IN VITRO FERTILIZATION

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1 FERTILITY AND STERILITY VOL. 82, NO. 4, OCTOBER 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. IN VITRO FERTILIZATION Prognostic use of mean ovarian volume in in vitro fertilization cycles: a prospective assessment John L. Frattarelli, M.D., a Andrew J. Levi, M.D., b Bradley T. Miller, M.D., c and James H. Segars, M.D. d Received October 17, 2003; revised and accepted February 26, This study, work unit #4415B-99, was supported by the Department of Clinical Investigation at Walter Reed Army Medical Center, Washington, DC. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Reprint requests: John L. Frattarelli, M.D., Tripler Army Medical Center, Department of Obstetrics and Gynecology, 1 Jarrett White Road, Honolulu, HI (FAX: ; a Tripler Army Medical Center. b Connecticut Fertility Associates. c Reproductive Medicine Associates of New Jersey. d Combined Federal Program in Reproductive Endocrinology and the Pediatric & Reproductive Endocrinology Branch, at Walter Reed Army Medical Center, National Naval Medical Center, Uniformed Services University for the Health Sciences, and the National Institutes of Health /04/$30.00 doi: /j.fertnstert Tripler Army Medical Center, Honolulu, Hawaii; Connecticut Fertility Associates, Bridgeport, Connecticut; Reproductive Medicine Associates of New Jersey, Morristown, New Jersey; and the Combined Federal Program in Reproductive Endocrinology and the Pediatric & Reproductive Endocrinology Branch, at Walter Reed Army Medical Center, National Naval Medical Center, Uniformed Services University for the Health Sciences, and the National Institutes of Health, Bethesda, Maryland Objective: To determine the predictive value and to define prognostic threshold measurements for mean ovarian volume (MOV) in patients undergoing IVF. Design: Prospective cohort analysis. Setting: Tertiary care center. Patient(s): Two hundred sixty-seven patients. Intervention(s): Transvaginal ultrasound before starting gonadotropins. Main Outcome Measure(s): Number of oocytes retrieved, basal hormone levels, and cycle outcomes. Result(s): The MOV for the population was cm 3 (range cm 3 ). The MOV significantly correlated with the majority of prestimulation and poststimulation IVF parameters. Threshold analysis demonstrated a lower pregnancy rate associated with a MOV of 2 cm 3 (31.6% vs. 55.6%). Threshold analysis revealed a trend toward higher cancellation rate associated with a MOV of 2cm 3 (21.1% vs. 7.3%). Conclusion(s): Although MOV correlated with IVF stimulation parameters, its use as an adjunct in counseling patients during IVF appears to be of limited value. A MOV 2 cm 3 was associated clinically with a higher cancellation rate (21.1%) and a lower pregnancy rate (31.6%) in those cycles not cancelled. However, these values do not deviate far from the mean national IVF outcome rates. There was no absolute MOV that was predictive of pregnancy outcome or cycle cancellation. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, mean ovarian volume, basal antral follicle count, pregnancy, cancellation rate, ovarian response, ovarian reserve, predictive value, clinical outcome, review Accurate methods of predicting IVF success allow for appropriate stimulation protocol selection. There are few noninvasive predictors of IVF outcome available to clinicians before starting an IVF cycle. Such predictors are helpful to counsel patients regarding the potential success expected when pursuing these financially and emotionally taxing treatments. It has been the clinical impression of some that women with small ovarian size on ultrasound respond poorly to ovarian stimulation (1 3). Measurement of ovarian volume by transvaginal ultrasonography is accurate and easily performed in most women. Interobserver variation among transvaginal ultrasound volume measurements has been shown to be very low (4). These findings enhance the value of this relatively noninvasive test. Various hormonal markers have also been used to predict ovarian response. Cycle day 3 serum levels of FSH, LH, and E 2, as well as provocative challenge tests have been studied. The predictive value of these hormonal tests has been demonstrated by a number of investigators (5 12). Despite their increasing acceptance in clinical practice, these tests have a variety of shortcomings. Principal among these has been the lack of predictive value of a normal result. A lack of test sensitivity has led investigators to continue the search for other markers that identify those 811

