, fertile women with endometriosis had a similar degree of staining for integrin as parous controls. In a recent case–control study, we reported aberrant expression in 48.6% of IVF patients with endometriosis and/or unexplained implantation failure despite adequate embryo development (10). Not all investigators have supported these conclusions, however. Two small series reported no statistically significant differences in endometrial avb3 expression in women with endometriosis in comparison to those with unexplained infertility or to fertile controls (8, 9). In a prospective observational study, Thomas et al. (20) described a group of 66 IVF patients who underwent precycle endometrial biopsy. A significantly higher percentage of women with positive avb3 integrin expression conceived than those without (47.1% vs. 26.7%; P<.027). However, only six of the overall patient cohort had a diagnosis of endometriosis. We are unaware of published studies that have demonstrated cycle-tocycle variation in integrin expression within individuals. Such variation, if it did occur, would represent a confounding variable given the design of the current investigation. The rationale behind linking aberrant avb3 integrin expression and successful response to a GnRH agonist is based on limited data. Lessey et al. (6) had previously reported a significantly greater return of endometrial integrin expression in a group of 22 women with stage I–II endometriosis treated with laparoscopic laser ablation of their disease followed by a 3-month course of a GnRH agonist than in a group of 25 similar women treated with surgery alone (64% vs. 35%; P<.05) (11). Trends, which did not achieve statistical significance toward higher pregnancy rates and shorter time Vol. 93, No. 2, January 15, 2010
In a retrospective case–control study, Surrey and coworkers (10) evaluated a group of patients at high risk for implantation failure and noted a trend toward more severe disease in those endometriosis patients who failed to express endometrial avb3 vitronectin. The subset of patients who did not express the integrin was treated with a 2-month course of a depot preparation of leuprolide acetate before IVF. Cycle outcomes were similar to a group of untreated controls with integrin expression. The lack of randomization was clearly a weakness in the study design.
Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
Results expressed as mean SD. No differences between Groups 1 and 2 or Groups A and B were statistically significant for any of these parameters.
0.42 0.9 0.25 0.46 0 0.2 0.42 41.7 37.5 57.2 30 1 2 A B
þ þ – –
– þ – þ
12 8 7 9
33.75 4.11 (Range: 28–40) 7.58 1.99 36.8 13.13 33.0 3.55 (Range: 28-39) 7.46 1.56 39.5 14.98 33.86 2.79 (Range: 31–39) 6.9 1.15 37.12 12.64 32.67 4.95 (Range: 23–40) 6.96 1.24 43.36 22.19
14.4 6.57 11.0 4.84 13.0 4.55 11.6 3.92
2.58 1.1 2.5 0.76 2.57 0.98 2.3 1.06
Prior Failed IVF Cycles III/IV (%) Mean Group aVb3
Prolonged GnRHa Patients
Age
Day 3 FSH (mIU/mL)
Day 3 E2 (pg/mL)
Antral follicles
Endometriosis stage Baseline Clinical Data.
TABLE 1
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intervals to conception, were noted in the group treated with the agonist as well. It is important to note that none of the patients in this trial underwent IVF so that it is not possible to determine whether the outcomes can be extrapolated to the model employed in the current investigation. Tei et al. (12) more recently evaluated 57 women with a history of repeated IVF cycle failures. avb3 integrin expression was lower in the group with unexplained infertility, although the incidence of endometriosis per se was not described. Nine of the patients randomly selected to receive a 12week course of oral danazol were noted to have a significant increase in integrin expression after completion of therapy (P¼.0076). However, the impact of this therapy on cycle outcome was not described.
