Ovarian reserve in women who remain premenopausal after chemotherapy for early stage breast cancer

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1 OVARY Ovarian reserve in women who remain premenopausal after chemotherapy for early stage breast cancer Ann H. Partridge, M.D., M.P.H., a,b Kathryn J. Ruddy, M.D., a,b Shari Gelber, M.S., a Lidia Schapira, M.D., c Mary Abusief, M.D., b Meghan Meyer, B.S., a and Elizabeth Ginsburg, M.D. a,b a Dana-Farber Cancer Institute; b Brigham and Women s Hospital; and c Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts Objective: To compare markers of ovarian reserve between women exposed to cytotoxic chemotherapy for early stage breast cancer and matched controls. Design: Cross-sectional evaluation of markers of ovarian reserve. Setting: Dana-Farber/Brigham and Women s Cancer Center, Massachusetts General Hospital, and Faulkner Hospital in Boston, MA. Patient(s): Breast cancer survivors with continued menses after chemotherapy were compared with age-matched, gravidity-matched controls. Main Outcome Measure(s): Antral follicle count (AFC), anti-m ullerian hormone (AMH), FSH, inhibin B (InB), and E 2 on day 2, 3, or 4 of the menstrual cycle. A Bonferroni correction was performed to correct for multiple comparisons. Result(s): Twenty survivors and 20 controls were evaluated; 50% of survivors were currently on tamoxifen. Median AFC was 6 for survivors and 9.5 for controls. There were significant differences between the two groups in AFC, AMH, and nonsignificant differences in FSH and InB, all indicating better ovarian reserve in controls. The AFC and AMH levels were highly correlated (r ¼ 0.72). Survivors on tamoxifen had lower AFC, AMH, InB, and higher E 2 than nontamoxifen treated survivors. Conclusion(s): Premenopausal breast cancer survivors have diminished ovarian reserve compared with controls. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Ovarian reserve, breast cancer, chemotherapy, premenopausal, fertility Breast cancer chemotherapy is associated with ovarian toxicity, which may diminish a woman s future fertility. Many young women with breast cancer are interested in having future biologic children and understanding their reproductive potential after treatment. Yet, at present, most research has used continued menses as an indicator of ovarian function, although menstruation is a poor surrogate for fertility, especially as women age. The increased incidence of amenorrhea after treatment is associated with older age at treatment and type of chemotherapy, with increased cumulative dose of alkylating agents being particularly ovarian toxic (1). A substantial proportion of young women do remain premenopausal after modern adjuvant chemotherapy for early stage breast cancer (2). But there are sparse data on actual fertility and pregnancy outcomes after chemotherapy in breast cancer Received November 26, 2008; revised March 6, 2009; accepted March 10, 2009; published online May 5, A.H.P. has nothing to disclose. K.J.R. has nothing to disclose. S.G. has nothing to disclose. L.S. has nothing to disclose. M.A. has nothing to disclose. M.M. has nothing to disclose. E.G. has nothing to disclose. Reprint requests: Ann H. Partridge, M.D., M.P.H., Dana-Farber Cancer Institute, 44 Binney St., Boston, MA (FAX: ; ahpartridge@partners.org). survivors. Even in the absence of rigorously defined ovarian failure, individuals may experience varying degrees of shortterm and long-term ovarian dysfunction after chemotherapy, presumably due to destruction of ovarian follicles or a natural decrease in ovarian function over the time required to take hormonal therapy. Measurement of ovarian reserve in breast cancer survivors may increase understanding of a woman s reproductive potential after cancer chemotherapy. Accurate identification of patients who are at risk for infertility or poor response to fertility treatments can help physicians to individualize counseling, and can help patients to understand their chances of achieving a pregnancy (3, 4). Reproductive potential is generally related both to the quantity and quality of ovarian primordial follicles. As women age, this pool diminishes, and rates of pregnancies decrease. However, in menstruating women, age alone is of limited value in predicting a successful pregnancy (3). Therefore, several other markers of ovarian reserve have been evaluated including early follicular phase serum E 2, FSH, anti-m ullerian hormone (AMH), and inhibin B (InB) levels, as well as measurements of antral follicle count (AFC) and ovarian volume. High E 2, high FSH, low InB, low AMH, and diminished AFC and ovarian volume 638 Fertility and Sterility â Vol. 94, No. 2, July /$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 