A prospective analysis of the changes in ovarian morphology during hormonal pituitary suppression before in vitro fertilization

A prospective analysis of the changes in ovarian morphology during hormonal pituitary suppression before in vitro fertilization John L. Frattarelli, M.D. Tripler Army Medical Center, Honolulu, Hawaii

Objective: To assess the change in ovarian morphology at three specific times integral to an IVF cycle, and to evaluate the factors associated with a change in ovarian morphology after pituitary desensitization. Design: Prospective cohort analysis. Setting: Academic IVF center. Patient(s): Infertile patients undergoing 85 IVF cycles. Intervention(s): Eighty-five patients had a transvaginal ultrasound to assess ovarian morphology on unstimulated menstrual day 3, 21 days after starting oral contraceptives (OCs), and 14 days after starting GnRH agonist (GnRH-a). Main Outcome Measure(s): Ovarian morphology. Result(s): Compared with the basal total ovarian volume, ovarian volume was significantly decreased after 21 days of OCs and after 14 days of GnRH-a. A significant decrease was noted in the total number of antral follicles from the basal state to after 21 days of OCs and after 14 days of GnRH-a treatment. There was no significant effect on pregnancy outcome rates. Univariate analysis showed a significant correlation between ovarian morphology and IVF stimulation parameters. Conclusion(s): Hormonal suppression significantly alters ovarian morphology. If the use of ovarian morphology is to be used to counsel patients and predict IVF success as suggested by some studies, it should be standardized for the day the ovarian morphology is evaluated. (Fertil Steril威 2006;86:577– 82. ©2006 by American Society for Reproductive Medicine.) Key Words: Ovarian morphology, ovarian volume, basal antral follicles, IVF, ovulation induction, infertility, transvaginal ultrasound, oral contraceptives, GnRH-a

The evaluation of ovarian reserve has been the focus of a substantial amount of clinical research during the past several years (1–5). A number of tests have been proposed and evaluated that may be used to prognosticate ovarian responsiveness to exogenous gonadotropin stimulation, the quality of the oocytes, and the subsequent implantation and pregnancy rates (PR) (1– 4). The prognostic value of these tests has been clearly demonstrated by a number of investigators in a wide variety of settings (4, 5). The assessment of ovarian reserve is valuable for determining stimulation protocols and predicting assisted reproductive technology (ART) outcome. Many tests have been evaluated to predict ART cycle outcome. The functional ovarian reserve as measured by antral follicle count and ovarian volume has been suggested to predict ovarian response in ART (6 –9). In recent investigations, antral follicle number correlated significantly with Received October 29, 2005; revised and accepted January 31, 2006. 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., Reproductive Medicine Associates of New Jersey, 100 Franklin Square Drive, Suite 200, Somerset, NJ 08873 (Office: 732-537-0631; FAX: 732-537-0134; E-mail: [email protected]).

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ovarian response and pregnancy results in patients undergoing ART cycles (6, 7). Likewise, groups have suggested that the measurement of ovarian volume significantly correlates with ovarian reserve and ART outcome (7–10). An effect of ovarian volume and antral follicle number on IVF cycles has recently been suggested; however, the current literature is not consistent in the timing of the ovarian measurements (6 –10). Although a few studies have evaluated the effect of GnRH agonist (GnRH-a) on ovarian morphology, no study in the literature was found that evaluated the subsequent and cumulative effect of pituitary downregulation by oral contraceptives (OCs) and GnRH-a on ovarian morphology before IVF. This effect is important as many programs use OCs and GnRH-a to desensitize the pituitary before gonadotropin start. Unfortunately, the timing of the ovarian ultrasound measurements is not standardized among or even within institutions. This study will better define the associations of pituitary down-regulation and ovarian morphology. If pituitary suppression produces an effect on ovarian morphology, the timing of the ovarian ultrasound measurements would need to be standardized before counseling patients on the reproductive prognosis of IVF.