There are several possible explanations for the failure to prove our hypotheses. One conclusion would be that any benefit derived by extended GnRH agonist therapy on IVF cycle outcome in infertile women with endometriosis does not occur by its action on avb3 vitronectin expression. The lack of statistically significant effects is compatible with this explanation. This possibility cannot be proven by the results of the current trial because endometrial biopsies would have had to have been repeated after therapy was completed and after return of ovulation. This would have necessitated a delay in initiation of an IVF cycle that could theoretically have been associated with a return to the abnormal state, which the agonist was designed to correct. As previously discussed, the issue of cycle-to-cycle variation in integrin expression has not been evaluated. A host of other factors that effect uterine receptivity have also been shown to differentially affect endometriosis patients and could certainly serve as confounding variables (21–26). A second explanation for the results in the current trial is the fact that, although prospective and randomized, this investigation is made of four groups of relatively small size with power that may be insufficient to detect significant differences between the treatment protocols. To demonstrate the 20% improvement in pregnancy rates described in our previously published trial, a sample size of at least 90 patients would have been necessary in each group pair (14). Even larger numbers would be required to detect a difference in treatment effects between integrin-positive and integrinnegative patients. It is also important to note that the patients previously evaluated by Lessey et al. (6), who demonstrated return on integrin expression after GnRH agonist therapy were exclusively those with stage I and II disease (11). 649
TABLE 2 Outcomes of Controlled Ovarian Hyperstimulation (COH). Group 1 2 A B
COH dose (units)
COH days
Oocytes retrieved
MII oocytes
2PN (%)
Embryos Embryos transferred cryopreserved
2638 1231 9.92 1.51 18.75 7.79 15.75 1.83 68.5 21.2 2.83 0.94 3560 709 9.5 0.93 11.63 5.73 8.88 4.82 69 28.2 2.5 1.23 2875 646.3 9.5 1.05 12.3 4.6 9.77 1.83 67.2 26.3 2.33 0.52 3558.8 963.1 9.6 1.07 13.78 9.22 11.56 8.9 75 17.4 2.56 0.73
3.5 3.92 0.88 1.13 0.67 1.21 1.78 2.28
Results expressed as mean SD. No differences between Groups 1 and 2 or Groups A and B were statistically significant for any of these parameters. Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
The preponderance of those endometriosis patients randomly assigned to receive GnRH agonist therapy in the previously published trial by Surrey et al. (14) had more severe disease and integrin biopsies were not performed. Thus, the effect of the agonist on integrin expression may differ based on the extent of the disease. A third explanation may have to do with the effect of the biopsies per se. Several investigative teams have suggested that the local endometrial injury induced by performing an endometrial biopsy in the cycle before IVF can improve outcomes (27, 28). In a prospective trial, Barash et al. (27) performed repeated endometrial biopsy on cycle days 8, 12, 21, and 26 during the menstrual cycle immediately before initiation of controlled ovarian hyperstimulation in a group of 45 randomly selected women who had failed to conceive during prior cycles and were felt to be good responders. Significantly higher live birth rates compared with a control group of 89 similar patients who did not undergo biopsy (48.9% vs. 22.5%, P¼.016) were reported. If in the current investigation
Biochemical (Bio), ongoing pregnancy (OPR), and implantation (IR) rates in Groups 1 and 2 with positive endometrial avb3 vitronectin expression.
1 75.0%
80% 2 62.5%
2 62.5% 1 41.6%
40%
2 35.0% 1 20.6%
20%
Bio OR 0.56 95% CI: 0.8-3.86
OPR OR 2.33 95% CI: 0.37-14.6
IR OR 2.08 95% CI: 0.6-7.14
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Surrey et al.
A 66.7%
B 77.8%
60%
60%
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Biochemical (Bio), ongoing pregnancy (OPR), and implantation (IR) rates in Groups A and B with negative avb3 vitronectin expression. 100%
100%
0%
Finally, it is important to note that most the patients in this trial did not have documented evidence of prior implantation failure. This contrasts with the experience of the integrinnegative group treated in a similar fashion in our previously published case–control trial that had undergone a mean of 1.5 prior to failed IVF cycles (10). Similarly, given the small sample sizes, parity was not assessed as an independent variable. One could speculate that the predictive value of this test may be greater if a specific group of infertile endometriosis
FIGURE 3
FIGURE 2
80%
the biopsy alone exerted a beneficial effect regardless of integrin expression, then the 3-month delay to await completion of GnRH agonist therapy in groups 2 and B would be a reason to assume that patients in the control groups would have improved outcomes given that these individual did not experience a delay in initiation of their IVF cycles. One means of controlling for this effect would have been to delay onset of controlled ovarian hyperstimulation for 3 months in control groups 1 and A.
Integrin and GnRH agonist response
A 66.7%
B 44.4%
A 42.8%
40%
B 31.6%
20% 0%
Bio
OPR
IR
OR 1.75 95% CI: 0.17-17.69
OR 0.40 95% CI: 0.05-3.42
OR 0.62 95% CI: 0.15-2.58
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Vol. 93, No. 2, January 15, 2010
patients with evidence of previous IVF cycle failure were evaluated. In conclusion, a high prevalence of aberrant endometrial avb3 vitronectin expression was noted in a group of infertile endometriosis patients who are IVF candidates. Endometrial avb3 integrin expression did not predict which patients would benefit from prolonged administration of a GnRH agonist before initiation of controlled ovarian hyperstimulation for IVF. Given that the vast majority of patients in this small prospective randomized pilot trial were undergoing their first IVF cycle, it is possible that the predictive value of avb3 integrin expression may be greater in those endometriosis patients with evidence of prior implantation failure. The limited sample size precludes drawing any formal conclusions, however. A definitive answer to this question requires the completion of an appropriately designed larger trial limited to this specific subset of patients. Acknowledgements: The authors would like to express their deep gratitude to the nursing and embryology teams of the Colorado Center for Reproductive Medicine without whose efforts, this work would not have been possible.
