Erasmus Medical Center, Rotterdam, The Netherlands

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1 FERTILITY AND STERILITY VOL. 78, NO. 2, AUGUST 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Women with regular menstrual cycles and a poor response to ovarian hyperstimulation for in vitro fertilization exhibit follicular phase characteristics suggestive of ovarian aging Nicole G. M. Beckers, M.D., a Nicholas S. Macklon, Ph.D., a Marinus J. C. Eijkemans, M.Sc., b and Bart C. J. M. Fauser, Ph.D. a Erasmus Medical Center, Rotterdam, The Netherlands Received October 2, 2001; revised and accepted January 28, Supported by Stichting Voortplantingsgeneeskunde Rotterdam, Rotterdam, The Netherlands. Presented at the 17th Annual European Society of Human Reproduction and Embryology (ESHRE) Meeting, Lausanne, Switzerland, July 3, Reprint requests: Bart C. J. M. Fauser, Ph.D., Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Erasmus Medical Center, Doctor Molewaterplein 40, 3015 GD Rotterdam, The Netherlands (FAX: ; fauser@gyna.azr.nl). a Division of Reproductive Medicine, Department of Obstetrics and Gynecology. b Department of Public Health /02/$22.00 PII S (02) Objective: To investigate whether follicular phase characteristics associated with ovarian aging can be observed in women of normal reproductive age, who had previously shown a poor response to ovarian hyperstimulation for IVF. Design: Observational, prospective study. Setting: Tertiary fertility center. Patient(s): Eleven regularly cycling, ovulatory women, aged years who previously presented with fewer than four dominant follicles after ovarian hyperstimulation for IVF. Intervention(s): Frequent serum hormone assessments and transvaginal ultrasound during the follicular phase of a spontaneous, unstimulated cycle. Main Outcome Measure(s): Duration of the follicular phase; serum LH, FSH, E 2, P, inhibin A, and inhibin B levels; and number of antral follicles observed by ultrasound. Results were compared with the cycle characteristics of a reference population of 38 healthy normo-ovulatory women aged years (as published elsewhere). Result(s): Poor responders had significantly fewer antral follicles than controls. Median FSH concentrations were significantly higher compared with controls, but the majority had FSH levels within the normal range. Follicular phase P levels were significantly higher in poor responders. Duration of the follicular phase, E 2, and inhibin A and inhibin B serum levels did not differ between poor responders and controls. Conclusion(s): Normo-ovulatory regularly cycling women with a previous poor response to ovarian hyperstimulation for IVF show follicular phase characteristics suggestive of ovarian aging. (Fertil Steril 2002;78: by American Society for Reproductive Medicine.) Key Words: Ovarian aging, IVF, poor response, follicular phase, follicles, FSH, progesterone, inhibin, ovary, menstrual cycle The process of ovarian aging begins before birth and continues until the menopause, when the primordial follicle pool becomes exhausted (1, 2). This process of follicle loss takes place at a rate of 1,000 per month and accelerates after 35 years of age. Before the menopause, follicle pool depletion (possibly associated with diminished oocyte quality) may cause a decline in fecundity (3). As shown previously, in a natural population in which the timing of having children was not influenced by contraception, the interval between last childbirth and onset of menopause was a fixed period of 10 years (4). Depletion in the size of the follicle pool is reflected in a smaller number of antral follicles visible by ultrasound (5); changes in serum E 2, FSH, and inhibin B levels (6); and a shortening of the menstrual cycle (7). Before the onset of cycle abnormalities, analysis of endocrine parameters such as early follicular phase FSH, E 2, and inhibin B levels may already reveal changes suggestive of aging (8, 9). 291

