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Repetitive oocyte donation does not decrease serum anti-Müllerian hormone levels
Repetitive oocyte donation does not decrease serum anti-M€ullerian hormone levels Orhan Bukulmez, M.D.,a Qin Li, M.S.,b Bruce R. Carr, M.D.,c Benjamin Leader, M.D., Ph.D.,d Kathleen M. Doody, M.D.,e and Kevin J. Doody, M.D.c,e a
Departments of Obstetrics and Gynecology; b Epidemiology & Health Policy Research, University of Florida College of Medicine, Gainesville, Florida; c Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas; d ReproSource, Bedford, Massachusetts; and e Center for Assisted Reproduction, Bedford, Texas
Objective: To determine if the anti-M€ullerian hormone (AMH), a proposed marker of ovarian aging, decreases with repetitive oocyte donation. Design: Retrospective cohort. Setting: Academic. Patient(s): Thirty-six young women who underwent three to seven oocyte donation cycles. Intervention(s): Assessor blind determination of AMH levels from serum samples collected during each treatment cycle. Main Outcome Measure(s): Cycle trends of serum AMH levels. Result(s): The AMH was the only predictor of oocyte yield in the first cycles. The AMH was negatively associated with donor age and follicle stimulating hormone (FSH) dose used. Serum AMH levels did not show any decrease per treatment cycle basis and per maximum number of oocyte donation cycles performed per woman. Whereas donors who underwent six cycles showed increasing AMH levels when controlled for studied covariates, the slopes of the multiple regression curves were not significantly different from donors who underwent three, four, and five cycles. Clinical outcome assessed by FSH dose/number of oocytes ratio did not show significant change over repetitive cycles. Intercycle variation of AMH in all patients over three cycles was found to be 12.5%, which was within the reported intermenstrual range. Conclusion(s): Serum AMH levels do not decrease over repetitive oocyte donation cycles, which may imply that accelerated ovarian aging may not occur in oocyte donors. (Fertil Steril 2010;94:905–12. 2010 by American Society for Reproductive Medicine.) Key Words: AMH, anti-mullerian hormone, oocyte donation, ovarian aging
The anti-M€ ullerian hormone (AMH) in females is produced in the granulosa cells from preantral and small antral follicles (1). Because AMH is solely produced in the growing ovarian follicles, the serum levels may be used as a marker of ovarian reserve (2). The higher early follicular phase serum AMH levels is associated with greater number of retrieved oocytes in in vitro fertilization (IVF) (3). The AMH has been reported to be the earliest marker to decrease with aging (4). AMH levels are unaffected by exogenous sex steroids used for contraception whether administered orally or vaginally (5). Because AMH shows modest intercycle and intracycle variability throughout the menstrual cycle, and exogenous Received January 21, 2009; revised May 6, 2009; accepted May 7, 2009; published online July 23, 2009. O.B. had nothing to disclose. Q.L. has nothing to disclose. B.R.C. has nothing to disclose. B.L. has nothing to disclose. K.M.D. has nothing to disclose. K.J.D. has nothing to disclose. Supported in part by UPN 07120257 from the University of Florida College of Medicine (Dr. Bukulmez). Presented in part at the American Society for Reproductive Medicine 64th Annual Meeting, November 8–12, 2008, San Francisco, CA. Reprint requests: Orhan Bukulmez, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0294 (FAX: 352-392-9724; E-mail: obukulmez@ ufl.edu).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.05.017
follicle stimulating hormone (FSH) administration does not affect its levels (6), it may be informative about the ovarian reserve even if it is randomly obtained. Anonymous oocyte donors are usually young women with excellent ovarian reserve, and they may undergo repetitive oocyte donation cycles. Whereas it has been reported that repetitive controlled ovarian hyperstimulation (COH) and oocyte harvesting, in the short term, may not affect ovarian response and oocyte yield in these young females (7), it is not known if repetitive procedures result in accelerated ovarian aging in the long term. It may be possible that the repeated procedure itself can lead to a subtle decline in ovarian reserve independent of chronologic aging without affecting shortterm clinical outcome, which may be assessed by determination of AMH levels. The primary aim of this study was to reveal if serum AMH levels significantly decreased by each oocyte donation cycle performed, which might suggest a potential detrimental affect of repetitive oocyte donation on ovarian aging. To assess the utility of AMH and to confirm prior reports on AMH as an ovarian reserve marker, we also investigated if AMH was associated with oocyte yield or clinical pregnancy in our cohort. Furthermore, we analyzed intercycle variation of serum AMH.
Fertility and Sterility Vol. 94, No. 3, August 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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MATERIALS AND METHODS Patients and Design Thirty-six women who had undergone 167 repetitive oocyte donation cycles were included. This study was determined to be exempt from review by the institutional review board of The University of Texas Southwestern Medical Center. The AMH levels were measured in an assessor-blind fashion from stored serum samples collected during each COH cycle on the day before commencing FSH stimulation. Hence, the first cycle serum samples of each patient reflected their baseline serum AMH levels before any stimulation or oocyte retrieval was performed. All donors were pretreated by combined oral contraceptive pills followed by daily recombinant FSH commenced 5 days after the last dose of the pill, with a gonadotropin releasing hormone antagonist protocol as published elsewhere (8). Then, per routine, their oocytes were retrieved and fertilized in an IVF laboratory (9) and the best two of the blastocyst stage embryos were transferred to the recipients. The recipient women were women with premature ovarian failure or diminished ovarian reserve aged between 29 and 45 years. All oocyte donors were between the ages of 19 and 30 years, and complied with the Food and Drug Administration criteria for the oocyte donation. Only the donors with three or more repetitive treatment cycles resulting in embryo transfers, and those having adequate frozen serum samples to determine AMH levels were included. The number of cycles for a given donor was attempted to be limited to six (10). Only one patient underwent seven cycles because of a special circumstance. Recipients needing menstrual suppression were down-regulated by daily leuprolide acetate, and they received oral micronized estradiol in incremental doses to develop endometrium. The day after the donor’s trigger for final oocyte maturation by human chorionic gonadotropin, the recipients were started with progesterone in oil intramuscularly to synchronize endometrium for embryo transfer. We assessed whether there was any significant decline in the serum AMH levels over repetitive treatment cycles in oocyte donors. The following parameters of ovarian response in each treatment cycle were also analyzed: [1] total dose of FSH (IU) used per cycle (FSH dose), [2] estradiol (E2) concentration on the day of hCG administration (peak E2), [3] number of oocytes retrieved per cycle (number of oocytes), [4] donor age at the time of each treatment cycle and time interval between treatment cycles calculated from this parameter. In addition, FSH dose (IU)/number of oocytes ratio for each cycle was calculated to be used as one of the measures of COH or clinical outcome (11). Clinical pregnancy was defined by the presence of at least one gestational sac with positive fetal heart tones detected after 6 weeks of gestation in recipients.
