Prognostic value of day 3 estradiol on in vitro fertilization outcome*

Transcription

1 FERTILITY AND STERILITY Vol. 64, No.6, December 1995 Copyright 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Prognostic value of day 3 estradiol on in vitro fertilization outcome* David B. Smotrich, M.D.H Eric A. Widra, M.D.t Paul R. GindofI, M.D.t Michael J. Levy, M.D. Jerry L. Hall, Ph.D. Robert J. Stillman, M.D.t The George Washington University, Washington, D.C., and The Shady Grove Fertility Center, Rockville, Maryland Objective: To evaluate the prognostic value of day 3 E2 levels, independent of day 3 FSH levels, on responses to ovulation induction and subsequent pregnancy rates (PRs) in IVF-ET patients. Design: Prospective, observational. Setting: University-based tertiary care and private reproductive endocrine-infertility units. Patients and Interventions: A total of 225 patients underwent 292 IVF cycles with luteal phase GnRH agonist suppression and hmg stimulation. Main Outcome Measures: We evaluated response and outcome data including age, day 3 FSH and E2levels from a menstrual cycle before IVF, ampules ofhmg used, maximum E21evel, cancellation rates, and clinical PR. Results: Despite similar age, number of ampules of hmg, and peak E2 levels, patients with an elevated E2 level (E2 :2: 80 pg/ml) (conversion factor to SI unit, 3.671) on day 3 of a cycle before IVF-ET achieved a lower PR per initiated cycle (14.8% versus 37.0%) and had a higher cancellation rate (18.5% versus 0.4%) compared with those with E2 levels < 80 pg/ml. Even when patients with elevated FSH levels (FSH :2: 15 miu/ml) (conversion factor to SI unit, 1.00) were excluded (leaving 279 cycles), those with an elevated day 3 E2 still had a lower PR per initiated cycle (14.8% versus 38.9%) and maintained a higher cancellation rate (18.5% versus 0.4%). When the day 3 E2 was :2:100 pg/ml there was a 33.3% cancellation rate and no pregnancies were achieved. Conclusion: Patients who presented with an elevated day 3 E2 (:2:80 pg/ml) in a cycle before IVF-ET had a higher cancellation rate and achieved a lower PR independent of FSH level. A day 3 E2 level, in addition to a day 3 FSH level, appears very helpful in prospectively counseling patients regarding cancellation risk and ultimate IVF-ET success. Fertil Steril 1995;64: Key Words: In vitro fertilization, serum E2, FSH A number of parameters have been evaluated to predict the response to stimulation and pregnancy outcome with IVF-ET. Muasher et al. (1) explored the prognostic value of cycle day 3 and stimulated serum gonadotropin levels on IVF -ET outcome. They concluded that day 3 concentrations of FSH and LH Received March 15, 1995; revised and accepted June 20,1995. * Presented in part at the 50th Annual Meeting of The AnIerican Fertility Society, San Antonio, Texas, November 5 to t Division of Reproductive Endocrinology, Fertility and IVF Programs. :j: Reprint requests: David B. Smotrich, M.D., The George Washington University, Division of Reproductive Endocrinology and Fertility, 2150 Pennsylvania Avenue, NW Room 6A-410, Washington, D.C. (FAX: ). The Shady Grove Fertility Center Smotrich et al. Day 3 E2 and NF outcome can help identify different populations of patients with distinct stimulation characteristics. Scott et al. (2) measured day 3 levels of FSH, LH, and E2 to determine their predictive value for stimulation quality and IVF-EToutcome. Their results indicated that elevated day 3 FSH (2:15 miu/ml) (conversion factor to SI unit, 1.00) concentrations predict poor stimulation response and pregnancy outcome, but day 3 LH and E2 do not improve the prognostic value beyond that provided by FSH alone. Toner et al. (3) investigated the use of day 3 FSH levels and age in predicting IVF-ET outcome and they concluded that day 3 FSH levels are a better predictor of IVF-ET outcome than age. In 1987, Navot et al. (4) described the clomiphene citrate (CC) challenge test, a test measuring serum