2 The incidence of poor response to ovarian hyperstimulation for IVF is unknown. This may be partly due to a lack of consensus as to what defines poor response (10, 11). Moreover, differences in indications for IVF and differences in age of the treated patients may further affect the observed incidence. Patients with a normal ovarian reserve, assessed by cycle day 3 FSH, may turn out to be poor responders to hyperstimulation (8, 12). Several investigators have aimed to establish whether ovarian response to a dynamic challenge test may improve the prediction of ovarian response to hyperstimulation (13 15), but as yet no reliable means for predicting poor response is available (12). Ovarian hyperstimulation itself may be viewed as an extended challenge test, and therefore a reduced ovarian response to stimulation may represent the earliest sign of ovarian aging. It can be postulated that patients with a regular menstrual cycle who show poor response to hyperstimulation for IVF may be manifesting the earliest sign of ovarian aging and may subsequently present with an early menopause. A number of studies have shown that adjusting treatment protocols in poor responders does not increase the poor pregnancy rates in these patients (10, 16 18). Moreover, recent observations show that the dose-dependent relationship between exogenous FSH and number of oocytes retrieved disappears beyond 36 years of age, suggesting that biological mechanisms behind poor response cannot be ameliorated by increasing the FSH dose (19). Collectively, these data support the concept that poor response to IVF hyperstimulation may be an early sign of ovarian aging (20). To test this hypothesis, we compared endocrine and sonographic features during the follicular phase of the unstimulated cycle in poor responders to those of healthy controls (21, 22). MATERIALS AND METHODS Patients and Controls The study was approved by the local ethics review committee, and written informed consent was obtained from all patients. Twelve IVF patients who fulfilled the following inclusion criteria participated in this study: [1] A poor response to ovarian hyperstimulation using 225 IU and with an increased dose of 450 IU hmg per day in a subsequent cycle, [2] regular menstrual cycles (cycle length between 24 days and 34 days), [3] proof of ovulation in the cycle preceding the study cycle (midluteal serum P levels of 20 nmol/l) (23), [4] age of 40 years, and [5] both ovaries present. Although no consensus exists as to the definition of poor response (11), it may be considered in terms of a response sufficient to yield at least two embryos for transfer. Given an average fertilization rate of 50% 60%, at least four oocytes will be required (12). We therefore defined poor response in the current patient population as the appearance of fewer than four preovulatory follicles ( 14 mm) in women treated with a so-called long protocol. Because pretreatment ovarian reserve test are not performed routinely in our clinic (24), no data such as cycle day 3 FSH before IVF treatment in these patients were available. Pituitary down-regulation was achieved by the administration of the GnRH agonist Decapeptyl (Ferring Nederland B.V., Hoofddorp, The Netherlands) from day 1 of the cycle, which was followed 2 3 weeks later by ovarian stimulation with hmg (Humegon; N.V. Organon, Oss, The Netherlands) (25). The time interval between the IVF attempt and the study cycle was 2 months. Thirty-eight paid volunteers aged years (median, 28 years) with a regular menstrual cycle (i.e., days), healthy body weight (body mass index, kg/m 2 ), and no history of infertility or any endocrine abnormalities served as controls, as published elsewhere (26). Study Protocol In the cycle preceding the study cycle, patients performed urine tests to detect the mid-cycle luteinizing hormone (LH) surge. Seven days after the positive LH test, a blood sample for P was taken to confirm ovulation; 12 days after the LH surge, daily ultrasound examinations and blood sampling were commenced, as described elsewhere (27). Transvaginal ultrasound of the ovaries was performed as described elsewhere (28) by a single observer (N.B.) using a 6-MHz transvaginal transducer (Tosbee, SSA-240 A, Toshiba Medical Systems, Zoeterneer, The Netherlands). All visible follicles of 2 mm were measured in both ovaries, as was the endometrial thickness. Both ultrasound and blood sampling were continued until ovulation occurred. The day of dominant follicle selection was defined as the day on which the dominant follicle reached a diameter of 10 mm. This day was determined by plotting cycle day against follicle diameter. In addition, the linear growth curve of the ovulated follicle was extrapolated backward until the day on which this follicle reached 10 mm, as described elsewhere (29). The day of the LH surge in the studied cycle was defined as the day on which the LH level peaked, or as the day on which the LH level was three times higher compared with the case of the previous day (30). Ovulation