REFERENCES 1. Lessey B, Damajanovich L, Coutifaris C, Castelbaum A, Albelda S, Buck C. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992;90:188–95. 2. Lessey B, Castelbaum A, Buck C, Lei Y, Yowell C, Sun J. Further characterization of endometrial integrins during menstrual cycle and in pregnancy. Fertil Steril 1994;67:497–506. 3. Campbell S, Swann H, Seif M, Kimber S, Aplin J. Cell adhesion molecules on the oocyte and preimplantation human embryo. Hum Reprod 1995;10:1571–8. 4. Lindhard A, Beutin-Ley U, Raun V, Islin H, Hoiid T, Rex S, et al. Biochemical evaluation of endometrial function at the time of implantation. Fertil Steril 2002;78:221–33. 5. Creus M, Ordi J, Fabregios F, Casainjana R, Carmona F, Cardesa A, et al. The effect of different hormone therapies on integrin expression and pinopode formation in the human endometrium: a controlled study. Hum Reprod 2003;18:683–93. 6. Lessey B, Castelbaum A, Sawin S, Sun J. Integrins as markers of uterine receptivity in women with primary unexplained infertility. Fertil Steril 1995;63:535–42. 7. Lessey B, Castelbaum A, Sawin S, Buck C, Schinnar R, Bilker W, et al. Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab 1994;79:643–9. 8. Hii L, Rogers P. Endometrial vascular and glandular expression of integrin aVb3 in women with and without endometriosis. Hum Reprod 1998;13:1030–5. 9. Ordi J, Creus M, Casamitjana R, Cardesa A, Vanrell J, Balasch J. Endometrial pinopode and aVb3 integrin expression is not impaired in women with endometriosis. J Assist Reprod Genet 2003;20:465–73. 10. Surrey E, Minjarez D, Schoolcraft W. The incidence of aberrant aVb3 vitronectin expression in a high risk infertility population. J Assist Reprod Genet 2007;24:553–6. 11. Lessey B. Medical management of endometriosis and infertility. Fertil Steril 2000;73:1089–96.
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12. Tei C, Maruyama T, Kuji N, Miyazaki T, Milkami M, Yoshimura Y. Reduced expression of aVb3 integrin in the endometrium of unexplained infertility patients with recurrent IVF-ET failures: improvement by danazol treatment. J Assist Reprod Genet 2003;20:13–20. 13. Sallam H, Garcia-Velasco J, Dias S, Arici A. Long-term pituitary downregulation before in vitro fertilization (IVF) for women with endometriosis. Cochrane Data Base 2006;CD004635. 14. Surrey E, Silverberg K, Surrey M, Schoolcraft W. Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization embryo transfer in patients with endometriosis. Fertil Steril 2002;78:699–704. 15. Gardner D, Schoolcraft W, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998;13:3434–40. 16. Practice Committee of the Society for Assisted Reproductive Technology and the Practice Committee of the American Society for Reproductive Medicine. Guidelines on number of embryos transferred. Fertil Steril 2006;86(Suppl 4):S51–2. 17. The Practice Committee of the Society for Assisted Reproductive Technology and the American Society for Reproductive Medicine. Guidelines on the number of embryos transferred. Fertil Steril 2004;82(Suppl 1):S1–2. 18. Remorgida V, Anserini P, Croce S, Costa M, Ferraiolo A, Capitanio G. Comparison of different stimulation protocols for gamete intrafallopian transfer in patients with minimal and mild endometriosis. Fertil Steril 1990;53:1060–3. 19. Dicker D, Goloman J, Levy T, Feldberg D, Ashkenazi J. The impact of long-term gonadotropin-releasing hormone analogue treatment on preclinical abortions in patients with severe endometriosis undergoing in vitro fertilization-embryo transfer. Fertil Steril 1992;57:597–600. 20. Thomas K, Thomson A, Wood S, Kingsland C, Vince G, Lewis-Jones I. Endometrial integrin expression in women undergoing in vitro fertilization and the association with subsequent treatment outcome. Fertil Steril 2003;80:502–7. 21. Wu Y, Halverson G, Basir Z, Strawn E, Yan P, Guo S. Aberrant methylation at HOXA 10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis. Am J Obstet Gynecol 2005;193:371–80. 22. Taylor H, Bagot C, Kardana A, Olive D, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod 1999;14:1328–31. 23. Matsuzaki S, Cams M, Vaurs-Barriere C, Boespflug-Tanguy O, Dastugne B, Mage G. DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection. Fertil Steril 2005;84:1180–90. 24. Burney R, Talbi S, Hamilton A, Vo K, Nyegaard M, Nezhat C, et al. Gene expression analysis of endometrium reveals progesterone resistance and candidate genetic loci in women with endometriosis. Endocrinology 2007;148:3814–26. 25. Kao L, Germeyer A, Tulac S, Lobo S, Yang J, Taylor R, et al. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology 2003;144:2870–81. 26. Fazleabas A, Brudney A, Chai D, Langoi D, Bulun S. Steroid receptor and aromatase expression in baboon endometriotic lesions. Fertil Steril 2003;80:820–7. 27. Barash A, Dekel N, Fieldust S, Segal I, Schechtman E, Granot I. Local injury to the endometrium doubles the incidence of successful pregnancies in patients undergoing in vitro fertilization. Fertil Steril 2003;79: 1317–22. 28. Raziel A, Schachter M, Strassburger D, Bern O, Ron-El R, Friedler S. Favorable influence of local injury to the endometrium in intracytoplasmic sperm injection patients with high-order implantation failure. Fertil Steril 2007;87:198–201.