in the early follicular phase of the ovulatory cycle have all been associated with poor response to IVF (3, 5 14). Although these measures have been evaluated most thoroughly in the setting of IVF programs, they may also have a role in determining ovarian reserve in breast cancer survivors interested in subsequent reproductive potential. However, it is unknown how these measures correlate with fertility in cancer survivors who are not undergoing ovarian stimulation and who do not have a history of infertility. Previous small studies have suggested diminished ovarian reserve in pediatric cancer survivors and adult survivor populations (15 20). We sought to evaluate measures of ovarian reserve in breast cancer survivors with continued menses after adjuvant chemotherapy compared with age-matched, gravidity-matched premenopausal controls in an effort to determine the impact of a history of chemotherapy on fertility potential. MATERIALS AND METHODS Data Collection We conducted a cross-sectional evaluation of ovarian reserve in women with a history of early breast cancer who remained premenopausal after cytotoxic chemotherapy and healthy age-matched (paired within 18 months), gravidity-matched (paired by 0 vs. R1 pregnancy) controls. Survivors were recruited from the patient population of the Breast Oncology Centers at the Dana-Farber/Brigham and Women s Cancer Center, Massachusetts General Hospital, and Faulkner Hospital in Boston, MA. All survivors were required to be R1 year from diagnosis, without evidence of recurrence, and to have had a menstrual period within 6 months of enrollment. Controls were recruited by word of mouth and using advertisements in print and online. The study received local Institutional Review Board (IRB) approval and participants signed informed consent before enrollment. All participants had blood drawn on day 2, 3, or 4 of their menstrual cycle. Serum was then stored and batched for assessment of AMH, FSH, InB, and E 2 at a standardized research laboratory. The AMH level was measured by a twosite ELISA (Diagnostic Systems Laboratory, Beckman-Coulter, Webster, TX). The minimum reportable concentration of this test is 0.03 ng/ml. Testing is monitored using quality control sera (two levels); the intra-assay coefficient of variation (CV) is <6% and the interassay CV is <12%. The FSH level was measured using a two-step, double monoclonal immunoassay using chemiluminescent microparticle immunoassay technology. The testing was done on a fully automated immunoanalyzer, the ARCHITECT (Abbott Diagnostics, Chicago, IL). The FSH levels are expressed in units of the first international standard preparation (World Health Organization 92/510). The limit of detection of the assay is 0.5 miu/ml. Testing is monitored using quality control sera. The interassay CVs are <5% for quality control sera (n ¼ 3) containing 5 75 miu/ml. Serum E 2 was also measured by ARCHITECT automated immunoassay. The minimum reportable concentration of this test is 10 pg/ml. The method has been standardized and calibrated against liquid chromatography/tandem mass spectrometry. Testing is monitored using quality control sera. The interassay CVs are 9.6%, 3.9%, and 2.0% for quality control sera containing 36, 184, and 378 pg/l, respectively. The InB assays were performed with a solid-phase sandwich ELISA (Serotec, Oxford, United Kingdom) based on the use of plates coated with a monoclonal antibody specific for the InB b-subunit with a second monoclonal antibody specific for the a-subunit for detection. The sensitivity of the assay is 7.0 pg/ml. The assay is controlled in triplicate using samples with mean concentrations of 155.3, 316.3, and pg/ml, with interassay CVs of 11.6%, 7.6%, and 9.7%, respectively. Participants also underwent transvaginal ultrasound (TVS) at Brigham and Women s Hospital with antral ovarian follicle count and ovarian volume measurement. Antral follicle counts were performed by two reproductive endocrinologists. Standardization of caliper placement for follicle measurement was confirmed before the start of the study, and was consistent with clinical AFC measurements used for standard follicle monitoring for fertility patients. Ovaries were scanned cephalad to caudad in the coronal plane, to ensure complete visualization of the ovary, and avoid missing follicles. Color Doppler was used on peripheral follicles to ensure that they were not vascular structures. The crosshairs of calipers were placed on the middle of the echogenic rim surrounding each follicle. Each patient was scanned by only one examiner, as is done in standard clinical practice, therefore interassay CVs cannot be calculated. The antral follicles were measured in the larger diameter per standard