was defined as either the disappearance or the 50% decrease in size of the largest follicle, if it was 15 mm (29). Seven days after ovulation, an additional blood sample was taken for P levels. Hormone Assays Blood samples were processed by centrifuge, and serum was frozen and stored at 20 C. Serum was assayed for FSH, LH, E 2, P, and inhibin A and B. From each patient, hormone assays were performed in the same run. Luteinizing hormone and FSH levels were measured by immunofluorometric assay (Immulite, Diagnostic Products Corp., Los Angelas, CA). Intra-assay and interassay variation were 5% and 7% for FSH and 5% and 15% for LH. Estradiol levels were measured by Coat-A-Count RIA (Immulite, Diagnostic Products Corp.). Intra-assay and interassay varia- 292 Beckers et al. The follicular phase in poor responders Vol. 78, No. 2, August 2002

3 TABLE 1 Clinical and cycle characteristics (median and range) of poor-response patients to ovarian hyperstimulation for IVF and controls. Characteristic Poor responders (n 11) Controls (n 38) P value a Age (y) 36 (29 40) 28 (20 36) Cycle length (d) 28 (25 30) 28 (24 31) 1.0 Follicular phase length (d) 13 (8 18) 15.5 (10 21) 0.22 Total number of antral follicles on cycle day 3 4 (1 9) 13 (4 20) Day of follicle dominance (cycle day) 6 (2 16) 8 (4 13) 0.31 Size at follicle dominance (mm) 11.0 ( ) 10.7 ( ) 0.74 Growth rate of dominant follicle (mm/d) 1.5 ( ) 1.5 ( ) 0.29 Size of largest preovulatory follicle (mm) 21.3 ( ) 20.6 ( ) 0.33 a P values (F test) after analysis of covariance (ANCOVA); parameters corrected for age. tion were 11% and 15%, respectively. P levels were measured by RIA (Immulite, Diagnostic Products Corp.). Intra-assay and interassay variation were 10% and 10%, respectively. Inhibin A and B were measured by immunoenzymatic assay (Serotec, Oxford, UK). Intra-assay and interassay variation were 8% and 15% for inhibin A and 8% and 15% for inhibin B, as published elsewhere (29). Statistical Analysis The data are presented as median and ranges. Differences in parameters between the study and control group were tested for statistical significance with the F test. P.05 was considered statistically significant. As the age of the healthy reference population was significantly lower compared with the age of the poor responders, an analysis of covariance was used to correct for age. In this analysis, two parallel regression lines (one for the study group, one for the control group) are fitted between age and an outcome parameter (e.g., number of follicles). The slope of these regression lines represents the association between age and the outcome parameter, whereas the vertical distance between the lines represents the systematic difference in outcome parameter between study and control group, corrected for age. Statistical analysis was performed using a commercially available software package (Statistical Package for Social Sciences, SPSS, SPSS Inc., Chicago, IL). Twelve patients were recruited in this study. In 1 patient, the studied cycle turned out to be nonovulatory, and this patient was excluded from further analysis. The data presented below are from the remaining 11 patients. The median number of observed follicles that were 14 mm during ovarian hyperstimulation for IVF was two (range, 0 3). Median midluteal P level in the cycle preceding the studied cycle was 43.5 nmol/l (range, ) and in the studied cycle, it was 56.4 nmol/l (range, ). Table 1 shows patient characteristics (age and cycle length) and sonographic data in both groups. All parameters were tested after correction for age. Poor responders had a significantly shorter duration of the follicular phase (P.03), but after correction for age this difference disappeared (P.22). Moreover compared with controls, poor responders had a significantly lower number of antral follicles through- FIGURE 1 The number of antral follicles present throughout the follicular phase is shown. The grey area depicts the 25th- and 75thpercentile amounts of the healthy controls, the black lines depict median and 25th- and 75th-percentile amounts of poor responders. RESULTS FERTILITY & STERILITY 293