Determination of Serum AMH Levels The serum samples kept at 20 C were shipped to the blinded reference laboratory (Repromedix, Woburn, MA). 906
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Serum AMH levels were determined using an enzymatically amplified two-site immunoassay (MIS/AMH ELISA, DSL10-14400, Diagnostic Systems Laboratories, Webster, TX). The intra- and interassay coefficients of variation were between 3% and 7%, with an average of 5%. The sensitivity of the assay was 0.017 ng/mL. Statistical Analysis SAS software (version 9.1, Cary, NC) was used unless stated otherwise. The AMH measurements were averaged over the assayed aliquots. Two-tailed significance level was P<.05. Analysis per oocyte donation cycle Initially, only the first cycles of 36 oocyte donors were analyzed to assess if baseline AMH levels predicted the number of oocytes retrieved. Multiple regression analysis of the number of oocytes retrieved on baseline AMH, donor age, FSH dose, peak E2, and clinical pregnancy outcome variables was performed. Logarithmic (log) transformation of the number of oocytes retrieved was used to satisfy Gaussian distribution. To compare each cycle specific variable over each repetitive cycle, repeated measure analysis with unstructured covariance structure was used including all patients. Log transformations were performed as needed. The time intervals between cycles in years were calculated by subtracting the ages of the donors, at the commencement of prior cycle from their ages at the start of the following cycle. Inverse transformation of these intervals was needed to run a repeated-measure analysis. To assess if serum AMH levels change over repetitive treatment cycles, a repeated-measure analysis of AMH levels was conducted to test for cycle trends while controlling for other covariates with an unstructured covariance matrix. The model treated the baseline (first cycle) AMH as covariate and treated the orderly additions of donor age, FSH dose, number of oocytes, peak E2, and clinical pregnancy as repeated covariates. An added-in-order (Type I) test was used for these variables. The square root of the AMH levels was performed. Six cycles including the first cycle with the baseline AMH level were used in the analysis. The quadratic and linear trends of the cycles were tested. The quadratic term was dropped because of its nonsignificance. A repeated measure with an unstructured covariance matrix (generalized estimated equations, GEE) was used to assess if AMH levels were associated with clinical pregnancy or nonpregnancy. Analysis according to maximum number of oocyte donation cycles performed per woman Because the patients who underwent three repetitive cycles (group 3) might be different than those who completed four, five, and six cycles (groups 4, 5, and 6, respectively), a repeated analysis of AMH levels was conducted to test for cycle trends by group allocation with unstructured covariance matrix. This model treated the baseline (first cycle) AMH as a covariate and treated the orderly additions of age, total FSH dose, number of oocytes, Vol. 94, No. 3, August 2010
peak E2 level, and clinical pregnancy as repeated covariates. An added-in-order (Type I) test was used for these variables. A square root of the AMH levels was necessary. Six cycles (first cycle includes baseline AMH level) were used for the analysis. The COH outcome was assessed, both per cycle and group basis, by a repeated-measure analysis of FSH dose/number of oocytes ratio, which was tested for cycle trends with an unstructured covariance matrix. The addition order of covariates was donor age, peak E2, and clinical pregnancy outcome, each of which was controlled for FSH dose/number of the oocytes ratio. Here, residual analysis was conducted to satisfy Gaussian distribution. Intraclass correlation (ICC) The ICC coefficient and its 95% confidence interval (CI) was calculated for first three cycles, which included all 36 patients and also for each oocyte donor group, to assess the intercycle variation of AMH levels (SPSS version 11, Chicago, IL). Then intravariation coefficients (¼1 ICC) were calculated. Pairwise ICC comparisons were performed among groups 3, 4, 5, and 6 by using a procedure based on Fisher’s transformation (12). RESULTS Cumulative clinical pregnancy rate per treatment cycle in the whole cohort was 53%. Mean (SD) values and their comparisons for all parameters studied per cycle basis are shown in Table 1. The time interval between cycles averaged 0.40 years (4.8 months), ranging between 0.1 and 2.2 years. The mean time intervals between each cycle were not significantly different from each other (Table 1). Median (range) AMH level of all cycles was 2.48 ng/mL (0.27–17.65). Of the analyzed cycles (n ¼ 166), the conventional IVF was done in 86 cycles (51.8%) and intracytoplasmic sperm injection (ICSI) was performed in 80 cycles (48.2%). Because there might be discrepancies between the assessment of oocyte maturity for IVF and ICSI, and the number of oocytes retrieved was used in the assessment of primary clinical outcome rather than the number of mature oocytes. Cumulative fertilization rates for IVF and ICSI were 62% 19% and 74% 16%, respectively, as expected. The AMH Predicts Number of Oocytes Retrieved When only the first cycles (n ¼ 36) were analyzed with multiple regression, AMH was found to be the only significant parameter (P¼.0035, parameter estimate .08) in prediction of total number of oocytes retrieved. Donor age, FSH dose, peak E2 and clinical pregnancy outcome were not found to be significant (Table 1 for parameter means). Advancing age of donors over repetitive cycles is not associated with a decrease in AMH levels In a repeated measure without controlling for other variables, as the number of cycles increased, donor age significantly advanced, as expected (Table 1). However, as the number Fertility and Sterility
of cycles increased, FSH dose, peak E2, number of oocytes, and AMH significantly increased but FSH dose/number of oocytes ratios stayed comparable (Table 1). There was no significant association between the number of repetitive oocyte donation cycles performed and the clinical pregnancy outcome (P¼.17, Table 1 for clinical pregnancy rates). The AMH levels do not decrease by repetitive cycles of oocyte donation Initial per cycle analysis demonstrated that the linear trend of the cycle was significantly associated with AMH levels obtained after the first cycle (P¼.035) when controlling for previous added variables (Table 2). Whereas baseline (first cycle) AMH, donor age, the number of oocytes, and FSH dose were significantly associated with AMH levels measured during repetitive cycles, peak E2 and the clinical pregnancy did not show any significant association with AMH. The positive coefficient estimates suggested that as the number of cycles increased the AMH levels increased (Table 2). This issue was further investigated for each donor group as detailed below. Although donor age and FSH dose showed significant negative association with AMH levels, number of oocytes retrieved showed positive association (Table 2). In donors with three, four, and five repetitive cycles (groups 3, 4, and 5), controlling for added covariates as mentioned in methods section, the cycle trends were not associated with AMH (P¼.93, P¼.20, and P¼.11, respectively, Fig. 1A–C). However, for donors who completed six repetitive cycles (group 6), the linear trend of cycle for AMH was found to be significant (P<.0001, estimate of coefficient ¼ .081, Fig. 1D). These results imply that groups 3, 4, or 5 did not show any significant change in AMH levels, whereas in group 6 AMH levels tended to increase by the number of cycles performed. We then tested if there were any significant differences among groups 3, 4, 5, and 6 in terms of AMH cycle trends (test for slope difference among groups). The linear trend of cycle was comparable among the groups (P¼.90). Therefore, comparing AMH trend of each group and cycle number did not show any significance (Fig. 1). The AMH does not predict clinical pregnancy Overall, AMH did not predict clinical pregnancy in recipients (GEE, P¼.90). The AMH shows nonsignificant and modest variation over repetitive treatment cycles Intravariation coefficients for donors who underwent three, four, five, and six repetitive cycles were 0.385 (95% CI 0.105–0.808), 0.267 (95% CI 0.08–0.548), 0.165 (95% CI 0.042–0.371), and 0.091 (95% CI 0.03–0.191), respectively. Whereas these results suggested that the group 6 with higher AMH levels (Fig. 2), may show lower variation in AMH levels than groups 3, 4, and 5, pairwise ICC comparisons among groups were not significant (P>.05 for all pair-wise comparisons) (12). The ICC coefficient of the AMH over 907