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Objective: To determine whether endometrial expression of the integrin avb3 vitronectin can predict which endometriosis patient subgroup will benefit from pre-IVF cycle prolonged GnRH agonist (GnRHa) therapy. Design: Prospective randomized institutional review board approved pilot trial. Setting: Private assisted reproductive technology program. Patient(s): IVF candidates with regular menses, surgically confirmed endometriosis, and normal ovarian reserve. Intervention(s): All patients underwent endometrial biopsy 9 to 11 days post-LH surge to evaluate avb3 integrin expression. Patients were randomized either to receive depot leuprolide acetate 3.75 mg every 28 days for three doses before controlled ovarian hyperstimulation (COH) or to proceed directly to COH and IVF. Group 1: integrin-positive controls (N ¼ 12); group 2: integrin-positive administered prolonged GnRHa (N ¼ 8). Group A: integrin-negative controls (N ¼ 7); group B: integrin-negative administered prolonged GnRHa (N ¼ 9). Main Outcome Measure(s): COH responses, ongoing pregnancy and implantation rates. Results: There were no significant effects of GnRH agonist treatment in either of the integrin expression strata regarding ongoing pregnancy or implantation rates, although these outcomes were more frequent in group 2 vs. 1 (62.5% vs. 41.6% and 35% vs. 20.6%, respectively). This effect may have because of limited sample size. The value of a negative integrin biopsy in predicting an ongoing pregnancy after prolonged GnRH agonist therapy was only 44.4%. Conclusion(s): Endometrial avb3 integrin expression did not predict which endometriosis patients would benefit from prolonged GnRHa therapy before IVF. (Fertil Steril 2010;93:646–51. 2010 by American Society for Reproductive Medicine.) Key Words: Implantation, integrin, endometriosis, GnRH agonist, infertility, in vitro fertilization
It has been previously demonstrated that endometrial expression of the integrin subtype avb3 vitronectin and its receptor appears to occur 6 to 10 days after ovulation, the presumed window of implantation (1, 2). This cell adhesion molecule has also been demonstrated on the surface of embryos (3). It has been proposed that this integrin and its receptor may act not only as a site of interaction between the embryo and the endometrium, but may also play a role in initiating trophoblast invasion of the endometrium (4, 5). Lessey et al. (6) reported that aberrant endometrial avb3 integrin expression in 39% of women with unexplained infertility and ‘‘in-phase’’ endometrial biopsies. Most of the patients in this trial were noted to have stage I and II endometriosis. These same investigators demonstrated aberrant expression in 44% of endometriosis patients (7). These findings, however, have not been consistently reported (8, 9). Received April 14, 2008; revised December 5, 2008; accepted December 11, 2008; published online January 27, 2009. E.S.S. has received grant support from TAP Pharmaceuticals. A.K.L. has nothing to disclose. R.L. Gustofson has nothing to disclose. D.A.M. has nothing to disclose. W.B.S. has nothing to disclose. Presented in part at the 63rd Annual Meeting of the American Society for Reproductive Medicine, Washington, DC, October 13–17, 2007. Supported in part by a grant from TAP Pharmaceuticals, Waukegan, IL. NIH Clinical Trials Registry Identifier: NCT00621179. Reprint requests: Eric S. Surrey, M.D., Colorado Center for Reproductive Medicine, 10290 RidgeGate Circle, Lone Tree, CO 80124 (FAX: 303-788-8310; E-mail: [email protected]).