2 patients whose ovarian reserve is insufficient to allow conception. Ovarian reserve as measured by mean ovarian volume (MOV) has been suggested to predict ovarian response in IVF. However, no consensus has been determined. There are data demonstrating a predictive benefit of measuring MOV (2, 13). Other studies have failed to reveal a predictive benefit (14 16). Cutoff values for MOV used in the previously published articles were arbitrary and not based on true threshold values. The purpose of this manuscript was to prospectively evaluate the predictive value of MOV in determining IVF outcome and to test for threshold values that might predict outcome. MATERIALS AND METHODS Population Patients meeting inclusion criteria (n 267) from October 1998 to April 2000 were counseled and consented to ultrasounds before pretreatment day 3. Inclusion criteria included: normal basal FSH per our laboratory (14 miu/ ml), presence of both ovaries, no prior history of ovarian surgery, ability to visualize both ovaries on transvaginal ultrasound, and absence of ovarian pathology or ovarian cysts 10 mm. Patients were included independent of their age, diagnoses, or prior reproductive history. Patients had the following primary etiologies for their infertility: tubal factor (29%), male factor (28%), unexplained (19%), anovulatory/ polycystic ovary syndrome (PCOS) (18%), and endometriosis (6%). Institutional Review Board approval from Walter Reed Army Medical Center was obtained. Experimental Design In this prospective cohort analysis, the main outcome measures were the number of mature oocytes recovered and pregnancy outcome. Transvaginal ultrasound was performed after 14 days of pituitary down-regulation with GnRH agonist (GnRH-a) and 4 days before starting gonadotropins. Transvaginal ultrasound was performed by two authors (J.L.F. and A.J.L.) using an Acuson 128 (Acuson Corporation, Mountain View, CA.) with a 7-MHz vaginal transducer. For volume calculations, the three planes measured were the longitudinal, anteroposterior, and transverse diameters. Ovarian volume was calculated using the prolate ellipsoid formula (V D 1 D 2 D ). The MOV is defined as the average volume of the two ovaries ([V 1 V 2 ]/2). Calculation of interobserver variation was performed using the ovarian measurements from the first 30 patients evaluated by the two authors (J.L.F. and A.J.L.). The first examiner measured the ovarian volume. The second examiner repeated the measurements using the same ultrasound machine and was blinded to the measurements from the first examiner. Interobserver variation was determined using measurements from these 30 patients. To assess ovarian responsiveness, the pretreatment ovarian ultrasound measurements were evaluated with respect to patient s age, number of follicles, number of mature oocytes, number of embryos, basal serum laboratory measures (E 2, FSH, and LH), peak E 2 level, ampules of gonadotropins, days of stimulation, pregnancy rate (PR), and cancellation rate. All patients received 35 g of oral contraceptive pills for days before ovarian down-regulation with GnRH-a. The patients were down-regulated with a GnRH-a (Lupron, TAP Pharmaceuticals, North Chicago, IL) followed by stimulation with exogenous gonadotropins. When at least two follicles reached 18 mm, 10,000 units of hcg was administered. Transvaginal follicular aspiration took place hours later. Embryos were transferred transvaginally under transabdominal ultrasound guidance using a Wallace transfer catheter (Cooper Surgical, Shelton, CT) on postretrieval day 3 or 5. Luteal support was provided with 50 mg of IM P in oil beginning on the night of the oocyte retrieval. Biochemical pregnancies were defined as having a positive pregnancy test on luteal day 16 with a rising titer confirmed by a second hcg level but with loss of the pregnancy before the development of sonographic evidence of the pregnancy. Clinical pregnancy loss was defined as a pregnancy loss after sonographic confirmation of an intrauterine pregnancy. Patients were canceled for failing to produce three expanding follicles or failure to respond to gonadotropins with an adequate E 2 response of at least 500 pg/ml by day 8 of gonadotropin stimulation. The physicians canceling the patients IVF cycles were blinded to the MOV and participation in