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TABLE 1 Distribution of nontransformed parameters per oocyte donation cycle performed. Parameter (mean ± SD)
Cycle1 (n [ 36)
Cycle 2 (n [ 36)
Cycle 3 (n [ 36)
Cycle 4 (n [ 28)
Cycle 5 (n [ 19)
Cycle 6 (n [ 11)
Cycle 7a (n [ 1) P valueb
Age (y) 24.74 3.38 25.17 3.31 25.57 3.34 25.92 3.29 26.22 3.08 25.16 2.82 24.34 2336.6 1275.2 2,789.2 1,597.6 2,510.1 1,142.1 2,574.6 787.0 2,773.4 1,635.3 2,882.1 1,191.6 3319.0 Peak E2 (pg/mL) FSH dose (IU) 1,947.9 713.7 2,114.6 758.7 2,162.5 869.8 2,325.0 849.0 2,208.3 944.9 2,072.7 774.5 1050.0 No. of oocytes 16.1 9.8 17.5 7.7 16.2 7.0 19.9 8.4 19.2 12.3 26.9 15.8 11 retrieved FSH dose/# 159.1105.3 157.9 117.9 161.3 92.9 146.3 101.7 147.4 92.4 112.0 89.0 95.5 oocytes AMH (ng/mL) 2.92 2.71 2.78 2.00 2.75 2.88 2.99 2.50 3.83 3.44 4.21 3.15 1.59 Time interval — 0.43 (0.09–2.51) 0.40 (0.10–1.95) 0.44 (0.10–2.11) 0.45 (0.13–2.22) 0.29 (0.15–0.96) 0.36* (mean y-range) Clinical pregnancy 22/36 (61.1%) 19/36 (52.8%) 19/36 (52.8%) 15/28 (53.6%) 8/18 (44.4%) 5/11 (45.5%) — rate a
Cycle 7 was not included in the analysis. Repeated measure for cycle trends was used to compare each log transformed variable without controlling for other covariates. c Inverse transformation was performed. NS ¼ not significant, P>.05. d Chi-square test. b
Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
< .0001 .005 .046 < .007 NS .006 .076c NSd
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TABLE 2 Repeated-measure multiple regression model and added in order (type I) test for serum AMH levels over repetitive oocyte donation cycles (n [ 166).
Variable Baseline AMH Donor age Number of oocytes FSH dose Peak E2 Pregnant—No versus Yes Cycle number
Estimate of coefficient Standard P (slope) error valuea 0.144 0.02 0.007 0.0001 0.00004 0.033 0.049
0.017 0.014 0.004
< .0001 .027 .013
0.00005 0.00003 0.057
.024 NS NS
0.021
.035
Note: AMH ¼ anti-Mullerian hormone. a The P-values suggest that the linear trend of the cycle was significant while controlling the previous variables added to the multiple regression model. As the number of cycles increased, AMH levels increased. Although donor age and FSH dose showed significant negative association with AMH levels, number of oocytes retrieved showed positive association. Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
the first three cycles was 0.875 (95% CI 0.929–0.795), which gives an intercycle variation of 0.125 (95% CI 0.071–0.205). Outcome of COH is comparable over repetitive oocyte donation cycles FSH dose/number of oocytes ratio analyzed both per cycle (P¼.07) and group basis was not significantly associated with the number of cycles performed. For groups 3, 4, 5, and 6, no significant trend of cycle was found after controlling for the studied covariates (P¼.33, .21, .20, .77, respectively). Whereas group 6 with higher AMH levels (Fig. 2) required a somewhat lower FSH dose/oocyte ratio, the comparisons of slopes between groups were not significant. DISCUSSION Our study found no decrease in AMH levels along with stable COH response in donors participating in as many as six oocyte donation cycles. Serum AMH levels in our population were associated with the number of oocytes retrieved, further confirming the reports on AMH from general infertility population (13). We also did not see any steady decline in serum AMH levels over the time frame of oocyte donation cycles. Although two earlier small longitudinal studies involving premenopausal women with somewhat more advanced mean ages than our population, have noted a steady decline Fertility and Sterility
of serum AMH levels over a period of 2.6 to 4 years (14, 15), a cross-sectional but larger study in subfertility population has reported that the mean AMH levels remain relatively stable between the ages of 18 to 29 (13). Although we are aware of the bias inherent to the retrospective properties of this report and limited sample size, we believe it still provides novel information regarding the trend of serum AMH levels over repetitive oocyte donation cycles. In addition, one recent systematic review on the role of AMH in prediction of IVF outcome has suggested that design of the study whether retrospective or prospective had no significant impact on the predictive performance of AMH (16). Over six repetitive cycles, baseline (first cycle) AMH, number of cycles, number of oocytes, donor age, and FSH dose were significantly associated with AMH levels obtained from repetitive cycles (Table 2). Negative coefficient estimates suggested that as donor age and FSH dose increased, AMH levels decreased confirming other studies (16). Whereas we found a significant trend that as the number of cycles increased. AMH levels also increased (Table 2). This was mainly applicable to group 6 (Figs. 1D and 2). Although this finding may be because of the limited sample size, it is possible that these donors underwent more cycles because their response were perceived to be better. Of note, because AMH levels were measured long after these cycles were completed, the management decisions of these donors were made without AMH information in hand. By all means, this finding also argues against any possible decline in AMH levels with oocyte donation. Whether or not AMH levels actually increase by repetitive COH in young females with higher AMH levels and its biologic plausibility need further elucidation. It is believed that repeated ovarian stimulation is unlikely to reduce the ovarian pool of primordial follicles given that these follicles do not possess receptors for FSH (17). However, FSH stimulation may hasten ovarian aging by increasing recruitment of small growing follicles, thereby accelerating the depletion of follicle reserve (18). A transgenic mouse model was developed that expressed pituitary-independent human FSH, which exhibit progressively increasing levels of circulating FSH independent to ovarian follicular feedback (19). In this model, increased basal FSH activity could advance the age-related onset of female infertility compared with wild type. Ovarian AMH mRNA expression and serum AMH levels were found to decrease at an earlier age in transgenic animals compared with the wild-type mice (19). Another alleged concern is possible ovarian damage with repeated ovarian puncture. It has been hypothesized that ovarian trauma induced by oocyte retrieval may diminish ovarian reserve by inducing the release of autoantigens (20, 21). This hypothesis has never been proven, and the potential long-term adverse effects of ovarian trauma have never been documented. The short-term effects of repetitive oocyte donation were assessed in 284 oocyte donors who had at least two repetitive 909
FIGURE 1 Slopes and the test of slope differences among groups 3 (A), 4 (B), 5 (C), and 6 (D) for AMH and cycle trends. Square root of AMH was used for statistics. Solid lines were obtained from repeated-measure multiple regression while controlling for baseline (first cycle) AMH, donor age at the time of each cycle, number of oocytes retrieved per cycle, FSH dose (IU) used per cycle, peak E2, and clinical pregnancy outcome in each cycle. Whereas AMH showed significant increase over repetitive cycles in group 6 (P< .0001), comparisons of the slopes between groups 3, 4, 5, and 6 were not significantly different from each other (P¼ .90). Therefore, the linear trend of the cycle was not significantly different among the groups.
Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
cycles of donation (7). The number and quality of oocytes were maintained, and there was no increase in the dose of gonadotropins over multiple cycles (7). Although this study seems to be reassuring for ovarian response to be unaffected with repetitive oocyte donation, the follow-up data of oocyte donors over a long period of time is lacking. AMH has been suggested to be an excellent marker for ovarian reserve not only in infertility patients but also in women during and after cancer treatment and in women who underwent uterine artery embolization (22–24). The AMH steadily decreases by advancing age (13, 25). Longitudinal studies have suggested that interval changes in conven910
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tional markers of ovarian reserve such as serum FSH, inhibin B and E2, or antral follicle count, do not remarkably change as AMH over the same time period (14, 15). Therefore, AMH has been demonstrated to be the earliest marker of ovarian aging (14, 15) and its levels may predict the age of menopause (26). Furthermore, the AMH levels do not show any significant variation throughout the menstrual cycle (27, 28). The AMH shows excellent correlation with the number of oocytes retrieved in IVF, and is a strong marker for poor COH response (29, 30). We showed an excellent association between serum AMH levels and ovarian response to FSH as reported before (3, 31). Vol. 94, No. 3, August 2010
FIGURE 2 Mean (SEM) of serum AMH levels over the mean time frame (years) of repetitive cycles according to the maximum number of oocyte donation cycles performed per woman (groups 3, 4, 5, and 6, as given by each line). Although groups showed various basal AMH levels, AMH levels do not decline over the time frame of oocyte donation cycles. The numbers of women in each group are given in parentheses.
Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
Although some investigators did not find AMH to be a useful predictor for pregnancy (32, 33), others reported a positive correlation between high AMH levels and pregnancy rates (27, 34). In our donor–recipient population, we did not demonstrate its association with clinical pregnancy. There is paucity of information on the variation of serum AMH levels over repetitive COH for IVF. Our data demonstrated that the intravariation coefficient of serum AMH levels calculated from oocyte donor groups ranged from 9.1% to 35.8%. The group with the highest number of repetitive cycles (group 6) had higher AMH levels and lower intravariation coefficient. The ICC comparisons of groups with each other, however, did not demonstrate any significant differences. Because FSH dose/number of oocyte ratios were not significantly different among groups, the variation of serum AMH levels within our reported range should not imply any short-term significant effects on the ovarian reserve. In a group of ovulatory infertile women, the ICC coefficient of AMH over three consecutive menstrual cycles have been reported to be 0.89; hence, the intravariation coefficient of 11% (35). In another study of young ovulatory women, intercycle variation of AMH between two cycles has been reported to be around 28% (36). Within a given menstrual cycle the average deviation of serum AMH levels from the individual cycle mean was reported to be about a modest 17.4% (37). In 36 patients over three repetitive oocyte donation cycles, we calculated intravariation coefficient as 12.5%. This number is comparable to the intercycle variation of AMH reported from normal menstrual cycles. Fertility and Sterility
None of our oocyte donors were poor responders clinically. Therefore, over a wide range of individual AMH levels with good ovarian response, our reported intercycle variations of AMH levels may be considered within the normal range for short-term implications. Long-term follow-up studies are needed to assess if the calculated intercycle AMH variations have any implications for eventual menopausal age. We measured AMH from serum collected on the day when basal hormone tests were performed before FSH stimulation. Comparable AMH levels have been reported when measured either day 3 of the cycle (basal state) or the day of the oocyte retrieval in the same patient (38). Therefore, our sampling day should not affect interpretation of our results. It is unknown whether repetitive oocyte donation causes earlier exhaustion of ovarian follicles, which may ultimately result in reaching menopause at a younger age. As a sensitive marker for the progressive decline in the ovarian reserve when measured over time, AMH levels determined during each oocyte donation cycle may have implications for accelerated ovarian aging. Whereas individual AMH levels of these young females were still found to be a strong predictor of oocyte yield in COH, our study did not show any decrease in AMH levels over repetitive oocyte donation cycles. Although a large-scale prospective cohort study is needed to determine if there is increased risk of early menopause in oocyte donors, our result do not suggest any subtle acceleration in ovarian aging as assessed by AMH levels. Acknowledgments: We thank Keith E. Muller, Ph.D., for his invaluable statistical advice. We also acknowledge Hulusi B. Zeyneloglu, M.D., for his initial assessment of the data.
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microdose HCG decreases clinical pregnancy rates without affecting embryo quality and blastulation. Reprod Biomed Online 2006;13:465–75. American Society for Reproductive Medicine. Repetitive oocyte donation. Fertil Steril 2008;90:S194–5. Al-Azemi M, Bernal AL, Steele J, Gramsbergen I, Barlow D, Kennedy S. Ovarian response to repeated controlled stimulation in in-vitro fertilization cycles in patients with ovarian endometriosis. Hum Reprod 2000;15: 72–5. Donner A, Bull S. Inferences concerning a common intraclass correlation coefficient. Biometrics 1983;39:771–5. Tremellen KP, Kolo M, Gilmore A, Lekamge DN. Anti-mullerian hormone as a marker of ovarian reserve. Aust N Z J Obstet Gynaecol 2005;45:20–4. de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. Antimullerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002;77:357–62. van Rooij IA, Broekmans FJ, Scheffer GJ, Looman CW, Habbema JD, de Jong FH, et al. Serum antimullerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: a longitudinal study. Fertil Steril 2005;83:979–87. Broer SL, Mol BW, Hendriks D, Broekmans FJ. The role of antimullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil Steril 2009;91:705–14. Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 1997;82:3748–51. Richardson SJ, Nelson JF. Follicular depletion during the menopausal transition. Ann N Y Acad Sci 1990;592:13–20. discussion 44–51. McTavish KJ, Jimenez M, Walters KA, Spaliviero J, Groome NP, Themmen AP, et al. Rising follicle-stimulating hormone levels with age accelerate female reproductive failure. Endocrinology 2007;148:4432–9. Gobert B, Barbarino-Monnier P, Guillet-May F, Bene MC, Faure GC. Anti-ovary antibodies after attempts at human in vitro fertilization induced by follicular puncture rather than hormonal stimulation. J Reprod Fertil 1992;96:213–8. Serna J, Garcia-Velasco JA. Effect of repeated assisted reproduction techniques on the ovarian response. Curr Opin Obstet Gynecol 2005;17:233–6. Themmen AP. Anti-Mullerian hormone: its role in follicular growth initiation and survival and as an ovarian reserve marker. J Natl Cancer Inst Monogr 2005;18–21. van Beek RD, van den Heuvel-Eibrink MM, Laven JS, de Jong FH, Themmen AP, Hakvoort-Cammel FG, et al. Anti-Mullerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin’s lymphoma during childhood. J Clin Endocrinol Metab 2007;92:3869–74. Hehenkamp WJ, Volkers NA, Broekmans FJ, de Jong FH, Themmen AP, Birnie E, et al. Loss of ovarian reserve after uterine artery embolization: a randomized comparison with hysterectomy. Hum Reprod 2007;22: 1996–2005. Shin SY, Lee JR, Noh GW, Kim HJ, Kang WJ, Kim SH, et al. Analysis of serum levels of anti-Mullerian hormone, inhibin B, insulin-like growth factor-I, insulin-like growth factor binding protein-3, and follicle-stimulating hormone with respect to age and menopausal status. J Korean Med Sci 2008;23:104–10.
