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Impact of pituitary suppression on antral follicle count and oocyte recovery after ovarian stimulation
Impact of pituitary suppression on antral follicle count and oocyte recovery after ovarian stimulation Nam D. Tran, M.D., Ph.D., Lusine Aghajanova, M.D., Ph.D., Chia-Ning Kao, M.S., Marcelle I. Cedars, M.D., and Mitchell P. Rosen, M.D. Center for Reproductive Health, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, California
Objective: To investigate the potential influence of short-term pituitary suppression on antral follicle count (AFC) and correlate the AFC with the number of oocytes retrieved after ovarian stimulation. Design: Retrospective study. Setting: University fertility center. Patient(s): A total of 1,479 infertile patients. Intervention(s): Patients had baseline AFC, when they were not on any medications known to cause pituitary suppression, and followup AFC (suppressed AFC) while on E2, GnRH agonist (GnRH-a), oral contraceptive (OC) pills, or OC pills/GnRH-a in preparation for ovarian stimulation, performed within 6 months of initial baseline AFC. Main Outcome Measure(s): The AFC at baseline, AFC during pituitary suppression, and the number of oocytes retrieved. Result(s): Although there was an average unadjusted decline of 0.4, 0.9, 2.2, and 3.0 in AFC while patients were on E2, GnRH-a, OC pills, and OC pills/GnRH-a, respectively, this decline was driven by age, baseline AFC, and the hormones used. Although baseline and suppressed AFC were found to be good predictors of the number of oocytes retrieved after ovarian stimulation, statistically, suppressed AFC was found to be a marginally better predictor. Conclusion(s): Short-term pituitary suppression has a negative impact on AFC. This decline in AFC may influence the number of oocytes retrieved, suggesting the suppressive impact of exogUse your smartphone enous hormones on the biological capacity of the ovary during stimulation. (Fertil SterilÒ to scan this QR code 2016;105:690–6. Ó2016 by American Society for Reproductive Medicine.) and connect to the Key Words: Pituitary suppression, antral follicle count, oocyte number Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/trann-impact-pituitary-suppression-afc/
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ontrolled ovarian hyperstimulation (COH) is an integral element of assisted reproductive technology (ART). The ovarian response to COH is best predicted with markers of ovarian reserve such as antral follicle count (AFC) and antimullerian hormone (1–3). Hence, an accurate evaluation of ovarian reserve has become an essential means for practitioners to predict the ovarian
response before stimulation and potentially select the stimulation protocol (4–6). At present, AFC is generally accepted as a good predictor of ovarian response to COH (1, 2), comparable with antimullerian hormone (3, 7). Antral follicles are fluid-filled structures, measuring 2–10 mm in diameter, noted on transvaginal ultrasound (8, 9). The AFC measurements appear to have
Received July 8, 2015; revised October 27, 2015; accepted November 17, 2015; published online December 13, 2015. N.D.T. has nothing to disclose. L.A. has nothing to disclose. C.-N.K. has nothing to disclose. M.I.C. has nothing to disclose. M.P.R. has nothing to disclose. Reprint requests: Nam D. Tran, M.D., Ph.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, 499 Illinois Street, San Francisco, California 94143 (E-mail: [email protected]). Fertility and Sterility® Vol. 105, No. 3, March 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.11.033 690
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good reproducibility between cycles and examiners (10, 11). With gonadotropin stimulation, growth of these structures is followed, and the AFC has been shown to predict ovarian response to gonadotropins (12), as well as the number of oocytes retrieved, and good quality embryos developed (13), suggesting an accurate assessment of reproductive capacity (3). Furthermore, AFC has been shown to decline with age (14–16) and correlate with antimullerian hormone (17, 18), suggesting that it may be an accurate reflection of the underlying remaining primordial follicular pool (19). During IVF treatment, it is often beneficial to regulate the hypothalamus-pituitary-ovarian axis VOL. 105 NO. 3 / MARCH 2016
Fertility and Sterility® either during the beginning of the follicular phase or the midluteal phase immediately preceding COH to minimize the early increase of endogenous gonadotropins, which could result in asynchronous recruitment of follicles (20, 21). Thus, shortterm pituitary suppression is often achieved with the use of E2, GnRH agonist (GnRH-a), oral contraceptive (OC) pills, or a combination of OC pills/GnRH-a before stimulation. In addition to being a predictor of COH response, AFC is also being used for decisions on gonadotropin dosing and IVF stimulation protocols (2, 22). Although studies have demonstrated that results of AFC assessment are similar when measured in the early follicular or the midluteal phase during natural cycles (11, 23), the effect of short-term pituitary suppression on AFC before ovarian stimulation remains controversial. Although some studies have shown no effect on AFC when patients were on GnRH-a (24–27), others have shown a decrease in AFC number after pituitary suppression with OC pills/GnRH-a or antral follicle size after suppression with OC pills (28, 29). Thus, it is unclear whether short-term pituitary suppression has an impact on AFC and ultimately the number of oocytes recovered after short-term pituitary suppression to prepare for IVF treatments. The aim of this study was to investigate the potential effect of different short-term pituitary suppression regimens on AFC and to correlate this change in AFC with the number of oocytes retrieved, a surrogate marker of ovarian response to COH.