Fertility and Sterility姞 Vol. 86, No. 3, September 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

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TABLE 1 Population demographics and ovarian morphology changes for the ART study group, which was further subdivided into patients who conceived and patients who did not conceive Variable

All patients N ⴝ 85

Pregnant N ⴝ 50

Not Pregnant N ⴝ 35

P valuea

Age (y) BMI (kg/m2) Basal FSH level (mIU/mL) Basal E2 level (pg/mL) Basal antral follicles Antral follicles after OCs Antral follicles after GnRH-a Basal ovarian volume (cm3) Ovarian volume after OCs (cm3) Ovarian volume after GnRH-a (cm3) Antral follicle change (basal to OCs) Antral follicle change (basal to GnRH-a) Antral follicle change (OCs to GnRH-a) Ovarian volume change (basal to OCs) (cm3) Ovarian volume change (basal to GnRH-a) (cm3) Ovarian volume change (OCs to GnRH-a) (cm3)

33.7 ⫾ 4.1 26.1 ⫾ 4.8 5.5 ⫾ 1.7 35.4 ⫾ 19.4 25.2 ⫾ 13.1 18.1 ⫾ 10.6 18.3 ⫾ 11.3 15.1 ⫾ 6.7 10.3 ⫾ 4.2 10.9 ⫾ 5.5 ⫺6.7 ⫾ 7.4 ⫺7.2 ⫾ 8.1 ⫺0.6 ⫾ 6.0 ⫺4.8 ⫾ 5.7 ⫺4.2 ⫾ 5.2 0.6 ⫾ 4.1

32.9 ⫾ 3.8 26.0 ⫾ 4.6 5.4 ⫾ 1.9 36.9 ⫾ 21.8 24.7 ⫾ 12.3 18.2 ⫾ 11.3 19.2 ⫾ 14.9 15.6 ⫾ 7.1 10.9 ⫾ 4.6 11.1 ⫾ 6.0 ⫺6.3 ⫾ 7.3 ⫺6.1 ⫾ 6.7 0.2 ⫾ 5.5 ⫺5.1 ⫾ 5.8 ⫺4.5 ⫾ 5.4 0.7 ⫾ 3.9

34.5 ⫾ 4.3 26.2 ⫾ 5.1 5.6 ⫾ 2.0 34.5 ⫾ 18.4 25.8 ⫾ 14.5 17.9 ⫾ 14.1 17.5 ⫾ 16.4 14.4 ⫾ 6.0 9.3 ⫾ 3.4 10.8 ⫾ 6.7 ⫺7.2 ⫾ 7.9 ⫺8.9 ⫾ 9.8 ⫺1.7 ⫾ 6.7 ⫺4.3 ⫾ 5.6 ⫺3.8 ⫾ 5.0 0.6 ⫾ 4.6

.63 .81 .76 .47 .37 .80 .69 .61 .34 .74 .71 .29 .33 .66 .67 .95

Note: Values are means ⫾ SD. P⬍.05 is considered statistically significant. a The pregnant patients were compared with those who were not pregnant using a t-test or a Wilcoxon-Mann Whitney rank sum test. Frattarelli. Pituitary suppression alters ovarian morphology. Fertil Steril 2006.

MATERIALS AND METHODS Population The patient population consisted of 85 couples undergoing fresh autologous IVF cycles. All infertile women undergoing IVF were eligible to participate in the study. Patients meeting inclusion criteria (n ⫽ 85) were counseled and consented to day 3 ultrasounds before treatment. Inclusion criteria included: normal basal FSH concentration per our laboratory (ⱕ12 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. Exclusion criteria included: patients unable to undergo transvaginal ultrasound on the specified dates, patients with a history of ovarian surgery, patients ⬎42 years, or use of hormonal contraception or hormonal infertility therapy within 3 months of study date. Experimental Design This is a prospective cohort analysis of 85 couples undergoing 85 IVF cycles at the Tripler Army Medical Center In Vitro Fertilization Institute. Institutional Review Board approval was obtained from the Tripler Army Medical Center Institutional Review Board, Honolulu, Hawaii. To document ovarian morphology, the patients underwent three transvaginal ultrasonograms; the first on menstrual day 3 of an 578

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unstimulated cycle immediately before starting OC pills that evening, the second 21 days after the start of OCs (30 ␮g ethinyl E2, 0.15 mg norgestrel), and the third 14 days after starting GnRH-a (500 ␮g) (luprolide acetate, Lupron; TAP Pharmaceuticals, North Chicago, IL). All transvaginal ultrasonograms were performed by the author (J.L.F.) using an ATL Ultramark HDI 3000 (Phillips Medical Systems, Bothell, WA) with a 7-MHz vaginal transducer. This machine is capable of measuring antral follicles ⬍2 mm in diameter. All antral follicles measuring 2–10 mm on both ovaries were counted. The total number of antral follicles per patient was used for calculations. Ovarian volume was determined by obtaining three perpendicular diameters and using the prolate ellipsoid formula (0.526 ⫻ D1 ⫻ D2 ⫻ D3). All patients started 30 ␮g of OCs the evening of menstrual day 3. The patients continued the OCs for 21 days before pituitary suppression with GnRH-a. There was a 7-day overlap between the OCs and GnRH-a administration. After 14 days of GnRH-a (500 ␮g), the dose was decreased to 250 ␮g, and stimulation with exogenous gonadotropins was initiated 5 days later. Outcome Measures The primary outcome measure was ovarian morphology defined as total antral follicle count and total ovarian vol-