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26. van Disseldorp J, Faddy MJ, Themmen AP, de Jong FH, Peeters PH, van der Schouw YT, et al. Relationship of serum anti-Mullerian hormone concentration to age of menopause. J Clin Endocrinol Metab 2008;93: 2129–34. 27. La Marca A, Giulini S, Tirelli A, Bertucci E, Marsella T, Xella S, et al. Anti-Mullerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum Reprod 2007;22:766–71. 28. Tsepelidis S, Devreker F, Demeestere I, Flahaut A, Gervy C, Englert Y. Stable serum levels of anti-Mullerian hormone during the menstrual cycle: a prospective study in normo-ovulatory women. Hum Reprod 2007;22:1837–40. 29. Muttukrishna S, McGarrigle H, Wakim R, Khadum I, Ranieri DM, Serhal P. Antral follicle count, anti-mullerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG 2005;112:1384–90. 30. McIlveen M, Skull JD, Ledger WL. Evaluation of the utility of multiple endocrine and ultrasound measures of ovarian reserve in the prediction of cycle cancellation in a high-risk IVF population. Hum Reprod 2007;22: 778–85. 31. van Rooij IA, Broekmans FJ, te Velde ER, Fauser BC, Bancsi LF, de Jong FH, et al. Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002;17:3065–71. 32. Penarrubia J, Fabregues F, Manau D, Creus M, Casals G, Casamitjana R, et al. Basal and stimulation day 5 anti-Mullerian hormone serum concentrations as predictors of ovarian response and pregnancy in assisted reproductive technology cycles stimulated with gonadotropin-releasing hormone agonist–gonadotropin treatment. Hum Reprod 2005;20: 915–22. 33. van Rooij IA, Broekmans FJ, Hunault CC, Scheffer GJ, Eijkemans MJ, de Jong FH, et al. Use of ovarian reserve tests for the prediction of ongoing pregnancy in couples with unexplained or mild male infertility. Reprod Biomed Online 2006;12:182–90. 34. Silberstein T, MacLaughlin DT, Shai I, Trimarchi JR, LambertMesserlian G, Seifer DB, et al. Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology. Hum Reprod 2006;21:159–63. 35. Fanchin R, Taieb J, Lozano DH, Ducot B, Frydman R, Bouyer J. High reproducibility of serum anti-Mullerian hormone measurements suggests a multi-staged follicular secretion and strengthens its role in the assessment of ovarian follicular status. Hum Reprod 2005;20:923–7. 36. Streuli I, Fraisse T, Chapron C, Bijaoui G, Bischof P, de Ziegler D. Clinical uses of anti-Mullerian hormone assays: pitfalls and promises. Fertil Steril 2009;91:226–30. 37. Hehenkamp WJ, Looman CW, Themmen AP, de Jong FH, Te Velde ER, Broekmans FJ. Anti-Mullerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J Clin Endocrinol Metab 2006;91:4057–63. 38. Fanchin R, Mendez Lozano DH, Frydman N, Gougeon A, di Clemente N, Frydman R, et al. Anti-Mullerian hormone concentrations in the follicular fluid of the preovulatory follicle are predictive of the implantation potential of the ensuing embryo obtained by in vitro fertilization. J Clin Endocrinol Metab 2007;92:1796–802.
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Departments of Obstetrics and Gynecology; b Epidemiology & Health Policy Research, University of Florida College of Medicine, Gainesville, Florida; c Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas; d ReproSource, Bedford, Massachusetts; and e Center for Assisted Reproduction, Bedford, Texas
Objective: To determine if the anti-M€ullerian hormone (AMH), a proposed marker of ovarian aging, decreases with repetitive oocyte donation. Design: Retrospective cohort. Setting: Academic. Patient(s): Thirty-six young women who underwent three to seven oocyte donation cycles. Intervention(s): Assessor blind determination of AMH levels from serum samples collected during each treatment cycle. Main Outcome Measure(s): Cycle trends of serum AMH levels. Result(s): The AMH was the only predictor of oocyte yield in the first cycles. The AMH was negatively associated with donor age and follicle stimulating hormone (FSH) dose used. Serum AMH levels did not show any decrease per treatment cycle basis and per maximum number of oocyte donation cycles performed per woman. Whereas donors who underwent six cycles showed increasing AMH levels when controlled for studied covariates, the slopes of the multiple regression curves were not significantly different from donors who underwent three, four, and five cycles. Clinical outcome assessed by FSH dose/number of oocytes ratio did not show significant change over repetitive cycles. Intercycle variation of AMH in all patients over three cycles was found to be 12.5%, which was within the reported intermenstrual range. Conclusion(s): Serum AMH levels do not decrease over repetitive oocyte donation cycles, which may imply that accelerated ovarian aging may not occur in oocyte donors. (Fertil Steril 2010;94:905–12. 2010 by American Society for Reproductive Medicine.) Key Words: AMH, anti-mullerian hormone, oocyte donation, ovarian aging
The anti-M€ ullerian hormone (AMH) in females is produced in the granulosa cells from preantral and small antral follicles (1). Because AMH is solely produced in the growing ovarian follicles, the serum levels may be used as a marker of ovarian reserve (2). The higher early follicular phase serum AMH levels is associated with greater number of retrieved oocytes in in vitro fertilization (IVF) (3). The AMH has been reported to be the earliest marker to decrease with aging (4). AMH levels are unaffected by exogenous sex steroids used for contraception whether administered orally or vaginally (5). Because AMH shows modest intercycle and intracycle variability throughout the menstrual cycle, and exogenous Received January 21, 2009; revised May 6, 2009; accepted May 7, 2009; published online July 23, 2009. O.B. had nothing to disclose. Q.L. has nothing to disclose. B.R.C. has nothing to disclose. B.L. has nothing to disclose. K.M.D. has nothing to disclose. K.J.D. has nothing to disclose. Supported in part by UPN 07120257 from the University of Florida College of Medicine (Dr. Bukulmez). Presented in part at the American Society for Reproductive Medicine 64th Annual Meeting, November 8–12, 2008, San Francisco, CA. Reprint requests: Orhan Bukulmez, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0294 (FAX: 352-392-9724; E-mail: obukulmez@ ufl.edu).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.05.017
follicle stimulating hormone (FSH) administration does not affect its levels (6), it may be informative about the ovarian reserve even if it is randomly obtained. Anonymous oocyte donors are usually young women with excellent ovarian reserve, and they may undergo repetitive oocyte donation cycles. Whereas it has been reported that repetitive controlled ovarian hyperstimulation (COH) and oocyte harvesting, in the short term, may not affect ovarian response and oocyte yield in these young females (7), it is not known if repetitive procedures result in accelerated ovarian aging in the long term. It may be possible that the repeated procedure itself can lead to a subtle decline in ovarian reserve independent of chronologic aging without affecting shortterm clinical outcome, which may be assessed by determination of AMH levels. The primary aim of this study was to reveal if serum AMH levels significantly decreased by each oocyte donation cycle performed, which might suggest a potential detrimental affect of repetitive oocyte donation on ovarian aging. To assess the utility of AMH and to confirm prior reports on AMH as an ovarian reserve marker, we also investigated if AMH was associated with oocyte yield or clinical pregnancy in our cohort. Furthermore, we analyzed intercycle variation of serum AMH.