Baseline AFC, AFC immediately after pituitary suppression (suppressed AFC), and the number of oocytes retrieved were measured, collected, and analyzed. The AFC was measured by attending physicians using Shimadzu SDU450XL ultrasound with a variable 5–7 mm transvaginal probe. All follicles measuring 2–10 mm on both ovaries were counted. To measure the potential effect of age, AFC, and drugs on ovarian suppression, an analysis of covariance (ANCOVA) model was used to analyze the difference in baseline and suppressed AFC yet controlling for age and baseline AFC. Pairwise comparisons were performed with the TukeyKramer pairwise comparison method. To determine whether baseline or suppressed AFC is a superior predictor of the number of oocytes retrieved, several methods were used. Initially, a Pearson correlation analysis was used to measure the relationship between AFC measurements and oocytes retrieved. Then a factor analysis confirmed the correlation findings. Finally, several ANCOVA models controlling for baseline AFC, age, and drugs were run and compared using wellaccepted fit statistics. Model comparisons were measured using the Akaike information criterion (AIC), Akaike information criterion with correction (AICC) for finite sample size and Bayesian information criterion to determine whether baseline or suppressed AFC was a superior predictor of oocytes retrieved.
RESULTS MATERIALS AND METHODS
Demographics
This study was performed under the approval of the University of California San Francisco Institutional Review Board. A chart review was done on all patients who presented to our clinic from 2000 to 2014. All patients undergoing their first IVF treatments were screened for the studies. Inclusion criteria included the presence of both ovaries, ability to visualize both ovaries with transvaginal ultrasound, and only patients with at least two AFC measurements by transvaginal ultrasound, performed by attending physicians, within 6 months of each other. Specifically, the first AFC (baseline AFC) was performed when patients were not on medications (E2, GnRH-a, or OC pills) known to interfere with the hypothalamus-pituitary-axis or natural follicular development for at least 3 months. The second AFC (suppressed AFC) was performed on cycle day 1–2 immediately after the cycle in which the patients were taking either E2, GnRH-a, OC pills, or OC pills/GnRH-a for pituitary suppression before COH for IVF. All patients were included independent of age or diagnoses pertaining to infertility. Pituitary suppression was achieved with either OC pills (30 mg ethinyl E2/0.15 mg desogestrel) for 14–21 days and SC GnRH-a (10 units/d, luprolide acetate; TAP Pharmaceuticals) starting 6 days after OC pills start, OC pills alone for 14– 21 days, GnRH-a (10 units/d) alone starting on day 7 after LH surge until menses, or E2 alone (Estrace 2 mg; Actavis) by mouth twice a day starting on day 7 after LH surge until menses. Patients started OC pills for either OC pills/GnRH-a or OC pills only protocols on cycle days 1–3. The choice of pituitary suppression protocol was typically dependent on baseline AFC, age, and physicians' preferences.
A total of 9,904 patients presented to our center for IVF treatment from 2000 to 2014 and these were reviewed. Of these patients, 1,479 met the inclusion criteria for the study. The distribution of the patients on different types of hormones for pituitary suppression and their mean age is demonstrated in Table 1. The mean age of all qualified patients was 37.5 4.2 years old. Although the mean ages of patients in the GnRH-a (37.4 years), OC pills (37.4 years), and OC pills/ GnRH-a (36.9 years) groups were similar, the mean age for the E2 group (38.7 years) was significantly higher (P< .001) than all other drug groups. The mean baseline AFC in all studied patients was 14.0 7.9. The mean AFC for patients in the E2 (8.3 3.8), GnRH-a (10.4 5.5), OC pills (15.8 9.3), and OC pills/GnRH-a (17.2 6.8) was significantly different (P< .0001) (Table 2). In
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TABLE 1 Patient distribution and mean age. Characteristic E2a GnRH-a OC pills OC pills/GnRH-a
No. of patients Mean age (y) ± SD Age range (y) 326 178 445 530
38.6 3.9 37.4 3.9 37.4 4.4 36.9 4.2
22.2–47.4 24.9–46.3 18.7–45.4 25.9–45.6
Note: Age was statistically different between groups by single factor analysis of variance (ANOVA) (P< .0001). a Tukey-Kramer pair-wise comparisons demonstrated only significant differences between E2 and GnRH-a (P< .008), oral contraceptive (OC) pills (P< .0002), and OC pills/GnRH-a (P< .0001). Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
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TABLE 2 Impact of short-term pituitary suppression on AFC and the number of oocytes retrieved. Characteristic
Baselinea AFC ± SD
Suppressed AFC ± SD
Oocytes retrieved ± SD
8.3 3.8 10.4 5.5 15.8 9.3 17.2 6.8
7.9 3.9 9.5 4.8 13.7 8.8 14.2 6.3
9.2 5.6 11.7 7.2 14.7 10.0 16.1 7.8
E2 GnRH-a oral contraceptive (OC) pills OC pills/GnRH-a
Note: Baseline antral follicle count (AFC) was statistically different between groups by single factor analysis of variance (ANOVA) (P< .0001). a Tukey-Kramer pair-wise comparisons demonstrated significant differences between all the four groups (all P values < .03). Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
addition, the mean baseline AFC of patients from each drug group was significantly different from each other based on Tukey-Kramer pairwise comparison (P< .03).