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FIGURE 1 Ovarian morphology, total antral follicles (bar graph), and total ovarian volume (line graph), measured on menstrual day 3 in the basal state, after 21 days of OC therapy, and after 14 days of GnRH-a therapy. Using a Friedman repeated measures one-way analysis of variance on ranks, a significant decrease in antral follicles and ovarian volume occurs after pituitary desensitization with OCs (*P⬍.01) and GnRH-a (**P⬍.01).

Frattarelli. Pituitary suppression alters ovarian morphology. Fertil Steril 2006.

ume. Secondary outcome measures were age, body mass index (BMI), IVF stimulation parameters, and pregnancy outcomes. Pregnancies and the accompanying rates were defined as follows. Initial pregnancy was documented by an increasing serum hCG concentration on luteal days 14 and 16. Biochemical pregnancy was defined as having a positive pregnancy test on luteal day 14 with a rising titer confirmed by a second hCG level but with loss of the pregnancy before sonographic evidence of the pregnancy. Spontaneous pregnancy loss was defined as a pregnancy loss after sonographic visualization of an intrauterine gestational sac at 6 weeks of gestation. Live-birth rate was defined as those pregnancies proceeding to deliver a viable infant. Statistical Analysis Based on a prior observational study, a sample size of 82 patients was estimated to be needed to detect a 20% difference in total ovarian volume with a power of 0.8 and an ␣-error of 0.05 by analysis of variance. A Wilcoxon-Mann Whitney rank sum test was used to compare nonparametric outcomes between two groups. For Fertility and Sterility姞

parametric data, a t-test was used to compare means between two groups. A Friedman repeated measures one-way analysis of variance on ranks was used to compare the paired changes in ovarian morphology after a series of different experimental treatments (basal state, OCs, and GnRH-a) on a single group of patients. For pairwise multiple comparison, Student-Newman-Keuls method was used. Differences in outcome rates were analyzed using a ␹2 or two-tailed Fisher’s exact test. Efficiency curves were constructed by grouping patients by ovarian volume beginning at ⱕ2 cm3 and extending to ⱖ12 cm3 at 1-cm3 increments. The patients also were grouped by antral follicle number beginning at ⱕ6 and extending to ⱖ30 at intervals of two follicles. The cancellation rates, clinical PRs, miscarriage rates, and live birth rates were then calculated for each increment. The outcome rates above and below each threshold were evaluated to determine whether there were any breakpoints at which there was a change in rates. Subsequently, contingency table analyses were used to evaluate the pregnancy and cancellation rates above and below the selected threshold values. 579

TABLE 2 Univariate and multivariate analysis comparing ovarian morphology in the basal unstimulated menstrual day 3 state, after oral contraceptive therapy for 21 days, and after GnRH-a therapy for 14 days to IVF prestimulation and stimulation parameters Basal antral follicles

Variables Age BMI (kg/m2) Peak E2 (pg/mL) Follicles Oocytes Ampules of gonadotropins used Days of stimulation Embryo number

r r r r r r r r

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

Antral follicles after OCs

⫺0.55, P⬍.001a 0.23, P⬍.05 0.55, P⬍.001 0.70, P⬍.001a 0.64, P⬍.001 ⫺0.59, P⬍.001 ⫺0.29, P⬍.01 0.57, P⬍.001

r r r r r r r r

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

⫺0.47, P⬍.001a 0.05, P⫽.65 0.72, P⬍.001a 0.74, P⬍.001 0.77, P⬍.001a ⫺0.57, P⬍.001 ⫺0.32, P⬍.01 0.67, P⬍.001

Antral follicles after GnRH-a r r r r r r r r

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

⫺0.27, P⬍.05 0.20, P⫽.10 0.79, P⬍.001a 0.77, P⬍.001a 0.63, P⬍.001 ⫺0.65, P⬍.001 ⫺0.36, P⬍.01 0.59, P⬍.001

Note: r-values and P-values reflect univariate analysis. a Variables deemed significant by multiple linear regression (P⬍.05). Frattarelli. Pituitary suppression alters ovarian morphology. Fertil Steril 2006.