practice. For breast cancer survivors only, a medical chart review was performed upon enrollment to obtain details of the medical history including treatment received. For the analysis presented here, only controls who were age- and graviditymatched to the 20 survivors were included. Statistical Analyses The primary objectives of this study were to estimate ovarian reserve in premenopausal breast cancer survivors using AFC as the primary measure and to compare the survivors with age- and gravidity-matched healthy controls. In other studies AFC has been found to reflect fertility potential in women without a history of cancer (21, 22). The study was initially designed to include 39 women per group to detect a mean difference of 2.2 follicles with an 80% power and a two-sided a of These calculations were based on a pooled estimate of the standard deviation (3.44) of the AFC from the two subsets reported on by Bath et al. (15). However, a preliminary evaluation of 17 survivors revealed that the mean AFC was substantially lower than that assumed when initially defining the sample size. Therefore, an interim analysis comparing the mean AFC of the groups was planned after 20 women were recruited to each group. The Wilcoxon sign rank test was used to compare AFC and AMH, E 2, FSH, and InB serum values for the two groups. To Fertility and Sterility â 639

3 TABLE 1 Characteristics of survivors and controls. Survivors (n [ 20) Controls (n [ 20) Total (n [ 40) P value a Age (y), mean (range) 36.8 (31 42) 36.9 (31 42) 36.8 (31 42) Race, n (%).14 White 16 (80) 10 (50) 26 (65.0) Black 1 (5) 4 (20) 5 (12.5) Asian 2 (10) 1 (5) 3 (7.5) Hispanic 0 4 (20) 4 (10.0) Other Latina 0 1 (5) 1 (2.5) Other West Indian 1 (5) 0 1 (2.5) Education, n (%).10 Post-college graduate 11 (55) 4 (20) 15 (37.5) College graduate 6 (30) 8 (40) 14 (35) Some college 1 (5) 5 (25) 6 (15.0) Tech/vocational 0 2 (10) 2 (5.0) High school graduate 2 (10) 1 (5) 3 (7.5) Pregnancies, n (%) (35) 8 (40) 15 (37.5) 1 5 (25) 6 (30) 11 (27.5) 2 6 (30) 3 (15) 9 (22.5) 3 1 (5) 0 1 (2.5) 4 1 (5) 2 (10) 3 (7.5) (5) 1 (2.5) Marital status, n (%).01 Divorced/separated 1 (5) 1 (5) 2 (5) Married 13 (65) 5 (25) 18 (45) Lives as married 2 (10) 0 2 (5) Never married 4 (20) 14 (70) 18 (45) Menstrual frequency, n (%).11 Once per mo 16 (80) 20 (100) 36 (90.0) Every 2 6 mo 4 (20) 0 4 (10) a Fisher s exact test P values. identify variables that distinguished the controls from survivors, conditional logistic regression based on the matched pairs was performed. Linear and logistic regression models were used to evaluate whether any of the serum values predicted AFC. The relationship between the AFC and the serum values was further explored using Spearman correlation. To correct for the multiple comparisons (Bonferroni), P values less than.01 were considered statistically significant. RESULTS At the interim analysis of 20 survivors compared with 20 controls, the criteria for stopping the trial was met, prompting cessation of recruitment. Data were analyzed for these ageand gravidity-matched pairs. Table 1 presents the demographics of the survivors and controls and Table 2 displays the disease characteristics and treatments of the survivors. One pair was mismatched on gravidity (a participant who initially reported no pregnancies eventually reported two previous abortions). Half of controls and 80% of survivors were white. The majority of both groups were college graduates (some with post-college education). Seventy-five percent of survivors were married or living as if married with a significant other, whereas 70% of controls were never married. Seventy-five percent of survivors and 100% of controls were menstruating once per month. Table 2 presents survivors disease and treatment characteristics. Most of the survivors had been diagnosed with stage II disease (55%), whereas 25% had stage I and 20% had stage III. Forty percent of survivors were less than 3 years from diagnosis, 35% were 3 to less than 6 years from diagnosis, and 25% were between 6 and 10 years from diagnosis. Most (90%) of survivors had no comorbidities and almost half (45%) had undergone mastectomy. Forty-five percent had received doxorubicin and cyclophosphamide alone, and an equal number had received paclitaxel in addition 640 Partridge et al. Ovarian reserve after chemotherapy Vol. 94, No. 2, July 2010