Effect of Short-term Pituitary Suppression on AFC In comparison to baseline AFC, when patients were not on medications known to affect the hypothalamic-pituitaryovarian axis, there was a general decline in AFC after shortterm pituitary suppression, regardless of the types of hormones used. The mean differences in AFC observed after short-term pituitary suppression with either E2, GnRH-a, OC pills, or OC pills/GnRH-a are shown in Figure 1. Specifically,
there was an unadjusted average decline of 0.4, 0.9, 2.2, and 3.0 in AFC when patients were on E2, GnRH-a, OC pills, and OC pills/GnRH-a, respectively (Table 2). Based on the mean differences in AFC at baseline and after pituitary suppression, E2 appeared to be the least suppressive, whereas the combination of OC pills/GnRH-a was the most suppressive on AFC. However, the ANCOVA model for AFC suppression was performed and revealed that age (P< .005), baseline AFC (P< .0001), and the type of hormones used for pituitary suppression (P< .002) all significantly contributed to the decline in AFC. Specifically, our study population of 18–47 years old showed an independent linear decline of 0.07 AFC per year after pituitary suppression.
FIGURE 1 45
40
AFC or Number of Oocytes Collected
35
30
25
20
15
10
5
0
Baseline Suppressed AFC AFC
Oocytes
Estradiol
Baseline Suppressed AFC AFC GnRHa
Oocytes
Baseline Suppressed AFC AFC OCP
Oocytes
Baseline Suppressed AFC AFC
Oocytes
v
Impact of short-term pituitary suppression on antral follicle count (AFC) and number of oocytes retrieved. A significant decrease in AFC after pituitary suppression (suppressed AFC) was observed in all treatment groups. Nonetheless, this decrease in AFC did not compromise the number of oocytes recovered after pituitary suppression, as both baseline and suppressed AFC were significant predictors of number of oocytes retrieved. GnRHa ¼ GnRH agonist; OCP ¼ oral contraceptive pills. Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
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Fertility and Sterility® Thus a 40-year- old patient will experience an additional decline of 1.4 AFC after pituitary suppression in comparison with a 20-year-old patient with the same baseline AFC and hormone used for pituitary suppression. Interestingly, baseline AFC also significantly contributed to the decline in AFC after suppression (P< .0001). However, this relationship is nonlinear. When baseline AFC was subgrouped into AFC %5, AFC 6–10, and AFC R11 to reflect patients with severe diminished ovarian reserve, diminished ovarian reserve, and normal reserve, respectively, only patients with AFC >5 experienced an additional decline in AFC after pituitary suppression, independent of age and types of hormones used. The ANCOVA model predicts an additional 0.4 and 2.7 AFC decline after pituitary suppression when patients have baseline AFC between 6 and 10, and R11, respectively. Thus, patients with baseline AFC of 10 and 20 will have an additional decline of 0.4 and 2.7 AFC, respectively, after suppression, in comparison with a patient with equivalent drug and age but with a baseline AFC of %5. Short-term pituitary suppression with E2, GnRH-a, OC pills, or OC pills/GnRH-a also contributed to an independent decline of 0.4, 0.6, 1.3, and 1.6 AFC, respectively, after adjustment for age and baseline AFC. Although there was no differences in the level of AFC suppression between E2 and GnRH-a (P¼ .96) groups or OC pills and OC pills/GnRH-a (P¼ .78) groups, the suppression was significantly different in both the OC pills and OC pills/GnRH-a groups versus the E2 and GnRH-a groups (P< .05). Figure 2 demonstrates the theoretical model predicting the decline in AFC after pituitary suppression factoring in age, baseline AFC, and types of hormones used. To simplify the model, baseline AFC was subgrouped into AFC %10 versus AFC R11 as the decline of AFC in patients with baseline AFC R11 was more profound (2.7) compared to patients with AFC %10 (<0.4).