Fertility and Sterility Vol. 94, No. 3, August 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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MATERIALS AND METHODS Patients and Design Thirty-six women who had undergone 167 repetitive oocyte donation cycles were included. This study was determined to be exempt from review by the institutional review board of The University of Texas Southwestern Medical Center. The AMH levels were measured in an assessor-blind fashion from stored serum samples collected during each COH cycle on the day before commencing FSH stimulation. Hence, the first cycle serum samples of each patient reflected their baseline serum AMH levels before any stimulation or oocyte retrieval was performed. All donors were pretreated by combined oral contraceptive pills followed by daily recombinant FSH commenced 5 days after the last dose of the pill, with a gonadotropin releasing hormone antagonist protocol as published elsewhere (8). Then, per routine, their oocytes were retrieved and fertilized in an IVF laboratory (9) and the best two of the blastocyst stage embryos were transferred to the recipients. The recipient women were women with premature ovarian failure or diminished ovarian reserve aged between 29 and 45 years. All oocyte donors were between the ages of 19 and 30 years, and complied with the Food and Drug Administration criteria for the oocyte donation. Only the donors with three or more repetitive treatment cycles resulting in embryo transfers, and those having adequate frozen serum samples to determine AMH levels were included. The number of cycles for a given donor was attempted to be limited to six (10). Only one patient underwent seven cycles because of a special circumstance. Recipients needing menstrual suppression were down-regulated by daily leuprolide acetate, and they received oral micronized estradiol in incremental doses to develop endometrium. The day after the donor’s trigger for final oocyte maturation by human chorionic gonadotropin, the recipients were started with progesterone in oil intramuscularly to synchronize endometrium for embryo transfer. We assessed whether there was any significant decline in the serum AMH levels over repetitive treatment cycles in oocyte donors. The following parameters of ovarian response in each treatment cycle were also analyzed: [1] total dose of FSH (IU) used per cycle (FSH dose), [2] estradiol (E2) concentration on the day of hCG administration (peak E2), [3] number of oocytes retrieved per cycle (number of oocytes), [4] donor age at the time of each treatment cycle and time interval between treatment cycles calculated from this parameter. In addition, FSH dose (IU)/number of oocytes ratio for each cycle was calculated to be used as one of the measures of COH or clinical outcome (11). Clinical pregnancy was defined by the presence of at least one gestational sac with positive fetal heart tones detected after 6 weeks of gestation in recipients.
Determination of Serum AMH Levels The serum samples kept at 20 C were shipped to the blinded reference laboratory (Repromedix, Woburn, MA). 906
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Serum AMH levels were determined using an enzymatically amplified two-site immunoassay (MIS/AMH ELISA, DSL10-14400, Diagnostic Systems Laboratories, Webster, TX). The intra- and interassay coefficients of variation were between 3% and 7%, with an average of 5%. The sensitivity of the assay was 0.017 ng/mL. Statistical Analysis SAS software (version 9.1, Cary, NC) was used unless stated otherwise. The AMH measurements were averaged over the assayed aliquots. Two-tailed significance level was P<.05. Analysis per oocyte donation cycle Initially, only the first cycles of 36 oocyte donors were analyzed to assess if baseline AMH levels predicted the number of oocytes retrieved. Multiple regression analysis of the number of oocytes retrieved on baseline AMH, donor age, FSH dose, peak E2, and clinical pregnancy outcome variables was performed. Logarithmic (log) transformation of the number of oocytes retrieved was used to satisfy Gaussian distribution. To compare each cycle specific variable over each repetitive cycle, repeated measure analysis with unstructured covariance structure was used including all patients. Log transformations were performed as needed. The time intervals between cycles in years were calculated by subtracting the ages of the donors, at the commencement of prior cycle from their ages at the start of the following cycle. Inverse transformation of these intervals was needed to run a repeated-measure analysis. To assess if serum AMH levels change over repetitive treatment cycles, a repeated-measure analysis of AMH levels was conducted to test for cycle trends while controlling for other covariates with an unstructured covariance matrix. The model treated the baseline (first cycle) AMH as covariate and treated the orderly additions of donor age, FSH dose, number of oocytes, peak E2, and clinical pregnancy as repeated covariates. An added-in-order (Type I) test was used for these variables. The square root of the AMH levels was performed. Six cycles including the first cycle with the baseline AMH level were used in the analysis. The quadratic and linear trends of the cycles were tested. The quadratic term was dropped because of its nonsignificance. A repeated measure with an unstructured covariance matrix (generalized estimated equations, GEE) was used to assess if AMH levels were associated with clinical pregnancy or nonpregnancy. Analysis according to maximum number of oocyte donation cycles performed per woman Because the patients who underwent three repetitive cycles (group 3) might be different than those who completed four, five, and six cycles (groups 4, 5, and 6, respectively), a repeated analysis of AMH levels was conducted to test for cycle trends by group allocation with unstructured covariance matrix. This model treated the baseline (first cycle) AMH as a covariate and treated the orderly additions of age, total FSH dose, number of oocytes, Vol. 94, No. 3, August 2010