Univariate analysis included regression and correlation coefficients examining the association of ovarian volume and antral follicle count with parameters of ovarian reserve and response. Multivariate analysis was then performed to assess which independent variables were most associated with ovarian morphology. An ␣ error of 0.05 was considered significant for all comparisons. Relative risk and 95% confidence intervals are displayed where appropriate. All data are reported as means with their associated standard deviations. RESULTS The demographics for the patient population are depicted in Table 1. Our patients had the following primary etiologies for their infertility: male factor (29%), tubal factor (27%), anovulatory or polycystic ovary syndrome (PCOS) (20%), unexplained (16%), endometriosis (6%), and uterine (2%). Antral follicle count decreased significantly by 28.2% after being treated with OCs (18.1 ⫾ 10.6) compared to the basal state (25.2 ⫾ 13.1) (P⬍.05). There was not a continued decrease in antral follicle count after being treated with GnRH-a for 14 days (18.3 ⫾ 11.3). The antral follicle count after GnRH-a therapy was significantly lower by 27.4% than the basal state (P⬍.01) (Table 1, Fig. 1). Compared to the basal state, there was a mean decrease in antral follicles after being treated with OCs of ⫺6.7 ⫾ 7.4 antral follicles (P⬍.01) and a mean decrease of ⫺7.2 ⫾ 8.1 antral follicles after being treated with GnRH-a (P⬍.01). There was no significant change in antral follicles from the OC treatment to the GnRH-a treatment (⫺0.6 ⫾ 6.0) (Table 1). Total ovarian volume decreased significantly by 31.8% after being treated with OCs (10.3 ⫾ 4.2 cm3) compared to 580

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the basal state (15.1 ⫾ 6.7 cm3) (P⬍.05). There was not a continued decrease in total ovarian volume after being treated with GnRH-a for 14 days (10.9 ⫾ 5.5 cm3). The total ovarian volume after GnRH-a therapy was significantly lower by 27.8% than the basal state (P⬍.01) (Table 1, Fig. 1). Compared to the basal state, there was a mean decrease in ovarian volume after being treated with OCs of ⫺4.8 ⫾ 5.7 cm3 (P⬍.01) and a mean decrease of ⫺4.2 ⫾ 5.2 cm3 after being treated with GnRH-a (P⬍.01). There was no significant change in ovarian volume from the OC treatment to the GnRH-a treatment (0.6 ⫾ 4.1 cm3) (Table 1). Table 1 also compares the pregnant patients to the patients not pregnant. When assessing the pregnancy outcomes, there was no difference in antral follicles or ovarian volume related to pregnancy outcome at the basal day 3 state, after OCs, or after GnRH-a therapy (Table 1). Likewise, there was not significant difference when evaluating the change in ovarian morphology during the IVF cycle. We attempted to find threshold values for antral follicle number and ovarian volume at the basal state, after OC therapy, and after GnRH-a therapy that would help predict ovarian response and ART success in terms of outcome rates (PR, biochemical pregnancy, spontaneous pregnancy loss, or live birth rate). Efficiency curves and contingency table analysis for outcome rates revealed no obvious differences throughout the full range of values for both ovarian volume and antral follicle number (data not shown). A univariate analysis was performed to evaluate the association of IVF prestimulation and stimulation parameters with ovarian morphology at the basal state, after OC therapy, and after GnRH-a therapy (Table 2). The BMI had little

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TABLE 2

Continued. Basal ovarian volume (cm3) r r r r r r r r

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

⫺0.37, P⬍.001 0.12, P⫽.29 0.35, P⬍.05 0.45, P⬍.001a 0.36, P⬍.01 ⫺0.48, P⬍.001 ⫺0.25, P⬍.05 0.32, P⬍.01

r r r r r r r r

Ovarian volume after OCs (cm3)

Ovarian volume after GnRH-a (cm3)

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

r r r r r r r r

⫺0.45, P⬍.001a 0.20, P⫽.07 0.37, P⬍.05 0.39, P⬍.001 0.46, P⬍.001 ⫺0.53, P⬍.001 ⫺0.41, P⬍.001 0.46, P⬍.001

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

⫺0.19, P⫽.11 0.26, P⬍.05 0.44, P⬍.01 0.52, P⬍.001 0.43, P⬍.001 ⫺0.60, P⬍.001a ⫺0.35, P⬍.01 0.43, P⬍.001

Frattarelli. Pituitary suppression alters ovarian morphology. Fertil Steril 2006.