Effect of Short-term Pituitary Suppression on Number of Oocytes Retrieved To determine whether baseline or suppressed AFC is a superior predictor of the number of oocytes retrieved after COH, Pearson correlation analyses were initially performed and showed a significant positive relationship between the numbers of oocytes retrieved with both baseline (r ¼ 0.60, P< .0001) and suppressed AFC (r ¼ 0.61, P< .0001). Factor analysis confirmed these findings, baseline and suppressed AFC accounted for 37.34% and 37.26% of the total variance (baseline AFC, suppressed AFC, age, drugs used, eggs collected), respectively. Two ANCOVA models were subsequently performed: one with baseline AFC and another with suppressed AFC. Both models were controlled for hormones used for pituitary suppression and age. Although baseline (AIC ¼ 9,868) and suppressed AFC (AIC ¼ 9,813) were found to be good predictors of the number of oocytes retrieved using the AIC criteria, suppressed AFC was slightly better, statistically, in predicting the number of oocytes retrieved based on the lower AIC, consistent with the Pearson correlation analyses. VOL. 105 NO. 3 / MARCH 2016
DISCUSSION Pituitary suppression during the preceding cycles immediately before IVF stimulation is an important part of ART. However, the potential impact of short-term pituitary suppression on ovarian reserve and eventual response to ovarian stimulation are unknown. The results from this study support that short-term pituitary suppression may have a negative impact on AFC primarily due to patient age, baseline AFC, and the type of hormones used. Furthermore, this shortterm suppression, to some degree, may alter the biological capacity of the ovary to respond to stimulation as suppressed AFC was found to be a statistically better predictor of the number of oocytes retrieved in comparison with baseline AFC. Prior studies have suggested that pituitary suppression has no impact on AFC. Yu Ng et al. (30) evaluated the ovarian volume, AFC, ovarian Doppler flow, pituitary and steroid hormones in patients undergoing IVF, before and after pituitary down-regulation with buserelin (Suprecur, Hoechst), and demonstrated no significant difference in any of the indices after treatment. Of note, the study population was small (n ¼ 85) and the age of the patients was not reported (30). Although statistically insignificant, there was a trend toward a decline in AFC after down-regulation. In addition to the differences between studies, it is possible the varied length of downregulation with nasal buserelin (5–20 days) versus SC leuprolide acetate (LA; 10–14 days) in our study may contribute to the discrepancy. Jayaprakasan et al. (25, 31) collected data from 114 patients and showed that pituitary suppression with GnRH-a increased antimullerian hormone, decreased ovarian volume and ovarian blood flow with a nonsignificant trend in AFC decline. It is possible the difference in age between the study (31) and ours (33.7 vs. 37.5 years) may contribute to the difference in decline in AFC after suppression, as we have shown in the present study that age directly correlated with the decline in AFC. In contrast, using a study population of 85 infertile patients undergoing IVF treatments, Frattarelli (28) demonstrated pituitary suppression with 21 days of OC pills significantly suppressed AFC, similar to our results. However, decline in AFC in Frattarelli's study was >7. It is possible the higher mean baseline AFC in that study (25.2) may contribute to the greater suppression, consistent with our findings of greater AFC suppression with higher baseline AFC independent of age and suppression drug. In addition, we investigated whether the degree of suppression has any effect on the number of oocytes retrieved. The overall baseline AFC and number of oocytes retrieved decreased from the OC pills/GnRH-a group, followed by OC pills, GnRH-a, and E2 group (Table 2). Similarly, the greatest decline in AFC after pituitary suppression was again noted with OC pills/GnRH-a, followed by OC pills, GnRH-a, and E2 (Table 2, Fig. 1). This trend is most likely due to our current clinical protocols in which decisions regarding the selection of stimulation protocol are often based largely on age and baseline AFC. We often use what are thought to be the least suppressive medications in older patients with low baseline AFC. This pattern of practice is best reflective by the low mean age and high baseline AFC in patients treated with OC pills/GnRH-a, in comparison with high mean age and low 693
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FIGURE 2
Age, baseline antral follicle count (AFC), and hormones independently affect the decrease in AFC after pituitary suppression. These plots demonstrate the theoretical predicted decrease in AFC after pituitary suppression in women between 30 and 49 years old taking E2 (estradiol), GnRH-a (GnRH agonist), OC pills (OCP), or OCP/GnRH-a. Negative value of Y-axis demonstrates the decrease in AFC after pituitary suppression factoring age and baseline AFC. (A) Theoretical model for patients with AFC %10. (B) Theoretical model for patients with AFC R11. Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
baseline in AFC in patients treated with E2. As demonstrated, the types of hormones contributed to the decline in AFC after pituitary suppression, confirming our clinical observations. 694
Nonetheless, baseline and suppressed AFC were found to be good predictors of the number of oocytes retrieved after COH by Pearson correlation analyses and ANCOVA models. VOL. 105 NO. 3 / MARCH 2016
Fertility and Sterility® Although suppressed AFC was a better fit based on ANCOVA models using AIC criteria, the absolute difference in AIC between baseline AFC (AIC ¼ 9,868) and suppressed AFC (AIC ¼ 9,813) was marginal at 55 indicating that although shortterm pituitary suppression may alter the biological capacity of the ovary during stimulation, the clinical significance of this suppression remains to be investigated. The strengths of our study include the large sample size (1,479 patients), broad age range (18–47 years old), characterization of ovarian reserve before and after suppression, and the number of oocytes retrieved after COH. Importantly, the baseline AFC was performed within 6 months before pituitary suppression, when patients were not taking any medications known to affect the hypothalamus-pituitary-ovarian axis, to assure minimal changes in AFC with time (32, 33). A weakness of this study is the retrospective design with a known practice bias of preferentially treating older patients with low AFC with either E2 or GnRH-a alone, and treating younger patients with higher AFC with OC pills or OC pills/ GnRH-a, which we addressed by controlling for age and baseline AFC with the ANCOVA models. In addition, as our clinic does not routinely perform COH without pituitary suppression, this only allows us to compare the change in AFC after pituitary suppression relative to different hormonal regimens rather than to no pituitary suppression. Similarly, we could only compare the number of oocytes recovered after pituitary suppression relative to different regimens and not to without suppression. However, we purposefully designed the time between baseline and suppressed AFC measurement to be within 6 months, as previous studies had demonstrated no changes in AFC during this time span (32, 33). The present data demonstrated that AFC declined during short-term pituitary suppression primarily due to patient age, baseline AFC, and type of hormones used. Furthermore, this decline in AFC appears to negatively impact the number of oocytes retrieved after COH. Although this decline in AFC after suppression was significant in comparison with the baseline AFC, baseline and suppressed AFC continue to be good predictors of the number of oocytes retrieved. However, based on ANCOVA modeling that suppressed AFC is marginally a better fit in predicting the number of oocytes retrieved after ovarian stimulation, we suggest using suppressed AFC as a main predictor of the number of oocytes retrieved, whereas baseline AFC is an acceptable alternative.