2 FSH levels before (day 3) and after (day 10) the administration of 100 mg of CC on cycle days 5 through 9. They found that patients with an exaggerated FSH response had diminished ovarian reserve (4). Later Loumaye et al. (5) found the CC challenge test to be a reliable method for predicting IVF -ET outcome. In 1992, Tanbo et al. (6) reported that an abnormal CC challenge test has a predictive value of 85% for cycle cancellation and 100% for conception failure. Scott et al. (7) further concluded that there was no significant benefit for measuring the E2 concentrations as a component of the CC challenge test. In contrast, Padilla et al. (8) evaluated the prognostic value of the early cycle FSH, LH, and E2 responses to GnRH agonist (GnRH-a) stimulation. They reported that the stimulated E2 response is a better indicator of IVF -ET outcome than the baseline or stimulated serum FSH or LH concentrations. Given these conflicting data regarding the significance ofe2levels, we evaluated the prognostic value of day 3 E2 and day 3 FSH concentrations in a cycle proximate but before the IVF -ET cycle, on responses to ovarian stimulation and subsequent pregnancy rates (PRs) in IVF-ET patients. MATERIALS AND METHODS Two hundred twenty-five patients initiating 292 IVF cycles between January 1993 and December 1994 at the George Washington University Medical Center and the Shady Grove Fertility Center were studied. We prospectively measured day 3 FSH and E2 concentrations in a cycle proximate but before suppression and ovarian stimulation. Similar stimulation protocols, the same culture media and laboratory protocols, and the same laboratory director and endocrine laboratory were used at both Centers throughout. Analysis was limited to those patients who underwent the same stimulation protocols at both centers using pituitary suppression with a GnRH-a and subsequent ovarian stimulation with hmg (9-12). All patients received 0.5 mg/d SC injections of leuprolide acetate (LA, Lupron; TAP Pharmaceuticals, Chicago, IL) beginning in the midluteal phase ofthe menstrual cycle. All patients had negative quantitative serum pregnancy tests before ovarian stimulation. Ovarian stimulation with hmg (Pergonal; Serono Laboratories, Inc., Norwell, MA) was initiated after the patient experienced withdrawal uterine bleeding, a baseline sonogram revealed no follicular cyst> 10 mm in diameter, and the serum E2level was <50 pg/ml (conversion factor to SI unit, 3.671). The daily LA dose was then decreased to 0.25 mg/d. The hmg dose was individualized based on the patients age (>40 or <40 years), day 3 FSH level (> 15 or < 15 miulml), and previous Vol. 64, No.6, December 1995 stimulation history, if applicable, but the day 3 E2 level did not influence the ovarian stimulation protocol. Patients underwent serial transvaginal sonography, and serum E2 measurements daily after 4 days ofhmg stimulation. Follicular maturation was triggered with hcg (Profasi; Serono Laboratories); 10,000 IU was administered when at least two follicles measured> 16 mm using a 5-MHz vaginal probe at a 40 mm focus (General Electric Medical Systems, Monterey Park, CA) as assessed by the mean of the two largest diameters combined with a maximum E2 level ~ 500 pg/ml. Transvaginal follicle aspiration was performed 35.5 hours after hcg administration. All oocytes were cultured in Human Oviductal Fluid media (Fertility Products, Inc., Rockville, MD) supplemented with 10% maternal serum. After 4 to 8 hours, oocytes were inseminated with 50,000 motile sperm per ml. Embryos were transferred 46 to 48 hours after retrieval. Luteal support was provided by P vaginal suppositories, 300 mg twice daily, and hcg, 3,300 IU given 1M on days 3, 6, and 9 after retrieval. No micromanipulation techniques were used in these patients. A quantitative serum,b-hcg was performed 17 days after retrieval. Transvaginal sonographic documentation of fetal viability was performed in all patients with a rising,b-hcg titer. Clinical pregnancy was defined as the presence of an intrauterine gestational sac documented by transvaginal sonography. We evaluated the following parameters: age, day 3 FSH and E2 concentrations, number of ampules of hmg used, maximum E2 levels, cancellation rates, and clinical PRs. All the serum from both Centers were assayed for FSH and E2 at Diagnostic Assay Services, Inc. (Gaithersburg, MD). The FSH concentration was assayed by RIA (Nichols Institute Radioisotopicassay, San Juan Capistrano, CA) with an interassay coefficient of variation (CV) of 3.2% to 4.5% and an intra-assay CV of 2.3% to 2.8%. The E2 concentration was assayed by RIA (Diagnostic Products Corp., Los Angeles, CA) with an interassay CVof6% to 7% and an intra-assay CV of 5% to 7%. Statistical analysis of data was performed using t-test, X 2, and the Fisher's exact test where appropriate, with significance at P < RESULTS We evaluated 292 initiated IVF cycles from 225 patients. Two hundred seven patients with day 3 E2 concentration < 80 pg/ml underwent 265 cycles, whereas 27 cycles were initiated in 18 patients who had had a day 3 E2 ~ 80 pg/ml. Table 1 shows the stimulation and cycle outcome data from these patient cycles. Cycles in which patients had had a day 3 E2 < 80 pg/ml resulted in a clinical PR per Smotrich et al. Day 3 E2 and NF outcome 1137