4 TABLE 2 Survivors disease and treatment characteristics. Disease and treatment characteristics No. (%) Time from diagnosis 6 mo 1 y 2 (10) 1 2 y 6 (30) 3 5 y 7 (35) 6 10 y 5 (25) Stage I 5 (25) II 11 (55) III 4 (20) Comorbidities None 18 (90) Preeclampsia PIH 1 (5) Lymphedema 1 (5) Surgery Mastectomy 9 (45) Less than mastectomy 11 (55) Radiotherapy Yes 14 (70) No 6 (30) Chemotherapy regimen CA 9 (45) CA -> T 1 (5) CA -> T dose dense 8 (40) High dose A -> CMF 1 (5) CAF 1 (5) Temporary ovarian suppression Yes 2 (10) No 18 (90) Current tamoxifen use Yes 10 (50) No 10 (50) Steps taken to avoid infertility Cryopreservation of oocytes 1 (5) Cryopreservation of embryos 1 (5) GnRH agonist 4 (20) Not sure 1 (5) None 13 (65) Did chemotherapy interrupt menses? Yes 16 (80) No 4 (20) Note: CA ¼ Cyclophosphamide and adriamycin; T ¼ paclitaxel; A ¼ adriamycin; CMF ¼ Cyclophosphamide methotrexate 5-fluorouracil; CAF ¼ Cyclophosphamide, adriamycin, 5-fluorouracil. (40% received doxorubicin and cyclophosphamide and then paclitaxel every 2 weeks with growth factor support and 5% received this chemotherapy regimen every 3 weeks). Eighty percent reported that menses had ceased temporarily during chemotherapy. Half were taking tamoxifen at the time of the study. Thirty percent reported that they had taken steps to protect fertility. One patient (5%) had preserved embryos, one (5%) had preserved oocytes, and four patients (20%) had used GnRH agonists to attempt to retain fertility after treatment. Measures of Ovarian Reserve Several measures of ovarian reserve distinguished survivors from controls. Median AFC was higher in controls than in survivors (9.50 vs. 6.00, P¼.004). Controls also had higher median InB levels (33.09 vs , P¼.02) and higher median AMH values (1.35 vs. 0.43, P¼.0004). In contrast, median FSH was lower in controls (6.90 vs. 9.00, P¼.02), as was median E 2 (38.00 vs , P¼.14) (Table 3). The outlier values for E 2 did not occur in the same patients as the outlier values for the FSH levels. In univariate logistic analyses, AMH, AFC, FSH, and InB levels were all found to be significantly different between survivors and controls. The AMH levels distinguished survivors from controls with the greatest statistical significance (odds ratio 7.63) in a multiple logistic regression model. Addition of the other factors did not significantly improve the distinction between survivors and controls in this model. The AFC and AMH levels, which both reflect the remaining pool of immature oocytes, were highly correlated (Spearman r ¼ 0.72, P<.0001). Survivors on tamoxifen had lower AFC, InB, AMH, and higher E 2 levels than nontamoxifen treated survivors, but the small size of our sample precluded formal analysis (Table 4). DISCUSSION Many young breast cancer survivors are concerned about their fertility potential and contraceptive needs. The experience or even threat of infertility may have significant psychosocial and medical implications for young breast cancer survivors. Data regarding fertility outcomes in young breast cancer survivors are limited. In women undergoing IVF, measures of ovarian reserve appear to be useful in predicting the likelihood that a woman can become pregnant. The AFC level is thought to be the most accurate reflection of ovarian reserve, although there has been interest in serum markers such as AMH, FSH, InB, and E 2 (18, 23). This study confirms that young breast cancer survivors who have undergone cytotoxic chemotherapy and remain premenopausal have diminished AFC levels when compared with healthy controls. Our study further reveals that a lower level of AMH appears to be the best serum predictor of diminished AFC, which is thought to reflect reduced likelihood of future pregnancy. Because AMH is a serologic test that does not fluctuate with the menstrual cycle, assessment of AMH may be a more practical factor for the measurement of fertility potential than AFC, which requires TVS. Fertility and Sterility â 641

5 TABLE 3 Differences in measures of ovarian reserve in controls compared with survivors. No. Mean Median Minimum Maximum P value a AFC b Controls Survivors AMH c Controls Survivors < FSH c Controls Survivors InB c Controls Survivors c E 2 Controls Survivors Note: AFC ¼ antral follicle count; AMH ¼ anti-m ullerian hormone; InB ¼ inhibin B. a Wilcoxon signed rank test P values. b Values rounded to the nearest whole number. c Values rounded to the nearest tenth. Our findings confirm previous work revealing that tamoxifen alters hormonal levels in premenopausal women, complicating assessment of fertility. Although the small sample size precluded formal analysis, the apparent trend toward lower AFC, InB, and AMH, as well as higher E 2 levels, in the survivors taking tamoxifen compared with the survivors not taking tamoxifen was consistent with previous findings of the endocrine impact of tamoxifen. However, unlike in TABLE 4 Serum values and AFC by current tamoxifen use. No. Mean Median Minimum Maximum AFC a Current tamoxifen use No current tamoxifen use AMH b Current tamoxifen use < No current tamoxifen use < FSH b Current tamoxifen use No current tamoxifen use InB b Current tamoxifen use No current tamoxifen use b E 2 Current tamoxifen use No current tamoxifen use Note: AFC ¼ antral follicle count; AMH ¼ anti-m ullerian hormone; InB ¼ inhibin B. a Values rounded to the nearest whole number. b Values rounded to the nearest tenth. 642 Partridge et al. Ovarian reserve after chemotherapy Vol. 94, No. 2, July 2010