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Objective: To investigate the potential influence of short-term pituitary suppression on antral follicle count (AFC) and correlate the AFC with the number of oocytes retrieved after ovarian stimulation. Design: Retrospective study. Setting: University fertility center. Patient(s): A total of 1,479 infertile patients. Intervention(s): Patients had baseline AFC, when they were not on any medications known to cause pituitary suppression, and followup AFC (suppressed AFC) while on E2, GnRH agonist (GnRH-a), oral contraceptive (OC) pills, or OC pills/GnRH-a in preparation for ovarian stimulation, performed within 6 months of initial baseline AFC. Main Outcome Measure(s): The AFC at baseline, AFC during pituitary suppression, and the number of oocytes retrieved. Result(s): Although there was an average unadjusted decline of 0.4, 0.9, 2.2, and 3.0 in AFC while patients were on E2, GnRH-a, OC pills, and OC pills/GnRH-a, respectively, this decline was driven by age, baseline AFC, and the hormones used. Although baseline and suppressed AFC were found to be good predictors of the number of oocytes retrieved after ovarian stimulation, statistically, suppressed AFC was found to be a marginally better predictor. Conclusion(s): Short-term pituitary suppression has a negative impact on AFC. This decline in AFC may influence the number of oocytes retrieved, suggesting the suppressive impact of exogUse your smartphone enous hormones on the biological capacity of the ovary during stimulation. (Fertil SterilÒ to scan this QR code 2016;105:690–6. Ó2016 by American Society for Reproductive Medicine.) and connect to the Key Words: Pituitary suppression, antral follicle count, oocyte number Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/trann-impact-pituitary-suppression-afc/
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ontrolled ovarian hyperstimulation (COH) is an integral element of assisted reproductive technology (ART). The ovarian response to COH is best predicted with markers of ovarian reserve such as antral follicle count (AFC) and antimullerian hormone (1–3). Hence, an accurate evaluation of ovarian reserve has become an essential means for practitioners to predict the ovarian
response before stimulation and potentially select the stimulation protocol (4–6). At present, AFC is generally accepted as a good predictor of ovarian response to COH (1, 2), comparable with antimullerian hormone (3, 7). Antral follicles are fluid-filled structures, measuring 2–10 mm in diameter, noted on transvaginal ultrasound (8, 9). The AFC measurements appear to have
Received July 8, 2015; revised October 27, 2015; accepted November 17, 2015; published online December 13, 2015. N.D.T. has nothing to disclose. L.A. has nothing to disclose. C.-N.K. has nothing to disclose. M.I.C. has nothing to disclose. M.P.R. has nothing to disclose. Reprint requests: Nam D. Tran, M.D., Ph.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, 499 Illinois Street, San Francisco, California 94143 (E-mail: [email protected]). Fertility and Sterility® Vol. 105, No. 3, March 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.11.033 690
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good reproducibility between cycles and examiners (10, 11). With gonadotropin stimulation, growth of these structures is followed, and the AFC has been shown to predict ovarian response to gonadotropins (12), as well as the number of oocytes retrieved, and good quality embryos developed (13), suggesting an accurate assessment of reproductive capacity (3). Furthermore, AFC has been shown to decline with age (14–16) and correlate with antimullerian hormone (17, 18), suggesting that it may be an accurate reflection of the underlying remaining primordial follicular pool (19). During IVF treatment, it is often beneficial to regulate the hypothalamus-pituitary-ovarian axis VOL. 105 NO. 3 / MARCH 2016
Fertility and Sterility® either during the beginning of the follicular phase or the midluteal phase immediately preceding COH to minimize the early increase of endogenous gonadotropins, which could result in asynchronous recruitment of follicles (20, 21). Thus, shortterm pituitary suppression is often achieved with the use of E2, GnRH agonist (GnRH-a), oral contraceptive (OC) pills, or a combination of OC pills/GnRH-a before stimulation. In addition to being a predictor of COH response, AFC is also being used for decisions on gonadotropin dosing and IVF stimulation protocols (2, 22). Although studies have demonstrated that results of AFC assessment are similar when measured in the early follicular or the midluteal phase during natural cycles (11, 23), the effect of short-term pituitary suppression on AFC before ovarian stimulation remains controversial. Although some studies have shown no effect on AFC when patients were on GnRH-a (24–27), others have shown a decrease in AFC number after pituitary suppression with OC pills/GnRH-a or antral follicle size after suppression with OC pills (28, 29). Thus, it is unclear whether short-term pituitary suppression has an impact on AFC and ultimately the number of oocytes recovered after short-term pituitary suppression to prepare for IVF treatments. The aim of this study was to investigate the potential effect of different short-term pituitary suppression regimens on AFC and to correlate this change in AFC with the number of oocytes retrieved, a surrogate marker of ovarian response to COH.