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We have recently described a 48.6% prevalence of absent integrin expression in a case–control study of 74 consecutive IVF candidates felt to be at high risk for implantation defects because of prior IVF failure but adequate embryo quality and/ or endometriosis (10). Of the patients who had previously undergone laparoscopy, 52.8% had been diagnosed with endometriosis, of which 57.1% was stage III–IV disease. Return of avb3 vitronectin expression with administration of a GnRH agonist or danazol has been described in prospective but nonrandomized trials (11, 12). This may represent an explanation for the improved pregnancy rates reported in endometriosis patients administered a prolonged course of a GnRH agonist before IVF-ET (13, 14). This approach has also been shown to result in IVF cycle outcomes that were no different than in integrin-positive patients who were not administered this agent (10). However, the question of whether endometrial avb3 vitronectin expression can predict which subgroup of endometriosis patients will benefit from prolonged pre-IVF cycle GnRH agonist therapy has not been assessed, and is the objective of the current investigation.
MATERIALS AND METHODS This investigation is a prospective randomized pilot trial approved by the HCA/HealthOne institutional review board. The study population consisted of patients with surgically
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documented endometriosis and no current sonographic evidence of an ovarian endometrioma >2 cm in mean diameter. All patients had evidence of normal ovarian reserve testing (day 3 serum FSH level <11 mIU/mL, E2 level <60 pg/ mL, and bilateral antral follicle count >4). A normal clomiphene challenge test was required for all women >38 years. All patients had a normal uterine cavity documented at precycle office hysteroscopy. Patients had received no depot preparations of a GnRH agonist, danazol, or other hormonal suppressive therapy for endometriosis within 6 months of study entry. Endometrial biopsies were performed on all patients 9 to 11 days after an LH surge was documented by urinary ovulation predictor kits. Tissue was evaluated for the presence or absence of avb3 vitronectin by commercial assay (Adeza, Sunnyvale, CA) using previously described techniques (1). All endometrial biopsy samples were confirmed to be in phase 2 days by standard histologic criteria. After the biopsy results were obtained, patients were randomized by two separate computer generated randomized number tables, one for women expressing the integrin and one for women who did not express the integrin, to either receive an intramuscular preparation of the GnRH agonist leuprolide acetate (TAP Pharmaceuticals, Waukegan, IL) 3.75 mg every 28 days for three injections before initiation of controlled ovarian hyperstimulation or to proceed directly to controlled ovarian hyperstimulation and IVF. This randomization scheme was designed to allow for more equal distribution of treatment regimens for both integrin-positive and -negative patients given the presumed unequal distribution of biopsy results on previously published data (10). Patients were randomized into one of four groups based on avb3 vitronectin expression and treatment protocol. Those integrin-positive patients randomized to the control group were assigned to group 1, and those who were integrin negative were assigned to group A. Patients who were randomly assigned to receive a prolonged course of the GnRH agonist who were integrin positive were assigned to group 2, whereas those who were integrin negative were assigned to group B. All patients subsequently underwent controlled ovarian hyperstimulation employing standard GnRH agonist downregulation or microdose flare protocols. The determination for which protocol was to be employed was based on ovarian reserve testing and response in previous cycles when appropriate. Indications for hCG administration, day 3 vs. 5 embryo transfer and numbers of embryos to transfer were based on previously published protocols and ASRM/SART guidelines (15–17). Remaining viable embryos were subsequently cryopreserved at the blastocyst stage 5 and/or 6 days after oocyte aspiration. Pregnancy tests were performed 14 days after oocyte aspiration. Biochemical pregnancy rate was defined as the number of positive serum pregnancy tests obtained 14 days after oocyte aspiration per embryo transfer procedure. Ongoing pregnancy rate was defined as the presence of an intrauterine gesFertility and Sterility
tational sac with fetal cardiac activity documented by ultrasound evaluation performed 4 to 5 weeks after a positive pregnancy test per embryo transfer procedure. Implantation rate was defined as the number of intrauterine gestational sacs with fetal cardiac activity as documented by ultrasound examination per number of embryos transferred. Data were analyzed by Student’s group t tests and chisquare analyses as appropriate. Values of P<.05 were considered to be statistically significant.