3 Table 1 Patient Stimulation and Outcome Characteristics by day 3 E2 Concentration for all Patients Regardless of Age or FSH* Factor E2 < 80 pg/ml t E2 ;", 80 pg/ml t E2 ;", 100 pg/ml No. of cycles No. of patients Age (y) Day 3 FSH (mid/ml) Ampules of hmg Peak E2 (pg/ml) Cancellation rate per initiated cycle (%) Clinical PR per initiated cycle (%) ::!: 0.3t 8.1::!: ::!: 1.2 1,646.5 ::!: ::!: ::!: ::!: 4.1 1,503.7 ::!: NS 0.01 NS NS < ::!: ::!: ::!: 5.6 1,305.0 ::!: o * Conversion factors of SI units are as follows: E2, 3-671; FSH, t Values are means ::!: SEM. :j: E2 < 80 versus ;",80 pg/ml. NS, not significant. initiated cycle of 37.0% whereas those with E2 ;;;,: 80 pg/ml had a 14.8% clinical PR per initiated cycle (P = 0.02). The clinical PRs per ET were 37.1% and 18.2% (P < 0.05), respectively. Cycles in patients with a day 3 E2 ;;;,: 80 pg/ml had a significantly higher cancellation rate due to poor ovarian stimulation compared with those with E2 < 80 pg/ml, (18.5% versus 0.4%, P < ). These higher cancellation rates and lower PRs in patients with E2 ;;;,: 80 pg/ml occurred despite a statistically significant lower mean day 3 FSH compared with the day 3 FSH concentration in patients with E2 < 80 pgl ml (5.7 ::!:: 0.5 versus 8.1 ::!:: 0.3 miu/ml, P = 0.01), although in both groups the mean day 3 FSH concentrations were <10 miu/ml. There was no difference in age (34.5 ::!:: 0.3 versus 35.8 ::!:: 0.9 years), number of ampules of hmg required (42.7 ::!:: 1.2 versus 46.9 ::!:: 4.1), and peak E2 concentrations at the time of hcg administration (1,646.5 ::!:: 50.1 versus 1,503.7 ::!:: pg/ml) between the two groups. We analyzed the 15 initiated IVF cycles in eight patients with a day 3 E2 ::!:: 100 pg/ml. There was a 33.3% cancellation rate per initiated cycle in these patients. In those patients not cancelled, none achieved a pregnancy, despite a mean age of (37.1 ::!:: 1.3 years) and a normal mean day 3 FSH concentration (6.6 ::!:: 0.7 miu/ml) (Table 1). When we restricted our analysis to the 279 initiated IVF cycles in patients with day 3 FSH levels < 15 miu/ml, the results were the same. Cycles from patients with a day 3 E2 < 80 pg/ml still yielded a higher clinical PR per initiated cycle (38.9% versus 14.8%, P = 0.01) and a significantly lower cancellation rate due to poor ovarian stimulation (0.4% versus 18.5%, P < ) than those cycles from patients with a day 3 E2 ;;;,: 80 pg/ml (Table 2). We also evaluated our data based on day 3 FSH levels ;;;,: 15 miu/ml. Our results support data reported elsewhere. No patient with a day 3 FSH level ;;;,: 15 miu/ml had a cycle resulting in pregnancy, 1138 Smotrich et al. Day 3 E2 and NF outcome whereas cycles with day 3 FSH levels < 15 miu/ ml had a clinical PR per initiated cycle of 36.6% (P = 0.007). The patients with elevated day 3 FSH levels were older (36.9 ::!:: 1.0 versus 34.4 ::!:: 0.3 years, P = 0.04) than the patients with normal day 3 FSH levels. Interestingly, none of the eight patients with a day 3 FSH level ;;;,: 15 miu/ml had a day 3 E2 concentration;;;,: 80 pg/ml. Further, no patient cycle with an elevated day 3 FSH level was cancelled due to poor ovarian stimulation, whereas 2.2% of the 279 cycles from patients with normal day 3 FSH levels were cancelled. To limit age as a confounding factor, we also analyzed our data restricted to patients < 40 years of age and to those < 40 years old with a normal day 3 FSH (Table 2). In the cohort of cycles from patients < 40 years of age with normal day 3 FSH ( < 15 miu/ ml) concentrations (n = 237), cycles from patients with day 3 E2levels ;;;,: 80 pg/ml again demonstrated a lower clinical PR per initiated cycle (19.0% versus 40.7%, P = 0.05) and