association with any of the ovarian morphology measurements and was only significantly correlated with the basal antral follicles and ovarian volume after GnRH-a therapy. Linear regression analysis revealed that antral follicles and ovarian volume in the basal state and after pituitary suppression had a significant positive correlation with peak E2 levels, number of follicles, number of oocytes, and number of embryos. Likewise, antral follicles and ovarian volume in the basal state and after pituitary suppression had a significant negative correlation with age, ampules of gonadotropins used, and days of stimulation. Multivariate analysis showed that age, peak E2, follicle number, oocyte number, and ampules of gonadotropins were most significantly associated with ovarian morphology (Table 2). DISCUSSION Protocols for ART vary among institutions. Pituitary desensitization before or during IVF is the standard of care at ART institutions. Although several studies have demonstrated the importance of ovarian morphology to reproductive outcome, no study in the literature has evaluated the effect of pituitary suppression with OCs and GnRH-a on ovarian volume and antral follicle number. Many IVF centers use OCs or GnRH-a for pituitary desensitizing. The transvaginal ultrasound is often obtained before the IVF cycle to aid in counseling the patient and tailoring the treatment protocol. The literature pertaining to ovarian morphology measurements and IVF is inconsistent regarding the timing of the ultrasound measurements. Some reports have documented an ultrasound on day 3 in the basal state (11–15). However, the majority of studies have evaluated ovarian morphology in the midluteal phase or after GnRH-a therapy immediately before initiation of gonadotropins (6 –10, 16 –25). Therefore, it is important to determine whether and how ovarian morphology changes with pituitary desensitization. Fertility and Sterility姞

These data reveal that ovarian morphology significantly changes after pituitary desensitization. A significant decrease in both antral follicles and ovarian volume was noted compared to the basal state after treatment with OC and GnRH-a. This finding may contribute to the lack of agreement among published studies concerning cutoff levels of ovarian morphology that demonstrate significance. Of note, there were no additional changes in ovarian morphology with the addition of GnRH-a to OCs. Although a few studies have evaluated a change in ovarian morphology after pituitary suppression, no study could be found that evaluated the basal state, the OC state, and the GnRH-a state (16, 26 –28). The data presented in this article agree with the data published by Sharara et al. (16), which revealed no change in ovarian volume or antral follicles when adding GnRH-a therapy after patients had been down-regulated with OCs. However, Sharara et al. did not evaluate ovarian morphology in the basal state. There have been three recent studies that have compared the ovarian morphology in the basal state to after GnRH-a therapy without the use of OCs (26 –28). Hansen et al. (26) in a prospective analysis of 20 patients found no significant decrease in antral follicles from the basal state to after GnRH-a therapy. Ng et al. (27) used a three-dimensional power Doppler ultrasound on 104 patients and found no significant change in antral follicle or ovarian volume from the basal state to after GnRH-a therapy. Yong et al. (28) found a change in ovarian volume but no change in antral follicles in 46 patients with ultrasound monitoring in the early follicular (days 3–5) phase, luteal (days 20 –25) phase, and after GnRH-a down-regulation. All of the ovarian morphology measurements correlated with IVF stimulation parameters (Table 2). Antral follicles more significantly correlated with IVF stimulation parameters than did ovarian volume. The antral follicle count seems 581

to reflect the pool of remaining primordial follicles available for recruitment and therefore reflects the reproductive age of the patient. Although the age of the patient was most significantly correlated with antral follicles in the basal state, Table 2 shows that many of the remaining stimulation parameters had the strongest correlation with antral follicles after GnRH-a therapy. Threshold levels for both antral follicle count and ovarian volume were evaluated. In evaluating pregnancy outcome along the continuum of antral follicles and ovarian volume, no obvious breakpoint was identified and no significant change in outcome rates was realized. Importantly, a recent study by Klinkert et al. (29) suggested that increasing the dose of gonadotropins in patients with expected poor response based on antral follicle count did not improve IVF outcomes. In summary, these data reveal that ovarian morphology significantly changes with pituitary desensitization. The use of ovarian morphology to counsel patients and tailor IVF stimulation protocols may be important but should be standardized for when the ovarian morphology is determined at each institution. Although no threshold for pregnancy outcome was realized, these data would suggest that antral follicles after GnRH-a therapy are most significantly correlated with IVF parameters. Therefore, larger studies evaluating ovarian morphology measurements after GnRH-a therapy are needed to assess pregnancy outcomes.

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