4 TABLE 2 Endocrine parameters (median and range) of poor-response patients to ovarian hyperstimulation for IVF and controls. Parameter Poor responders (n 11) Controls (n 38) a P value b Early follicular phase c LH (IU/L) 3.5 ( ) 2.6 ( ) 0.75 FSH (IU/L) 10.3 ( ) 5.1 ( ) E 2 (pmol/l) 117 (74 177) 120 (65 297) 0.03 Inhibin B (ng/l) 65 (0 235) 120 (19 195) 0.18 P (nmol/l) 3.4 ( ) 1.1 ( ) 0.02 Late follicular phase d E 2 (pmol/l) 545 ( ) 592 (303 1,254) 0.02 Inhibin A (ng/l) 32 (22 45) 34 (17 88) 0.48 P (nmol/l) 2.9 ( ) 0.9 ( ) Areas under the curve e LH (IU/L) 73 (36 212) 73 (32 120) 0.50 FSH (IU/L) 100 (78 453) 69 (42 116) E 2 (pmol/l) 101 (13 174) 105 (37 157) 0.09 P (nmol/l) 49 (38 85) 16 (17 88) 0.02 a As published previously (Macklon and Fanser [(24)]). b P values (F test) after analysis of covariance (ANCOVA): parameters corrected for age. c Cycle days 2 3. d One to two days before LH surge. e Areas under the curve are calculated from the period 14 days before the LH surge until 1 day after the LH surge. out the follicular phase. On cycle day 3, the median number of follicles was 4 (range, 1 9) compared with 13 (range, 4 20) in control subjects (P.001; Table 1). The median area under the curve for total number of follicles in the entire follicular phase was 59 for poor responders, whereas the median area under the curve in control subjects was 218 (P.001). Number of follicles throughout the follicular phase is depicted in Figure 1. Table 2 shows baseline and late follicular phase endocrine characteristics and areas under the curve for FSH, LH, E 2, and P throughout the follicular phase. Again, all parameters were tested for significance after correcting for age. Figure 2 depicts day-to-day hormone concentrations in poor responders and healthy controls. The median cycle day 3 FSH in the poor-responder patients was 10.3 IU/L (range, ) compared with 5.1 IU/L (range, ) in controls (P.001). The maximum follicular FSH level (FSH max ) was also higher in poor responders: 11.6 IU/L ( ) vs. 6.2 IU/L ( ) in the control subjects (P.001). In the poor responders, we observed a significantly higher area under the curve for P levels in the follicular phase compared with the case of controls (P.001; Table 2). DISCUSSION In this study, a reduced number of small follicles throughout the entire follicular phase was found in women who had shown a poor response during preceding IVF treatment compared with controls. This observation is consistent with recent data from regularly cycling women aged years that showed a high correlation between number of antral follicles and reproductive age (30). The current control group was not matched for age with the study group. However after correcting for age, the observed difference in amount of antral follicles remains highly significant (P.0002). To illustrate this, Figure 3 shows the association between age and number of antral follicles, overall and for the two groups separately. In the middle age range (30 37), where both groups are represented, a large difference in number of antral follicles is demonstrated (see boxed area of Fig. 3). The straight line gives the best fit to the total data when group membership is ignored. Almost all poor responders are below this line, indicating that a separate, lower regression line for them would be more appropriate, as shown by the analysis of covariance (line not shown in Fig. 3). Normo-ovulatory regularly cycling women with no overt sign of ovarian aging can unexpectedly turn out to be poor responders to ovarian hyperstimulation for IVF. Their chances of getting pregnant are extremely low. However the present study shows that preclinical signs of ovarian aging may be detectable by ultrasound examination in the form of a relatively low number of antral follicles. This finding is consistent with recent observations indicating that ovarian response can be predicted by assessing ovarian volume (31) and the total number of antral follicles present in the early 294 Beckers et al. The follicular phase in poor responders Vol. 78, No. 2, August 2002

5 FIGURE 2 Follicular phase serum levels of FSH, LH, E 2, and P. The gray areas depict the 25th- and 75th-percentile levels of the healthy controls; the black lines depict median and 25th- and 75th-percentile levels of poor responders. follicular phase of the spontaneous menstrual cycle (12) or after downregulation (32, 33). Sonographic features seem to be better predictors of ovarian response than chronological age, baseline FSH (12), or inhibin levels, which change later in the process of ovarian aging (20). In our study, the sonographic findings were indeed much more striking than the differences in endocrine parameters between poor responders and controls. In 5 of the 11 poor responder patients, cycle day 3 FSH levels were within the normal range for our