Baseline AFC, AFC immediately after pituitary suppression (suppressed AFC), and the number of oocytes retrieved were measured, collected, and analyzed. The AFC was measured by attending physicians using Shimadzu SDU450XL ultrasound with a variable 5–7 mm transvaginal probe. All follicles measuring 2–10 mm on both ovaries were counted. To measure the potential effect of age, AFC, and drugs on ovarian suppression, an analysis of covariance (ANCOVA) model was used to analyze the difference in baseline and suppressed AFC yet controlling for age and baseline AFC. Pairwise comparisons were performed with the TukeyKramer pairwise comparison method. To determine whether baseline or suppressed AFC is a superior predictor of the number of oocytes retrieved, several methods were used. Initially, a Pearson correlation analysis was used to measure the relationship between AFC measurements and oocytes retrieved. Then a factor analysis confirmed the correlation findings. Finally, several ANCOVA models controlling for baseline AFC, age, and drugs were run and compared using wellaccepted fit statistics. Model comparisons were measured using the Akaike information criterion (AIC), Akaike information criterion with correction (AICC) for finite sample size and Bayesian information criterion to determine whether baseline or suppressed AFC was a superior predictor of oocytes retrieved.
RESULTS MATERIALS AND METHODS
Demographics
This study was performed under the approval of the University of California San Francisco Institutional Review Board. A chart review was done on all patients who presented to our clinic from 2000 to 2014. All patients undergoing their first IVF treatments were screened for the studies. Inclusion criteria included the presence of both ovaries, ability to visualize both ovaries with transvaginal ultrasound, and only patients with at least two AFC measurements by transvaginal ultrasound, performed by attending physicians, within 6 months of each other. Specifically, the first AFC (baseline AFC) was performed when patients were not on medications (E2, GnRH-a, or OC pills) known to interfere with the hypothalamus-pituitary-axis or natural follicular development for at least 3 months. The second AFC (suppressed AFC) was performed on cycle day 1–2 immediately after the cycle in which the patients were taking either E2, GnRH-a, OC pills, or OC pills/GnRH-a for pituitary suppression before COH for IVF. All patients were included independent of age or diagnoses pertaining to infertility. Pituitary suppression was achieved with either OC pills (30 mg ethinyl E2/0.15 mg desogestrel) for 14–21 days and SC GnRH-a (10 units/d, luprolide acetate; TAP Pharmaceuticals) starting 6 days after OC pills start, OC pills alone for 14– 21 days, GnRH-a (10 units/d) alone starting on day 7 after LH surge until menses, or E2 alone (Estrace 2 mg; Actavis) by mouth twice a day starting on day 7 after LH surge until menses. Patients started OC pills for either OC pills/GnRH-a or OC pills only protocols on cycle days 1–3. The choice of pituitary suppression protocol was typically dependent on baseline AFC, age, and physicians' preferences.
A total of 9,904 patients presented to our center for IVF treatment from 2000 to 2014 and these were reviewed. Of these patients, 1,479 met the inclusion criteria for the study. The distribution of the patients on different types of hormones for pituitary suppression and their mean age is demonstrated in Table 1. The mean age of all qualified patients was 37.5 4.2 years old. Although the mean ages of patients in the GnRH-a (37.4 years), OC pills (37.4 years), and OC pills/ GnRH-a (36.9 years) groups were similar, the mean age for the E2 group (38.7 years) was significantly higher (P< .001) than all other drug groups. The mean baseline AFC in all studied patients was 14.0 7.9. The mean AFC for patients in the E2 (8.3 3.8), GnRH-a (10.4 5.5), OC pills (15.8 9.3), and OC pills/GnRH-a (17.2 6.8) was significantly different (P< .0001) (Table 2). In
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TABLE 1 Patient distribution and mean age. Characteristic E2a GnRH-a OC pills OC pills/GnRH-a
No. of patients Mean age (y) ± SD Age range (y) 326 178 445 530
38.6 3.9 37.4 3.9 37.4 4.4 36.9 4.2
22.2–47.4 24.9–46.3 18.7–45.4 25.9–45.6
Note: Age was statistically different between groups by single factor analysis of variance (ANOVA) (P< .0001). a Tukey-Kramer pair-wise comparisons demonstrated only significant differences between E2 and GnRH-a (P< .008), oral contraceptive (OC) pills (P< .0002), and OC pills/GnRH-a (P< .0001). Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
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TABLE 2 Impact of short-term pituitary suppression on AFC and the number of oocytes retrieved. Characteristic
Baselinea AFC ± SD
Suppressed AFC ± SD
Oocytes retrieved ± SD
8.3 3.8 10.4 5.5 15.8 9.3 17.2 6.8
7.9 3.9 9.5 4.8 13.7 8.8 14.2 6.3
9.2 5.6 11.7 7.2 14.7 10.0 16.1 7.8
E2 GnRH-a oral contraceptive (OC) pills OC pills/GnRH-a
Note: Baseline antral follicle count (AFC) was statistically different between groups by single factor analysis of variance (ANOVA) (P< .0001). a Tukey-Kramer pair-wise comparisons demonstrated significant differences between all the four groups (all P values < .03). Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
addition, the mean baseline AFC of patients from each drug group was significantly different from each other based on Tukey-Kramer pairwise comparison (P< .03).