peak E2 level, and clinical pregnancy as repeated covariates. An added-in-order (Type I) test was used for these variables. A square root of the AMH levels was necessary. Six cycles (first cycle includes baseline AMH level) were used for the analysis. The COH outcome was assessed, both per cycle and group basis, by a repeated-measure analysis of FSH dose/number of oocytes ratio, which was tested for cycle trends with an unstructured covariance matrix. The addition order of covariates was donor age, peak E2, and clinical pregnancy outcome, each of which was controlled for FSH dose/number of the oocytes ratio. Here, residual analysis was conducted to satisfy Gaussian distribution. Intraclass correlation (ICC) The ICC coefficient and its 95% confidence interval (CI) was calculated for first three cycles, which included all 36 patients and also for each oocyte donor group, to assess the intercycle variation of AMH levels (SPSS version 11, Chicago, IL). Then intravariation coefficients (¼1 ICC) were calculated. Pairwise ICC comparisons were performed among groups 3, 4, 5, and 6 by using a procedure based on Fisher’s transformation (12). RESULTS Cumulative clinical pregnancy rate per treatment cycle in the whole cohort was 53%. Mean (SD) values and their comparisons for all parameters studied per cycle basis are shown in Table 1. The time interval between cycles averaged 0.40 years (4.8 months), ranging between 0.1 and 2.2 years. The mean time intervals between each cycle were not significantly different from each other (Table 1). Median (range) AMH level of all cycles was 2.48 ng/mL (0.27–17.65). Of the analyzed cycles (n ¼ 166), the conventional IVF was done in 86 cycles (51.8%) and intracytoplasmic sperm injection (ICSI) was performed in 80 cycles (48.2%). Because there might be discrepancies between the assessment of oocyte maturity for IVF and ICSI, and the number of oocytes retrieved was used in the assessment of primary clinical outcome rather than the number of mature oocytes. Cumulative fertilization rates for IVF and ICSI were 62% 19% and 74% 16%, respectively, as expected. The AMH Predicts Number of Oocytes Retrieved When only the first cycles (n ¼ 36) were analyzed with multiple regression, AMH was found to be the only significant parameter (P¼.0035, parameter estimate .08) in prediction of total number of oocytes retrieved. Donor age, FSH dose, peak E2 and clinical pregnancy outcome were not found to be significant (Table 1 for parameter means). Advancing age of donors over repetitive cycles is not associated with a decrease in AMH levels In a repeated measure without controlling for other variables, as the number of cycles increased, donor age significantly advanced, as expected (Table 1). However, as the number Fertility and Sterility
of cycles increased, FSH dose, peak E2, number of oocytes, and AMH significantly increased but FSH dose/number of oocytes ratios stayed comparable (Table 1). There was no significant association between the number of repetitive oocyte donation cycles performed and the clinical pregnancy outcome (P¼.17, Table 1 for clinical pregnancy rates). The AMH levels do not decrease by repetitive cycles of oocyte donation Initial per cycle analysis demonstrated that the linear trend of the cycle was significantly associated with AMH levels obtained after the first cycle (P¼.035) when controlling for previous added variables (Table 2). Whereas baseline (first cycle) AMH, donor age, the number of oocytes, and FSH dose were significantly associated with AMH levels measured during repetitive cycles, peak E2 and the clinical pregnancy did not show any significant association with AMH. The positive coefficient estimates suggested that as the number of cycles increased the AMH levels increased (Table 2). This issue was further investigated for each donor group as detailed below. Although donor age and FSH dose showed significant negative association with AMH levels, number of oocytes retrieved showed positive association (Table 2). In donors with three, four, and five repetitive cycles (groups 3, 4, and 5), controlling for added covariates as mentioned in methods section, the cycle trends were not associated with AMH (P¼.93, P¼.20, and P¼.11, respectively, Fig. 1A–C). However, for donors who completed six repetitive cycles (group 6), the linear trend of cycle for AMH was found to be significant (P<.0001, estimate of coefficient ¼ .081, Fig. 1D). These results imply that groups 3, 4, or 5 did not show any significant change in AMH levels, whereas in group 6 AMH levels tended to increase by the number of cycles performed. We then tested if there were any significant differences among groups 3, 4, 5, and 6 in terms of AMH cycle trends (test for slope difference among groups). The linear trend of cycle was comparable among the groups (P¼.90). Therefore, comparing AMH trend of each group and cycle number did not show any significance (Fig. 1). The AMH does not predict clinical pregnancy Overall, AMH did not predict clinical pregnancy in recipients (GEE, P¼.90). The AMH shows nonsignificant and modest variation over repetitive treatment cycles Intravariation coefficients for donors who underwent three, four, five, and six repetitive cycles were 0.385 (95% CI 0.105–0.808), 0.267 (95% CI 0.08–0.548), 0.165 (95% CI 0.042–0.371), and 0.091 (95% CI 0.03–0.191), respectively. Whereas these results suggested that the group 6 with higher AMH levels (Fig. 2), may show lower variation in AMH levels than groups 3, 4, and 5, pairwise ICC comparisons among groups were not significant (P>.05 for all pair-wise comparisons) (12). The ICC coefficient of the AMH over 907
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TABLE 1 Distribution of nontransformed parameters per oocyte donation cycle performed. Parameter (mean ± SD)
Cycle1 (n [ 36)
Cycle 2 (n [ 36)
Cycle 3 (n [ 36)
Cycle 4 (n [ 28)
Cycle 5 (n [ 19)
Cycle 6 (n [ 11)
Cycle 7a (n [ 1) P valueb
Age (y) 24.74 3.38 25.17 3.31 25.57 3.34 25.92 3.29 26.22 3.08 25.16 2.82 24.34 2336.6 1275.2 2,789.2 1,597.6 2,510.1 1,142.1 2,574.6 787.0 2,773.4 1,635.3 2,882.1 1,191.6 3319.0 Peak E2 (pg/mL) FSH dose (IU) 1,947.9 713.7 2,114.6 758.7 2,162.5 869.8 2,325.0 849.0 2,208.3 944.9 2,072.7 774.5 1050.0 No. of oocytes 16.1 9.8 17.5 7.7 16.2 7.0 19.9 8.4 19.2 12.3 26.9 15.8 11 retrieved FSH dose/# 159.1105.3 157.9 117.9 161.3 92.9 146.3 101.7 147.4 92.4 112.0 89.0 95.5 oocytes AMH (ng/mL) 2.92 2.71 2.78 2.00 2.75 2.88 2.99 2.50 3.83 3.44 4.21 3.15 1.59 Time interval — 0.43 (0.09–2.51) 0.40 (0.10–1.95) 0.44 (0.10–2.11) 0.45 (0.13–2.22) 0.29 (0.15–0.96) 0.36* (mean y-range) Clinical pregnancy 22/36 (61.1%) 19/36 (52.8%) 19/36 (52.8%) 15/28 (53.6%) 8/18 (44.4%) 5/11 (45.5%) — rate a
Cycle 7 was not included in the analysis. Repeated measure for cycle trends was used to compare each log transformed variable without controlling for other covariates. c Inverse transformation was performed. NS ¼ not significant, P>.05. d Chi-square test. b
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< .0001 .005 .046 < .007 NS .006 .076c NSd
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TABLE 2 Repeated-measure multiple regression model and added in order (type I) test for serum AMH levels over repetitive oocyte donation cycles (n [ 166).