the protocol. All data were collected prospectively and entered into a computerized database. Laboratory Assays The LH, FSH, and E 2 assays were all done with the Immulite immunoassay system (Diagnostic Products Corporation, Los Angeles, CA). The inter- and intra-assay coefficients of variation were 5.2% and 4.9% for FSH, 6.2% and 5.4% for LH, and 5.9% and 5.0% for E 2, respectively. Statistical Analysis and Sample Size Controlling the probability of a type I error at alpha 0.05, a sample of 260 patients would have a 90% power to detect a 30% difference in MOV between those subjects pregnant and those not pregnant. Efficiency curves were constructed by grouping MOV beginning at 2 cm 3 and extending to 8 cm 3 at intervals of 1 cm 3. The cancellation rate and PR were then calculated for each increment. The PRs and cancellation rates above each threshold were evaluated to determine whether there were any breakpoints at which there was a change in outcome. Subsequently, contingency table analyses were used to evaluate the PRs and cancellation rates above and below the selected threshold values. A Wilcoxon-Mann Whitney ranksum test was used to compare outcomes between two groups. 812 Frattarelli et al. Ovarian volume in IVF cycles Vol. 82, No. 4, October 2004

3 A Kruskal-Wallis one-way analysis of variance was used to compare outcomes among multiple groups. For pairwise multiple comparison, Dunn s method was used. Univariate analysis included regression and correlation coefficients examining the association of MOV with parameters of ovarian reserve and response. Logistic regression was used when comparing binomial outcome rates with MOV. The correlation of the ovarian measurement made by each examiner was calculated using Pearson s correlation coefficient. All data were reported as means with their associated standard deviations. An alpha error of 0.05 was considered significant for all calculations. RESULTS TABLE 1 Correlation of mean ovarian volume with ovarian reserve and ART stimulation parameters. Variable Linear regression Multiple linear regression Age (y) r 0.17, P.01 P.75 Parity r 0.47, P.26 Basal FSH level (miu/ml) r 0.18, P.01 P.51 Basal LH level (miu/ml) r 0.24, P.10 Basal E 2 level (pg/ml) r 0.008, P.26 Peak E 2 level (pg/ml) r 0.21, P.001 P.68 Ampules of gonadotropins used r 0.22, P.001 P.05 a Days of stimulation r 0.36, P.12 No. of follicles r 0.38, P.001 P.001 a No. of mature oocytes retrieved r 0.24, P.001 P.74 No. of embryos r 0.25, P.001 P.72 Basal antral follicle number r 0.57, P.001 P.001 a Note: P.05 is considered statistically significant. a Using multiple linear regression analysis number of ampules of gonadotropins, number of follicles, and basal antral follicle number most significantly correlated with mean ovarian volume. In total, 289 patients underwent baseline ultrasound evaluation after being counseled and having consented to the study. Twenty-two patients were ultimately excluded. Seven patients had ovarian cysts 10 mm and 15 patients had poor visualization of one or both ovaries preventing accurate ovarian measurements. Therefore, 267 patients were included in the study. Twenty-three cycles (8.6%) were canceled at any time after initiating gonadotropins and 244 patients went to oocyte retrieval. There were 142 pregnancies, an overall PR of 53.2% per cycle start and 58.2% per retrieval. Of these, 15 were biochemical (10.6%) and 24 were clinical losses (16.9%); thus, a total of 103 deliveries (38.6% per cycle and 42.6% per retrieval) resulted. The MOV for the entire patient population was cm 3 (range 0.9 to 21.1 cm 3 ). The correlation coefficient (0.91) between the 30 pairs of ovarian volume measurements demonstrated a high degree of correlation and low interobserver variability in the transvaginal ultrasound measurements. There was no difference in MOV when canceled cycles ( ) were compared with retrieved cycles ( ) (P.19). Likewise, there was no significant difference in MOV when the patients were further divided into those cancelled ( ), those achieving pregnancy ( ), and those not pregnant ( ) (P.37). To determine whether the MOV correlated with ovarian reserve and assisted reproductive technology (ART) stimulation performance, we evaluated the stimulation parameters with linear regression analysis. The results of the univariate analysis are demonstrated in Table 1. There was a direct linear correlation between MOV and basal antral follicle number, number