RESULTS Forty patients were initially recruited to participate in the trial. The overall incidence of absent endometrial integrin expression was 45%. Thirty-seven underwent randomization after three dropped out: two for personal reasons and one who elected to proceed with oocyte donation because of multiple failed IVF cycles with uniformly compromised embryo quality. A summary of the randomization scheme is displayed in Figure 1. Baseline clinical characteristics are displayed in Table 1. There were no significant differences between group 1 and 2 or groups A and B with regard to age, characteristics of ovarian reserve, extent of endometriosis, or number of prior failed cycles. It is interesting to note that only 18.9% of the patients who underwent randomization had previously undergone an unsuccessful IVF cycle. Of these 7 patients, only one had failed more than one prior cycle. Outcomes of controlled ovarian hyperstimulation are displayed in Table 2.There were no statistically significant differences between group 1 and 2 or groups and B for any of the parameters assessed. However, higher gonadotropin dose requirements that did not reach statistical significance were noted for both groups 2 and B, who received prolonged courses of the GnRH agonist. Similarly, there were no significant differences between group 1 vs. group 2 and group A vs. group B with regard to the incidence of day 3 vs. 5 embryo transfer, use of intracytoplasmic sperm injection, or assisted hatching. One group B cycle was cancelled before oocyte aspiration for poor response. One patient randomized to group A patient conceived spontaneously before initiation of therapy. Cycle outcomes are displayed in Figures 2 and 3. There were no significant differences between groups 1 and 2 or groups A and B with regard to biochemical pregnancy, ongoing pregnancy, or implantation rates. An apparent trend toward higher ongoing pregnancy and implantation rates in group 2 vs. 1 did not achieve statistical significance (odds ratio [OR] 2.33; 95% confidence interval [CI]: 0.37–14.61 and OR 2.08; 95% CI: 0.60–7.14, respectively). Based on the small patient numbers evaluated in this study, the value of a negative integrin biopsy in predicting an ongoing pregnancy after a prolonged course of GnRH agonist was 44.4%. The value of a positive integrin biopsy in predicting 647
FIGURE 1 Randomization summary of patients eligible to participate in the current trial. 40 Patients Eligible
3 Drop-Outs Prior to Randomization1
Randomization
Group 1 12 Patients
Group 2 8 Patients
Group A 7 Patients
Group B 10 Patients
Completed Therapy: 12 Patients
Completed Therapy: 8 Patients
Completed Therapy: 6 Patients2
Completed Therapy: 9 Patients3
1. 2 drop-out for personal reasons, 1 proceeded with oocyte donation 2. 1 patient conceived pre-therapy 3. 1 patient cancelled due to a poor response
Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
an ongoing pregnancy without prolonged GnRH agonist therapy was 41.7%. DISCUSSION In the current investigation, we hypothesized that endometrial avb3 vitronectin expression might predict response to prolonged GnRH agonist therapy before an IVF cycle in endometriosis patients. The randomization scheme in this prospective pilot trial was designed to assign relatively equal numbers of patients with either positive or negative biopsies to each of the two treatment groups given the unequal distribution of aberrant integrin expression. Unfortunately, we noted that there were no significant differences in ongoing pregnancy or implantation rates in those patients who failed to express avb3 vitronectin who were treated with a 3-month course of a GnRH agonist before an IVF cycle in comparison to untreated controls. The predictive values of biopsy results in determining response to prolonged GNRH agonist therapy was uniformly poor, with a 44.4% likelihood that a negative biopsy would predict IVF pregnancy after GnRH agonist therapy and a 41.7% likelihood that a positive biopsy would predict pregnancy if prolonged GnRH agonist therapy was not administered. The use of prolonged courses of GnRH agonists has been shown to enhance assisted reproductive technology cycle outcomes in patients with endometriosis. Three prospective randomized trials demonstrated significantly higher clinical pregnancy rates after a 3- to 6-month course of therapy before 648
Surrey et al.
Integrin and GnRH agonist response