a significantly higher cancellation rate than those with day 3 E2 levels < 80 pgl ml (14.3% versus 0.5%, P < ) (Table 2). When we examined our data by the patient's first cycle, an E2 < 80 pg/ml remained predictive of a higher PR per initiated cycle (34.8% versus 16.7%) but no longer reached statistical significance. However, when the data were analyzed by individual patient, stratified by the number of cycles, an E2 < 80 pg/ml was strongly predictive of higher PR (44.4% versus 16.7%, P = 0.02) and lower cancellation rate (0.5% versus 22.2%, P < 0.001), independent of the number of cycles performed. This effect was not due to repeated cycles by the poor prognosis patients with an E2 ;;;,: 80 pg/ml, but rather by the occurrence of pregnancy in 20 patients with E2 < 80 pg/ml in their subsequent IVF-ET cycles. DISCUSSION Several investigators have evaluated precycle day 3 FSH concentrations to predict the response to stim-

4 Table 2 Patient Stimulation and Outcome Data by Day 3 E2 Concentration, Age, and Day 3 FSH Concentration* All patients < 40 years with All patients with FSH < 15 m1u/mlt All patients < 40 years FSH < 15 m1ulml Ez < 80 E2 '" 80 E2 < 80 pg/ml pg/ml Pt pg/ml E2 '" 80 E2 < 80 E2 ", 80 pg/ml Pt pg/ml pg/ml Pt No. of cycles Age (y) 34.3 ± ± 0.9 NS 33.3 ± 0.2 Days FSH 7.4 ± ± ± 0.3 No of ampules 42.3 ± ± 4.1 NS 40.7 ± 1.3 MaximumE 2 1,658.6 ± ,503.7 ± 209 NS 1,691.5 ± 56.1 Cancellation rate per initiated cycle (%) < PR per initiated cycle (%) ± 0.7 NS 33.9 ± ± 0.2 NS 5.8 ± 0.7 NS 7.2 ± ± ± 4.6 NS 44.9 ± ± 1.3 NS 1,557.1 ± 234 NS 1,557.1 ± 234 1,692 ± 57.4 NS 14.3 < < NS (0.06) * Conversion factors to S1 units are as follows: FSH, 1.00; E2, t Values expressed in means ± SEM. t E2 < 80 pg/ml versus ",80 pg/ml. ulation and pregnancy outcome in IVF-ET patients (1-3, 13). Muasher et al. (1) reported a 21.3% PR per initiated cycle in 80 consecutive IVF-ET patients. They found the day 3 FSH level was significantly lower in the patients that achieved pregnancy (9.09 ± 2 versus 12.5 ± 5.9 mlvlml, P < 0.05) (1). Scott et al. (2) retrospectively studied 758 IVF-ET cycles to evaluate the predictive value of day 3 FSH values. They reported that patients with day 3 FSH levels < 15 mlu/ml had higher clinical PR per initiated cycle (24.0%) than those with intermediate FSH levels (15 to 24.9 mlu/ml, 13.6%), both of which were higher than the patients with high FSH levels (2::25 mlu/ml, 10.7%) (2). Toner et al. (3) have reported that day 3 FSH levels more strongly predicted cancellation and PRs than age in 1,478 IVF-ET cycles. The pulsatile nature of FSH and the potential for intercycle and lab assay variation led Scott et al. (14) to evaluate the reproducibility of day 3 FSH concentrations. They reported on data from 81 patients who had undergone at least three or more IVF-ET attempts with repeated FSH testing over a 2-year period. Patients with day 3 FSH concentrations < 15 mlu/ml had lower intercycle variation than those patients with day 3 FSH levels 2:: 15 mlvl ml (2.6 ± 0.2 versus 7.4 ± 0.9 mlvlml, P < 0.01). They also found that slight changes in day 3 FSH levels do not predict changes in ovarian responsiveness (14). In their review article, Scott and Hofmann (15) reconfirmed elevated day 3 FSH levels as highly prognostic of diminished ovarian reserve but, although citing numerous studies that found differences between high and low responders, they could find no other single ovarian product correlated with day 3 FSH levels or clinical outcome. Scott et al. (2) reported that day 3 E2 levels do not correlate with either day 3 FSH levels or PRs, using an E2 concentration of 2:: 50 pg/ml as the cutoff. We evaluated the day 3 E2 values at intervals of 5 pg/ml Vol. 64, No.6, December 1995 from 50 pg/ml to > 100 pg/ml. Our data, although confirming the utility of FSH levels, demonstrate that day 3 E2 levels> 80 pg/ml in a cycle proximate but before IVF-ET strongly and independently predicted poor IVF-ET response and outcome. We found clear differences in patients using a discriminatory E2 value of 80 pg/ml, independent of age and FSH level; when these two potentially confounding variables were eliminated, patients with day 3 E2 levels 2:: 80 pg/ml still had a significantly higher cancellation rate and lower clinical PRo Moreover, patients with an E2 2:: 100 pg/ml