of follicles, number of mature oocytes, number of embryos, peak E 2, and gravidity. The MOV demonstrated a negative linear correlation with patient s age, day 3 FSH level, and ampules of gonadotropins. The MOV did not correlate with parity, basal LH, basal E 2, or days of stimulation. Using a multiple linear regression analysis number of ampules of gonadotropins used (P.05), number of follicles (P.001), and basal antral follicle number (P.001) were most significantly associated with MOV (Table 1). We also sought to determine threshold values for the prestimulation MOV that might help predict ovarian responsiveness and IVF success. Efficiency curves demonstrated an increased risk for cancellation in patients with a MOV 2 cm 3. Contingency table analysis confirmed that a mean MOV of 2 cm 3 was associated with a clinically significant increased risk in cycle cancellation (21.1% vs. 7.3%) (P.07, odds ratio [OR] 3.21, 95% confidence interval [CI] ) (Fig. 1). Efficiency curves demonstrated poorer overall pregnancy success (PR per cycle start) in all patients with a MOV 2 cm 3. Contingency table analysis for PRs confirmed that a MOV 2 cm 3 was associated with a clinically significantly lower PR (31.6% vs. 55.6%) (P.05, OR 0.38, 95% CI ) (Fig. 2). When evaluating only those patients with MOV 2 cm 3 who underwent oocyte retrieval (PR per retrieval), the PRs were lower than those with MOV 2 cm 3 (35.3% vs. 59.4%) (P.05, OR 0.37, 95% CI ). Efficiency curves demonstrated no significant trends in live birth, miscarriage, or multiple birth rates associated with MOV. DISCUSSION Some studies have demonstrated poorer outcome in patients with MOV of 3cm 3 (2, 13). Other studies have revealed that ovarian volume is less predictive (14 16). These studies did not describe contingency tables or threshold analysis in determining a value for ovarian volume. In our prospective study, we observed a 47.2% PR and a 15.1% cancellation rate for patients with ovarian volumes 3cm 3 (Figs. 1 and 2) compared with a 54.7% PR and a 6.5% cancellation rate for ovarian volumes 3 cm 3. These values do not reflect statistical differences or demonstrate clinically significant threshold values in patients undergoing IVF procedures. Our data suggest that a MOV 2 cm 3 after pituitary down-regulation but before gonadtropin initiation was asso- FERTILITY & STERILITY 813

4 FIGURE 1 Graph demonstrating cancellation rates at incremental mean ovarian volumes (MOV). Yellow bars demonstrate the cancellation rate for patients with a MOV less than that listed under the graph. Blue bars demonstrate the cancellation rate equal to or greater than the MOV listed under the graph. The mean cancellation rate for the population studied is demonstrated with the red bar. Contingency table analysis revealed that a MOV 2 cm 3 was associated with an increase in cycle cancellation (21.1% for 2 cm 3 vs. 7.3% for 2 cm 3 )(P.07, odds ratio 3.21, 95% confidence interval ). ciated with a high cancellation rate and a low PR during IVF (Figs. 1 and 2). The P values for PR per cycle start and per oocyte retrieval are listed as.05. However, the 95% confidence intervals cross 1.0. The actual P values are.051 and.053 for PR per cycle start and PR per oocyte retrieval, respectively. A post-hoc power analysis revealed that 10 additional patients would have been needed to demonstrate statistical significance. The data demonstrated that MOV correlated with the accepted clinically predictive measures of ovarian reserve and stimulation parameters. It is important to note that although these parameters correlate, no definite causative association has been confirmed. The population studied is fairly heterogeneous and therefore other variables may be affecting the outcome of the correlation analysis. We also attempted to define threshold levels for the MOV in predicting IVF outcome. This concept had not been explored adequately by other investigators. Although no absolute threshold value can be discerned, we did determine a value at which there was a decrease in ovarian response as demonstrated by an increase in cycle cancellation and a decrease in PRs. We have previously demonstrated that patients with a basal antral follicle count of 4 benefited from additional counseling and individualization of treatment protocols during ART cycles to maximize their stimulation response (17). In that prospective study, 4 antral follicles was associated with a lower PR (23.5% vs. 