IVF or gamete intrafallopian transfer (14, 18, 19). In a recent meta-analysis, Sallam et al. (13) noted that the clinical pregnancy rate in endometriosis patients administered a GnRH agonist for 3 to 6 months before the assisted reproductive technologies was increased fourfold in comparison to controls (OR 4.28; 95% CI 2–9.15). In the current study, a 3month course of agonist administration was selected because this was the shortest duration demonstrated to be efficacious in previously published trials (11, 14). However, it is important to note that ongoing pregnancy and implantation rates in the control group reported more recently by Surrey and coworkers (14) were still rather high (53.80% and 30.38%, respectively). Therefore, it would be advantageous to be able to predict which endometriosis patient groups might better benefit from prolonged GnRH agonist therapy to minimize the additional expense and delay associated with this treatment. Aberrant endometrial avb3 integrin expression was reported by Lessey and coworkers (6) in 39% of women with unexplained infertility and an ‘‘in-phase’’ endometrial biopsy performed between cycle days 20 to 24. Sixty-four percent of these women had a prior diagnosis of minimal to mild endometriosis. These investigators also demonstrated that 44% of infertile women with endometriosis failed to express this integrin subtype (7). It is interesting to note that in this trial, fertile women with endometriosis had a similar degree of staining for integrin as parous controls. In a recent case–control study, we reported aberrant expression in 48.6% of IVF patients with endometriosis and/or unexplained implantation failure despite adequate embryo development (10). Not all investigators have supported these conclusions, however. Two small series reported no statistically significant differences in endometrial avb3 expression in women with endometriosis in comparison to those with unexplained infertility or to fertile controls (8, 9). In a prospective observational study, Thomas et al. (20) described a group of 66 IVF patients who underwent precycle endometrial biopsy. A significantly higher percentage of women with positive avb3 integrin expression conceived than those without (47.1% vs. 26.7%; P<.027). However, only six of the overall patient cohort had a diagnosis of endometriosis. We are unaware of published studies that have demonstrated cycle-tocycle variation in integrin expression within individuals. Such variation, if it did occur, would represent a confounding variable given the design of the current investigation. The rationale behind linking aberrant avb3 integrin expression and successful response to a GnRH agonist is based on limited data. Lessey et al. (6) had previously reported a significantly greater return of endometrial integrin expression in a group of 22 women with stage I–II endometriosis treated with laparoscopic laser ablation of their disease followed by a 3-month course of a GnRH agonist than in a group of 25 similar women treated with surgery alone (64% vs. 35%; P<.05) (11). Trends, which did not achieve statistical significance toward higher pregnancy rates and shorter time Vol. 93, No. 2, January 15, 2010
In a retrospective case–control study, Surrey and coworkers (10) evaluated a group of patients at high risk for implantation failure and noted a trend toward more severe disease in those endometriosis patients who failed to express endometrial avb3 vitronectin. The subset of patients who did not express the integrin was treated with a 2-month course of a depot preparation of leuprolide acetate before IVF. Cycle outcomes were similar to a group of untreated controls with integrin expression. The lack of randomization was clearly a weakness in the study design.
Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
Results expressed as mean SD. No differences between Groups 1 and 2 or Groups A and B were statistically significant for any of these parameters.
0.42 0.9 0.25 0.46 0 0.2 0.42 41.7 37.5 57.2 30 1 2 A B
þ þ – –
– þ – þ
12 8 7 9
33.75 4.11 (Range: 28–40) 7.58 1.99 36.8 13.13 33.0 3.55 (Range: 28-39) 7.46 1.56 39.5 14.98 33.86 2.79 (Range: 31–39) 6.9 1.15 37.12 12.64 32.67 4.95 (Range: 23–40) 6.96 1.24 43.36 22.19
14.4 6.57 11.0 4.84 13.0 4.55 11.6 3.92
2.58 1.1 2.5 0.76 2.57 0.98 2.3 1.06
Prior Failed IVF Cycles III/IV (%) Mean Group aVb3
Prolonged GnRHa Patients
Age
Day 3 FSH (mIU/mL)
Day 3 E2 (pg/mL)
Antral follicles
Endometriosis stage Baseline Clinical Data.
TABLE 1
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intervals to conception, were noted in the group treated with the agonist as well. It is important to note that none of the patients in this trial underwent IVF so that it is not possible to determine whether the outcomes can be extrapolated to the model employed in the current investigation. Tei et al. (12) more recently evaluated 57 women with a history of repeated IVF cycle failures. avb3 integrin expression was lower in the group with unexplained infertility, although the incidence of endometriosis per se was not described. Nine of the patients randomly selected to receive a 12week course of oral danazol were noted to have a significant increase in integrin expression after completion of therapy (P¼.0076). However, the impact of this therapy on cycle outcome was not described.