6 some prior research, there was no increase in FSH level observed in participants on tamoxifen in our study. Several small studies have shown that tamoxifen-treated women tend to have normal-to-high FSH, with supraphysiologic E 2 levels, from one to eight times normal (24 28). Additional research is needed to determine how hormone levels and AFC during tamoxifen therapy relate to levels before and after tamoxifen therapy. This study adds to previous evidence that ovarian function may be impaired in women who remain premenopausal after breast cancer chemotherapy (17, 18, 29). Oktay et al. (18) reported that AMH levels declined after each course of chemotherapy for three breast cancer patients. Likewise, Anderson et al. (17) reported that breast cancer chemotherapy induced rapid deceases in AMH, InB, and AFC levels in 50 premenopausal women, without a change in E 2 level. Two previous studies compared measures of ovarian reserve in cancer survivors with controls, but there was a wide array of cancers treated and ages included, and controls were not as well matched. Still, our results are consistent with a study that found that a group of survivors of a variety of childhood cancers differed in FSH and AMH levels from controls (15). Evaluating measures of ovarian reserve in 10 premenopausal pediatric cancer survivors and 11 controls, that study reported that serum FSH levels were significantly higher ( vs IU/L; P¼.02), and ovarian volumes were significantly lower ( vs ml, P<.05) in cancer survivors. Among women receiving combined oral contraceptive (OC) pills, AFC was significantly lower in the pediatric cancer survivors than in controls ( vs ; P¼.02). However, those controls were not matched by age or gravidity, regimens received by those patients were dissimilar from breast cancer regimens, and the time since chemotherapy was much longer for those survivors of pediatric cancers than for the breast cancer patients in the current study. In a more invasive study sampling, ovarian tissue at the time of cryopreservation for fertility preservation in 26 women preparing for chemotherapy, 10 patients who had already received chemotherapy in the past (mostly for hematologic malignancies) were found to have lower follicle counts. Only seven of the participating patients had breast cancer (29). Our research is most concordant in design and in results with a study in which AFC, FSH, AMH, and InB were found to differ between 22 menstruating breast cancer patients and 24 age-matched controls (16). After chemotherapy, the patients were found to have lower AFC, higher FSH (11.32 vs. 6.62, P <.001), lower AMH (0.95 vs. 7.89, P <.0001), and lower InB levels (19.24 vs , P <.0001) (16).However, that study did not match on gravidity (all controls but the minority of patients had at least one previous childbirth). Therefore, patients may have had lower ovarian function even before chemotherapy due to the requirement that all included controls have proven fertility. Furthermore, the ovarian reserve testing was done much earlier after chemotherapy in that study. Because our study required that all survivors be at least 1 year out from the diagnosis of breast cancer, our results more strongly suggest permanent changes rather than temporary alterations in hormonal patterns and ovarian follicle count. Fertility is an area of great concern for the many young women diagnosed with breast cancer each year. Our findings may also have implications for survivors of other cancers who remain premenopausal after chemotherapy. However, conclusions from this study are limited by its small sample size, as well as by the multiple comparisons that were performed in this statistical analysis. Despite efforts to match controls to survivors on gravidity and age, there were differences between the groups, most notably by race, and we cannot be sure that the groups did not differ in ovarian reserve even before chemotherapy. Furthermore, there are no pregnancy outcomes recorded in this cross-sectional study. Nevertheless, these findings provide important preliminary data needed for future research. The ability to predict a woman s reproductive potential would be of considerable value for many young breast cancer survivors for their treatment decision-making, family planning, and contraception concerns in follow-up. 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