laboratory. This is in agreement with other publications showing normal cycle day 3 FSH levels to be an unreliable predictor for response to ovarian hyperstimulation. In 67% of patients with an increased FSH, a poor response was observed, whereas 16% of patients with normal FSH levels showed a poor response (8). Other studies have also shown a relatively high prevalence of normal FSH levels in poor responders, as reflected by the large standard deviation within this patient population (12). Early follicular phase FSH levels may therefore represent a late indication of ovarian aging (20). The median cycle day at which FSH max was reached was day 4 (range, days 2 9). Thus in 5 of 11 patients, FSH max was reached later than cycle day 3. Only in patients showing extremely few follicles (one or two follicles) on day 3 was the FSH max level consistently above the normal range ( IU/L). Both on cycle day 3 and on cycle day 5, inhibin B levels were within the normal ranges in all but 2 of the 11 patients. Inhibin A levels 1 day before the LH surge were also within normal limits. These findings do not support the concept of inhibin A or inhibin B as a good predictor for poor response. It has been shown that a decrease in cycle day 3 inhibin B preceded the rise in cycle day 3 FSH levels in aging women and poor responders (9). In our study, however, more patients were found to have an increased FSH max or increased day 3 FSH compared with decreased inhibin B. This apparent discrepancy may be due in part to the small number of patients studied. The unexpected finding of significantly increased P levels throughout the follicular phase of the spontaneous unstimulated cycle in poor responders has not been reported in earlier studies. In a single study describing hormonal changes in inhibins and steroids during the menstrual cycle of older women, relatively high follicular P levels were reported (34). It can be postulated that P is produced by luteinized granulosa cells either from the corpora lutea remaining from preceding cycles or from premature luteinized granulosa cells from the follicles in the present cycle. As the P levels are already significantly higher in the early follicular phase, continued production by the corpus luteum seems the most likely explanation. A correlation between P concentrations and the time interval between the study cycle and the preceding IVF attempt could not be detected (data not shown). The possibility of premature luteinization seems unlikely because LH levels were similar in both groups. FERTILITY & STERILITY 295

6 FIGURE 3 The association between age and number of follicles overall and for poor responders ( ) and healthy controls ( ). The gray area indicates the overlap of age in the poor-responder and the healthy-control groups. In a previous study, 13% of 400 women with unexplained infertility showed premature luteinization and elevated P levels in their spontaneous cycle (35). When these patients were treated with exogenous gonadotropins, premature luteinization did not occur and the resulting pregnancy rates were high (58%) (35), excluding the possibility of ovarian aging in these women. Premature luteinization has been more frequently described in patients undergoing ovarian hyperstimulation for IVF. Although the clinical relevance remains uncertain, recent observations have indicated that premature luteinization in IVF cycles may be an early manifestation of low ovarian reserve (36). Recent data suggested that in older women with increased early follicular FSH levels and decreased inhibin B levels, follicular phase P level is normal. However, in this group, luteal phase P is depressed (15, 34). The physiological background and clinical implication of elevated follicular phase P levels remain uncertain. In conclusion, the results of the present study support the premise that poor response to ovarian hyperstimulation for IVF may indeed represent the first clinical sign of ovarian aging, preceding the onset of endocrine changes or subtle cycle abnormalities. However, this phenomenon appears to be accompanied, and possibly preceded, by detectable abnormalities in the follicular phase of the spontaneous cycle. Ovarian aging remains a challenging clinical problem for which therapeutic options are limited. Improving our understanding of the mechanisms behind ovarian aging may ultimately lead to more effective and timely therapeutic interventions. References 1. 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