Effect of Short-term Pituitary Suppression on AFC In comparison to baseline AFC, when patients were not on medications known to affect the hypothalamic-pituitaryovarian axis, there was a general decline in AFC after shortterm pituitary suppression, regardless of the types of hormones used. The mean differences in AFC observed after short-term pituitary suppression with either E2, GnRH-a, OC pills, or OC pills/GnRH-a are shown in Figure 1. Specifically,
there was an unadjusted average decline of 0.4, 0.9, 2.2, and 3.0 in AFC when patients were on E2, GnRH-a, OC pills, and OC pills/GnRH-a, respectively (Table 2). Based on the mean differences in AFC at baseline and after pituitary suppression, E2 appeared to be the least suppressive, whereas the combination of OC pills/GnRH-a was the most suppressive on AFC. However, the ANCOVA model for AFC suppression was performed and revealed that age (P< .005), baseline AFC (P< .0001), and the type of hormones used for pituitary suppression (P< .002) all significantly contributed to the decline in AFC. Specifically, our study population of 18–47 years old showed an independent linear decline of 0.07 AFC per year after pituitary suppression.
FIGURE 1 45
40
AFC or Number of Oocytes Collected
35
30
25
20
15
10
5
0
Baseline Suppressed AFC AFC
Oocytes
Estradiol
Baseline Suppressed AFC AFC GnRHa
Oocytes
Baseline Suppressed AFC AFC OCP
Oocytes
Baseline Suppressed AFC AFC
Oocytes
v
Impact of short-term pituitary suppression on antral follicle count (AFC) and number of oocytes retrieved. A significant decrease in AFC after pituitary suppression (suppressed AFC) was observed in all treatment groups. Nonetheless, this decrease in AFC did not compromise the number of oocytes recovered after pituitary suppression, as both baseline and suppressed AFC were significant predictors of number of oocytes retrieved. GnRHa ¼ GnRH agonist; OCP ¼ oral contraceptive pills. Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
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Fertility and Sterility® Thus a 40-year- old patient will experience an additional decline of 1.4 AFC after pituitary suppression in comparison with a 20-year-old patient with the same baseline AFC and hormone used for pituitary suppression. Interestingly, baseline AFC also significantly contributed to the decline in AFC after suppression (P< .0001). However, this relationship is nonlinear. When baseline AFC was subgrouped into AFC %5, AFC 6–10, and AFC R11 to reflect patients with severe diminished ovarian reserve, diminished ovarian reserve, and normal reserve, respectively, only patients with AFC >5 experienced an additional decline in AFC after pituitary suppression, independent of age and types of hormones used. The ANCOVA model predicts an additional 0.4 and 2.7 AFC decline after pituitary suppression when patients have baseline AFC between 6 and 10, and R11, respectively. Thus, patients with baseline AFC of 10 and 20 will have an additional decline of 0.4 and 2.7 AFC, respectively, after suppression, in comparison with a patient with equivalent drug and age but with a baseline AFC of %5. Short-term pituitary suppression with E2, GnRH-a, OC pills, or OC pills/GnRH-a also contributed to an independent decline of 0.4, 0.6, 1.3, and 1.6 AFC, respectively, after adjustment for age and baseline AFC. Although there was no differences in the level of AFC suppression between E2 and GnRH-a (P¼ .96) groups or OC pills and OC pills/GnRH-a (P¼ .78) groups, the suppression was significantly different in both the OC pills and OC pills/GnRH-a groups versus the E2 and GnRH-a groups (P< .05). Figure 2 demonstrates the theoretical model predicting the decline in AFC after pituitary suppression factoring in age, baseline AFC, and types of hormones used. To simplify the model, baseline AFC was subgrouped into AFC %10 versus AFC R11 as the decline of AFC in patients with baseline AFC R11 was more profound (2.7) compared to patients with AFC %10 (<0.4).
Effect of Short-term Pituitary Suppression on Number of Oocytes Retrieved To determine whether baseline or suppressed AFC is a superior predictor of the number of oocytes retrieved after COH, Pearson correlation analyses were initially performed and showed a significant positive relationship between the numbers of oocytes retrieved with both baseline (r ¼ 0.60, P< .0001) and suppressed AFC (r ¼ 0.61, P< .0001). Factor analysis confirmed these findings, baseline and suppressed AFC accounted for 37.34% and 37.26% of the total variance (baseline AFC, suppressed AFC, age, drugs used, eggs collected), respectively. Two ANCOVA models were subsequently performed: one with baseline AFC and another with suppressed AFC. Both models were controlled for hormones used for pituitary suppression and age. Although baseline (AIC ¼ 9,868) and suppressed AFC (AIC ¼ 9,813) were found to be good predictors of the number of oocytes retrieved using the AIC criteria, suppressed AFC was slightly better, statistically, in predicting the number of oocytes retrieved based on the lower AIC, consistent with the Pearson correlation analyses. VOL. 105 NO. 3 / MARCH 2016
DISCUSSION Pituitary suppression during the preceding cycles immediately before IVF stimulation is an important part of ART. However, the potential impact of short-term pituitary suppression on ovarian reserve and eventual response to ovarian stimulation are unknown. The results from this study support that short-term pituitary suppression may have a negative impact on AFC primarily due to patient age, baseline AFC, and the type of hormones used. Furthermore, this shortterm suppression, to some degree, may alter the biological capacity of the ovary to respond to stimulation as suppressed AFC was found to be a statistically better predictor of the number of oocytes retrieved in comparison with baseline AFC. Prior studies have suggested that pituitary suppression has no impact on AFC. Yu Ng et al. (30) evaluated the ovarian volume, AFC, ovarian Doppler flow, pituitary and steroid hormones in patients undergoing IVF, before and after pituitary down-regulation with