57.6%) (P.01) and a higher cancellation rate (41% vs. 6.4%) (P.001). Although MOV correlated with the parameters currently used to assess ovarian reserve and to predict cycle outcome, it did not seem to correlate as well or be as predictive as basal antral follicle number (17). We attempted to limit bias in this study by multiple methods. All data were collected in a prospective manner after obtaining patient consent. Ultrasound assessment was performed by two of the authors. Variability between the two researchers was assessed and was minimal. Patients were cancelled by physicians who were blinded to both patient participation in the study and MOV. In summary, no threshold value for MOV in predicting cycle cancellation or IVF success could be determined. There was no MOV that predicted absolute success or failure. A MOV 2 cm 3 was associated with an increase in cycle cancellation (21.1% vs. 7.3%) and a decrease in preg- 814 Frattarelli et al. Ovarian volume in IVF cycles Vol. 82, No. 4, October 2004

5 FIGURE 2 Graph demonstrating pregnancy rates per cycle start at incremental mean ovarian volumes (MOV). Yellow bars demonstrate the pregnancy rate for patients with a MOV less than that listed under the graph. Blue bars demonstrate the pregnancy rate equal to or greater than the MOV listed under the graph. The mean pregnancy rate for the population studied is demonstrated with the red bar. Contingency table analysis revealed that a MOV 2 cm 3 was associated with a decrease in pregnancy rate (31.6% for 2 cm 3 vs. 55.6% for 2 cm 3 )(P.051, odds ratio 0.38, 95% confidence interval ). nancy success (31.6% vs. 55.6%). A post-hoc power analysis revealed that only 10 additional patients would be needed to reach statistical significance. However, because these values were well within the normal range for PRs and cancellation rates, ovarian volume offers little additional information when assessing the prognostic success of IVF. References 1. Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 1995;64: Lass A, Skull J, McVeigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in vitro fertilization can predict poor response. Hum Reprod 1997;12: Frattarelli JL, Miller BT, Lauria-Costa D, Bergh PA, Scott RT. Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles. Fertil Steril 2000;74: Higgins RV, Van Nagell JR, Woods CH, Thompson EA, Kryscio RJ. Interobserver variation in ovarian measurements using transvaginal sonography. Gynecol Oncol 1990;39: Scott RT, Toner JF, Muasher SJ, Oehninger SC, Robinson S, Rosenwaks Z. Follicle stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989;51: Smotrich DB, Widra EA, Gindoff PR, Levy MJ, Hall JL, Stillman RJ. Prognostic value of day 3 estradiol on in vitro fertilization outcome. Fertil Steril 1995;64: Loumaye E, Billion JM, Mine JM, Psalit I, Pensis M, Thomas K. Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril 1990;53: Scott RT, Leonardi MR, Hofmann GE, Illions EH, Neal GS, Navot D. A prospective evaluation of clomiphene citrate challenge test screening in the general infertility population. Obstet Gynecol 1993;82: Tanbo T, Dale PO, Abyhom T, Stokke KT. Follicle-stimulating hormone as a prognostic indicator in clomiphene citrate/human menopausal gonadotrophin-stimulated cycles for in vitro fertilization. Hum Reprod 1989;6: Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995; 64: Scott RT, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril 1990;53: Frattarelli JL, Bergh PA, Sable DB, Drews MR, Sharara FI, Scott RT. Evaluation of the prognostic significance of day 3 estradiol levels in assisted reproductive technology cycles. Fertil Steril 2000;74: Syrop CH, Dawson JD, Husman KJ, Sparks AE, Van Voorhis BJ. Ovarian volume may predict assisted reproductive outcomes better than follicle stimulating hormone concentration on day 3. Hum Reprod 1999;14: Sharara FI, McClamrock HD. Use of microdose GnRH agonist protocol in women with low ovarian volumes undergoing IVF. Hum Reprod 2001;16: Banesi LF, Brockmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002;77: Schild RL, Knobloch C, Dorn, Fimmers R, van der Ven H, Hansmann M. The role of ovarian volume in an in vitro fertilization programme as assessed by 3D ultrasound. Arch Gynecol Obstet 2001;265: Frattarelli JL, Levi AJ, Miller BT, Segars JH. A prospective assessment of the predictive value of basal antral follicles in in vitro fertilization cycles. Fertil Steril 2003;80: FERTILITY & STERILITY 815