Variable Baseline AMH Donor age Number of oocytes FSH dose Peak E2 Pregnant—No versus Yes Cycle number
Estimate of coefficient Standard P (slope) error valuea 0.144 0.02 0.007 0.0001 0.00004 0.033 0.049
0.017 0.014 0.004
< .0001 .027 .013
0.00005 0.00003 0.057
.024 NS NS
0.021
.035
Note: AMH ¼ anti-Mullerian hormone. a The P-values suggest that the linear trend of the cycle was significant while controlling the previous variables added to the multiple regression model. As the number of cycles increased, AMH levels increased. Although donor age and FSH dose showed significant negative association with AMH levels, number of oocytes retrieved showed positive association. Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
the first three cycles was 0.875 (95% CI 0.929–0.795), which gives an intercycle variation of 0.125 (95% CI 0.071–0.205). Outcome of COH is comparable over repetitive oocyte donation cycles FSH dose/number of oocytes ratio analyzed both per cycle (P¼.07) and group basis was not significantly associated with the number of cycles performed. For groups 3, 4, 5, and 6, no significant trend of cycle was found after controlling for the studied covariates (P¼.33, .21, .20, .77, respectively). Whereas group 6 with higher AMH levels (Fig. 2) required a somewhat lower FSH dose/oocyte ratio, the comparisons of slopes between groups were not significant. DISCUSSION Our study found no decrease in AMH levels along with stable COH response in donors participating in as many as six oocyte donation cycles. Serum AMH levels in our population were associated with the number of oocytes retrieved, further confirming the reports on AMH from general infertility population (13). We also did not see any steady decline in serum AMH levels over the time frame of oocyte donation cycles. Although two earlier small longitudinal studies involving premenopausal women with somewhat more advanced mean ages than our population, have noted a steady decline Fertility and Sterility
of serum AMH levels over a period of 2.6 to 4 years (14, 15), a cross-sectional but larger study in subfertility population has reported that the mean AMH levels remain relatively stable between the ages of 18 to 29 (13). Although we are aware of the bias inherent to the retrospective properties of this report and limited sample size, we believe it still provides novel information regarding the trend of serum AMH levels over repetitive oocyte donation cycles. In addition, one recent systematic review on the role of AMH in prediction of IVF outcome has suggested that design of the study whether retrospective or prospective had no significant impact on the predictive performance of AMH (16). Over six repetitive cycles, baseline (first cycle) AMH, number of cycles, number of oocytes, donor age, and FSH dose were significantly associated with AMH levels obtained from repetitive cycles (Table 2). Negative coefficient estimates suggested that as donor age and FSH dose increased, AMH levels decreased confirming other studies (16). Whereas we found a significant trend that as the number of cycles increased. AMH levels also increased (Table 2). This was mainly applicable to group 6 (Figs. 1D and 2). Although this finding may be because of the limited sample size, it is possible that these donors underwent more cycles because their response were perceived to be better. Of note, because AMH levels were measured long after these cycles were completed, the management decisions of these donors were made without AMH information in hand. By all means, this finding also argues against any possible decline in AMH levels with oocyte donation. Whether or not AMH levels actually increase by repetitive COH in young females with higher AMH levels and its biologic plausibility need further elucidation. It is believed that repeated ovarian stimulation is unlikely to reduce the ovarian pool of primordial follicles given that these follicles do not possess receptors for FSH (17). However, FSH stimulation may hasten ovarian aging by increasing recruitment of small growing follicles, thereby accelerating the depletion of follicle reserve (18). A transgenic mouse model was developed that expressed pituitary-independent human FSH, which exhibit progressively increasing levels of circulating FSH independent to ovarian follicular feedback (19). In this model, increased basal FSH activity could advance the age-related onset of female infertility compared with wild type. Ovarian AMH mRNA expression and serum AMH levels were found to decrease at an earlier age in transgenic animals compared with the wild-type mice (19). Another alleged concern is possible ovarian damage with repeated ovarian puncture. It has been hypothesized that ovarian trauma induced by oocyte retrieval may diminish ovarian reserve by inducing the release of autoantigens (20, 21). This hypothesis has never been proven, and the potential long-term adverse effects of ovarian trauma have never been documented. The short-term effects of repetitive oocyte donation were assessed in 284 oocyte donors who had at least two repetitive 909
FIGURE 1 Slopes and the test of slope differences among groups 3 (A), 4 (B), 5 (C), and 6 (D) for AMH and cycle trends. Square root of AMH was used for statistics. Solid lines were obtained from repeated-measure multiple regression while controlling for baseline (first cycle) AMH, donor age at the time of each cycle, number of oocytes retrieved per cycle, FSH dose (IU) used per cycle, peak E2, and clinical pregnancy outcome in each cycle. Whereas AMH showed significant increase over repetitive cycles in group 6 (P< .0001), comparisons of the slopes between groups 3, 4, 5, and 6 were not significantly different from each other (P¼ .90). Therefore, the linear trend of the cycle was not significantly different among the groups.
Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
cycles of donation (7). The number and quality of oocytes were maintained, and there was no increase in the dose of gonadotropins over multiple cycles (7). Although this study seems to be reassuring for ovarian response to be unaffected with repetitive oocyte donation, the follow-up data of oocyte donors over a long period of time is lacking. AMH has been suggested to be an excellent marker for ovarian reserve not only in infertility patients but also in women during and after cancer treatment and in women who underwent uterine artery embolization (22–24). The AMH steadily decreases by advancing age (13, 25). Longitudinal studies have suggested that interval changes in conven910
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tional markers of ovarian reserve such as serum FSH, inhibin B and E2, or antral follicle count, do not remarkably change as AMH over the same time period (14, 15). Therefore, AMH has been demonstrated to be the earliest marker of ovarian aging (14, 15) and its levels may predict the age of menopause (26). Furthermore, the AMH levels do not show any significant variation throughout the menstrual cycle (27, 28). The AMH shows excellent correlation with the number of oocytes retrieved in IVF, and is a strong marker for poor COH response (29, 30). We showed an excellent association between serum AMH levels and ovarian response to FSH as reported before (3, 31). Vol. 94, No. 3, August 2010
FIGURE 2 Mean (SEM) of serum AMH levels over the mean time frame (years) of repetitive cycles according to the maximum number of oocyte donation cycles performed per woman (groups 3, 4, 5, and 6, as given by each line). Although groups showed various basal AMH levels, AMH levels do not decline over the time frame of oocyte donation cycles. The numbers of women in each group are given in parentheses.
Bukulmez. AMH and repetitive oocyte donation. Fertil Steril 2010.
Although some investigators did not find AMH to be a useful predictor for pregnancy (32, 33), others reported a positive correlation between high AMH levels and pregnancy rates (27, 34). In our donor–recipient population, we did not demonstrate its association with clinical pregnancy. There is paucity of information on the variation of serum AMH levels over repetitive COH for IVF. Our data demonstrated that the intravariation coefficient of serum AMH levels calculated from oocyte donor groups ranged from 9.1% to 35.8%. The group with the highest number of repetitive cycles (group 6) had higher AMH levels and lower intravariation coefficient. The ICC comparisons of groups with each other, however, did not demonstrate any significant differences. Because FSH dose/number of oocyte ratios were not significantly different among groups, the variation of serum AMH levels within our reported range should not imply any short-term significant effects on the ovarian reserve. In a group of ovulatory infertile women, the ICC coefficient of AMH over three consecutive menstrual cycles have been reported to be 0.89; hence, the intravariation coefficient of 11% (35). In another study of young ovulatory women, intercycle variation of AMH between two cycles has been reported to be around 28% (36). Within a given menstrual cycle the average deviation of serum AMH levels from the individual cycle mean was reported to be about a modest 17.4% (37). In 36 patients over three repetitive oocyte donation cycles, we calculated intravariation coefficient as 12.5%. This number is comparable to the intercycle variation of AMH reported from normal menstrual cycles. Fertility and Sterility
None of our oocyte donors were poor responders clinically. Therefore, over a wide range of individual AMH levels with good ovarian response, our reported intercycle variations of AMH levels may be considered within the normal range for short-term implications. Long-term follow-up studies are needed to assess if the calculated intercycle AMH variations have any implications for eventual menopausal age. We measured AMH from serum collected on the day when basal hormone tests were performed before FSH stimulation. Comparable AMH levels have been reported when measured either day 3 of the cycle (basal state) or the day of the oocyte retrieval in the same patient (38). Therefore, our sampling day should not affect interpretation of our results. It is unknown whether repetitive oocyte donation causes earlier exhaustion of ovarian follicles, which may ultimately result in reaching menopause at a younger age. As a sensitive marker for the progressive decline in the ovarian reserve when measured over time, AMH levels determined during each oocyte donation cycle may have implications for accelerated ovarian aging. Whereas individual AMH levels of these young females were still found to be a strong predictor of oocyte yield in COH, our study did not show any decrease in AMH levels over repetitive oocyte donation cycles. Although a large-scale prospective cohort study is needed to determine if there is increased risk of early menopause in oocyte donors, our result do not suggest any subtle acceleration in ovarian aging as assessed by AMH levels. Acknowledgments: We thank Keith E. Muller, Ph.D., for his invaluable statistical advice. We also acknowledge Hulusi B. Zeyneloglu, M.D., for his initial assessment of the data.
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Vol. 94, No. 3, August 2010