had a further decrement in response and outcome: none conceived even when the day 3 FSH was < 15 mlvlml. Thus, a day 3 E2 concentration appears to be a valuable additional precycle screening tool to predict IVF-ET cycle response and outcome. We found a significant difference in prestimulation FSH for patients categorized by E2 levels 2:: 80 pg/ml with the FSH actually being lower in patients with higher day 3 E2 concentrations. However, these differences were not clinically useful because both mean levels were <10.0 mlu/ml and the ranges overlapped (SD = 4.6). Further, in considering any FSH levels, we should note that patients with the highest concentrations often were excluded from initiating IVF-ET cycles. Supporting those authors discussed above, day 3 FSH levels in our population also prospectively identified patients with poor IVF ET outcome: patients with normal day 3 FSH levels achieved a 36.6% clinical PR per initiated cycle, whereas no patient with a day 3 FSH level 2:: 15 mlu/ml who had an IVF-ET cycle became pregnant (1-3, 15). Several factors mediate the transition from the late luteal phase to the initiation of the next cycle. The demise of the corpus luteum normally results in a decline of inhibin levels. This allows circulating FSH levels to rise in the late luteal and early follicu- Smotrich et al. Day 3 E2 and NF outcome 1139

5 lar phase (16). At the same time, an increased frequency of GnRH pulsatile secretion selectively augments FSH secretion (17). This increase in FSH initiates follicular recruitment. In women with decreased ovarian reserve and function, diminished inhibin production in the late luteal-early follicular phase is suggested to be the mechanism for an abnormally increased day 3 FSH level The FSH increases as an attempt to stimulate stroma and granulosa cell response and may do so to a limited degree until the menopause. Our hypothesis regarding elevated day 3 E2 levels is that these patients may represent a group with an intermediate stage of decreased ovarian responsiveness between the normal response and that seen with elevated FSH levels. In this hypothesis, lower inhibin production before day 3 results in a transiently elevated FSH. Here, the FSH elevation is followed by a granulosa cell response, producing relatively more E2. This higher estrogen feeds back centrally to decrease FSH secretion, resulting in the combination of findings we see in these patients: higher E2 but normal FSH. The ultimate poor IVF-ET response and outcome are a reflection of diminished ovarian-oocyte function. Interestingly, in support of this theory, we found a highly significant (P < ) negative correlation between E2 and FSH for our study group (data not shown) that supports the presence of an intact negative feedback loop. Although our data presented here cannot further validate this hypothesis, they may be an avenue for further investigation. Our data support the premise that day 3 E2 concentration is a simple, inexpensive, and effective screening tool-independent of FSH and of agewhich is quite helpful in prospectively counselling patients regarding IVF-ET cancellation risk and ultimate IVF-ET success. Even patients with a normal day 3 FSH who had an elevated day 3 E2 level (E2 ~ 80 pg/ml) had an increased risk of cycle cancellation and had a distinctly diminished chance of pregnancy. Each IVF center, using their own endocrine laboratory, might establish E2 values that predict these outcome differences. Based on such values, patients with an elevated day 3 E2 could be counseled about their poorer prognosis and then may decide not to proceed with an IVF-ET cycle. Because no pregnancies were achieved in patients with the highest day 3 E2 concentrations-in our laboratory E2 ~ 100 pg/ml-we believe that those patients can be recommended strongly not to embark on IVF. REFERENCES 1. Muasher SJ, Oehninger S, Simonetti S, Matta J, Ellis LM, Liu H-C, et al. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988;50: Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. 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J Clin Endocrinol Metab 1992;69: Hall JE, Schoenfeld DA, Martin KA, Crowley WF Jr. Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal-follicular transition. J Clin Endocrinol Metab 1992;74: Smotrich et al. Day 3 E2 and IVF outcome