buserelin (Suprecur, Hoechst), and demonstrated no significant difference in any of the indices after treatment. Of note, the study population was small (n ¼ 85) and the age of the patients was not reported (30). Although statistically insignificant, there was a trend toward a decline in AFC after down-regulation. In addition to the differences between studies, it is possible the varied length of downregulation with nasal buserelin (5–20 days) versus SC leuprolide acetate (LA; 10–14 days) in our study may contribute to the discrepancy. Jayaprakasan et al. (25, 31) collected data from 114 patients and showed that pituitary suppression with GnRH-a increased antimullerian hormone, decreased ovarian volume and ovarian blood flow with a nonsignificant trend in AFC decline. It is possible the difference in age between the study (31) and ours (33.7 vs. 37.5 years) may contribute to the difference in decline in AFC after suppression, as we have shown in the present study that age directly correlated with the decline in AFC. In contrast, using a study population of 85 infertile patients undergoing IVF treatments, Frattarelli (28) demonstrated pituitary suppression with 21 days of OC pills significantly suppressed AFC, similar to our results. However, decline in AFC in Frattarelli's study was >7. It is possible the higher mean baseline AFC in that study (25.2) may contribute to the greater suppression, consistent with our findings of greater AFC suppression with higher baseline AFC independent of age and suppression drug. In addition, we investigated whether the degree of suppression has any effect on the number of oocytes retrieved. The overall baseline AFC and number of oocytes retrieved decreased from the OC pills/GnRH-a group, followed by OC pills, GnRH-a, and E2 group (Table 2). Similarly, the greatest decline in AFC after pituitary suppression was again noted with OC pills/GnRH-a, followed by OC pills, GnRH-a, and E2 (Table 2, Fig. 1). This trend is most likely due to our current clinical protocols in which decisions regarding the selection of stimulation protocol are often based largely on age and baseline AFC. We often use what are thought to be the least suppressive medications in older patients with low baseline AFC. This pattern of practice is best reflective by the low mean age and high baseline AFC in patients treated with OC pills/GnRH-a, in comparison with high mean age and low 693
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FIGURE 2
Age, baseline antral follicle count (AFC), and hormones independently affect the decrease in AFC after pituitary suppression. These plots demonstrate the theoretical predicted decrease in AFC after pituitary suppression in women between 30 and 49 years old taking E2 (estradiol), GnRH-a (GnRH agonist), OC pills (OCP), or OCP/GnRH-a. Negative value of Y-axis demonstrates the decrease in AFC after pituitary suppression factoring age and baseline AFC. (A) Theoretical model for patients with AFC %10. (B) Theoretical model for patients with AFC R11. Tran. Impact of pituitary suppression on AFC. Fertil Steril 2016.
baseline in AFC in patients treated with E2. As demonstrated, the types of hormones contributed to the decline in AFC after pituitary suppression, confirming our clinical observations. 694
Nonetheless, baseline and suppressed AFC were found to be good predictors of the number of oocytes retrieved after COH by Pearson correlation analyses and ANCOVA models. VOL. 105 NO. 3 / MARCH 2016
Fertility and Sterility® Although suppressed AFC was a better fit based on ANCOVA models using AIC criteria, the absolute difference in AIC between baseline AFC (AIC ¼ 9,868) and suppressed AFC (AIC ¼ 9,813) was marginal at 55 indicating that although shortterm pituitary suppression may alter the biological capacity of the ovary during stimulation, the clinical significance of this suppression remains to be investigated. The strengths of our study include the large sample size (1,479 patients), broad age range (18–47 years old), characterization of ovarian reserve before and after suppression, and the number of oocytes retrieved after COH. Importantly, the baseline AFC was performed within 6 months before pituitary suppression, when patients were not taking any medications known to affect the hypothalamus-pituitary-ovarian axis, to assure minimal changes in AFC with time (32, 33). A weakness of this study is the retrospective design with a known practice bias of preferentially treating older patients with low AFC with either E2 or GnRH-a alone, and treating younger patients with higher AFC with OC pills or OC pills/ GnRH-a, which we addressed by controlling for age and baseline AFC with the ANCOVA models. In addition, as our clinic does not routinely perform COH without pituitary suppression, this only allows us to compare the change in AFC after pituitary suppression relative to different hormonal regimens rather than to no pituitary suppression. Similarly, we could only compare the number of oocytes recovered after pituitary suppression relative to different regimens and not to without suppression. However, we purposefully designed the time between baseline and suppressed AFC measurement to be within 6 months, as previous studies had demonstrated no changes in AFC during this time span (32, 33). The present data demonstrated that AFC declined during short-term pituitary suppression primarily due to patient age, baseline AFC, and type of hormones used. Furthermore, this decline in AFC appears to negatively impact the number of oocytes retrieved after COH. Although this decline in AFC after suppression was significant in comparison with the baseline AFC, baseline and suppressed AFC continue to be good predictors of the number of oocytes retrieved. However, based on ANCOVA modeling that suppressed AFC is marginally a better fit in predicting the number of oocytes retrieved after ovarian stimulation, we suggest using suppressed AFC as a main predictor of the number of oocytes retrieved, whereas baseline AFC is an acceptable alternative.
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