There are several possible explanations for the failure to prove our hypotheses. One conclusion would be that any benefit derived by extended GnRH agonist therapy on IVF cycle outcome in infertile women with endometriosis does not occur by its action on avb3 vitronectin expression. The lack of statistically significant effects is compatible with this explanation. This possibility cannot be proven by the results of the current trial because endometrial biopsies would have had to have been repeated after therapy was completed and after return of ovulation. This would have necessitated a delay in initiation of an IVF cycle that could theoretically have been associated with a return to the abnormal state, which the agonist was designed to correct. As previously discussed, the issue of cycle-to-cycle variation in integrin expression has not been evaluated. A host of other factors that effect uterine receptivity have also been shown to differentially affect endometriosis patients and could certainly serve as confounding variables (21–26). A second explanation for the results in the current trial is the fact that, although prospective and randomized, this investigation is made of four groups of relatively small size with power that may be insufficient to detect significant differences between the treatment protocols. To demonstrate the 20% improvement in pregnancy rates described in our previously published trial, a sample size of at least 90 patients would have been necessary in each group pair (14). Even larger numbers would be required to detect a difference in treatment effects between integrin-positive and integrinnegative patients. It is also important to note that the patients previously evaluated by Lessey et al. (6), who demonstrated return on integrin expression after GnRH agonist therapy were exclusively those with stage I and II disease (11). 649
TABLE 2 Outcomes of Controlled Ovarian Hyperstimulation (COH). Group 1 2 A B
COH dose (units)
COH days
Oocytes retrieved
MII oocytes
2PN (%)
Embryos Embryos transferred cryopreserved
2638 1231 9.92 1.51 18.75 7.79 15.75 1.83 68.5 21.2 2.83 0.94 3560 709 9.5 0.93 11.63 5.73 8.88 4.82 69 28.2 2.5 1.23 2875 646.3 9.5 1.05 12.3 4.6 9.77 1.83 67.2 26.3 2.33 0.52 3558.8 963.1 9.6 1.07 13.78 9.22 11.56 8.9 75 17.4 2.56 0.73
3.5 3.92 0.88 1.13 0.67 1.21 1.78 2.28
Results expressed as mean SD. No differences between Groups 1 and 2 or Groups A and B were statistically significant for any of these parameters. Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
The preponderance of those endometriosis patients randomly assigned to receive GnRH agonist therapy in the previously published trial by Surrey et al. (14) had more severe disease and integrin biopsies were not performed. Thus, the effect of the agonist on integrin expression may differ based on the extent of the disease. A third explanation may have to do with the effect of the biopsies per se. Several investigative teams have suggested that the local endometrial injury induced by performing an endometrial biopsy in the cycle before IVF can improve outcomes (27, 28). In a prospective trial, Barash et al. (27) performed repeated endometrial biopsy on cycle days 8, 12, 21, and 26 during the menstrual cycle immediately before initiation of controlled ovarian hyperstimulation in a group of 45 randomly selected women who had failed to conceive during prior cycles and were felt to be good responders. Significantly higher live birth rates compared with a control group of 89 similar patients who did not undergo biopsy (48.9% vs. 22.5%, P¼.016) were reported. If in the current investigation
Biochemical (Bio), ongoing pregnancy (OPR), and implantation (IR) rates in Groups 1 and 2 with positive endometrial avb3 vitronectin expression.
1 75.0%
80% 2 62.5%
2 62.5% 1 41.6%
40%
2 35.0% 1 20.6%
20%
Bio OR 0.56 95% CI: 0.8-3.86
OPR OR 2.33 95% CI: 0.37-14.6
IR OR 2.08 95% CI: 0.6-7.14
Surrey. Integrin and GnRH agonist response. Fertil Steril 2010.
Surrey et al.
A 66.7%
B 77.8%
60%
60%
650
Biochemical (Bio), ongoing pregnancy (OPR), and implantation (IR) rates in Groups A and B with negative avb3 vitronectin expression. 100%
100%
0%
Finally, it is important to note that most the patients in this trial did not have documented evidence of prior implantation failure. This contrasts with the experience of the integrinnegative group treated in a similar fashion in our previously published case–control trial that had undergone a mean of 1.5 prior to failed IVF cycles (10). Similarly, given the small sample sizes, parity was not assessed as an independent variable. One could speculate that the predictive value of this test may be greater if a specific group of infertile endometriosis
FIGURE 3
FIGURE 2
80%
the biopsy alone exerted a beneficial effect regardless of integrin expression, then the 3-month delay to await completion of GnRH agonist therapy in groups 2 and B would be a reason to assume that patients in the control groups would have improved outcomes given that these individual did not experience a delay in initiation of their IVF cycles. One means of controlling for this effect would have been to delay onset of controlled ovarian hyperstimulation for 3 months in control groups 1 and A.
Integrin and GnRH agonist response
A 66.7%
B 44.4%
A 42.8%
40%
B 31.6%
20% 0%
Bio
OPR
IR
OR 1.75 95% CI: 0.17-17.69
OR 0.40 95% CI: 0.05-3.42
OR 0.62 95% CI: 0.15-2.58
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Vol. 93, No. 2, January 15, 2010
patients with evidence of previous IVF cycle failure were evaluated. In conclusion, a high prevalence of aberrant endometrial avb3 vitronectin expression was noted in a group of infertile endometriosis patients who are IVF candidates. Endometrial avb3 integrin expression did not predict which patients would benefit from prolonged administration of a GnRH agonist before initiation of controlled ovarian hyperstimulation for IVF. Given that the vast majority of patients in this small prospective randomized pilot trial were undergoing their first IVF cycle, it is possible that the predictive value of avb3 integrin expression may be greater in those endometriosis patients with evidence of prior implantation failure. The limited sample size precludes drawing any formal conclusions, however. A definitive answer to this question requires the completion of an appropriately designed larger trial limited to this specific subset of patients. Acknowledgements: The authors would like to express their deep gratitude to the nursing and embryology teams of the Colorado Center for Reproductive Medicine without whose efforts, this work would not have been possible.
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