- Email: [email protected]
Early follicle development alters the relationship between antral follicle counts and inhibin B and follicle-stimulating hormone levels on cycle day 3
REPRODUCTIVE ENDOCRINOLOGY Early follicle development alters the relationship between antral follicle counts and inhibin B and follicle-stimulating hormone levels on cycle day 3 Michael Grynberg, M.D.,a,b,c Estelle Feyereisen, M.D.,a Juliano Brum Scheffer, M.D.,a Panayotis Koutroubis, M.D.,a Rene Frydman, M.D.,a,b,c and Renato Fanchin, M.D., Ph.D.a,b,c a
Assistance Publique-H^opitaux de Paris (AP-HP), Service de Gynecologie-Obstetrique et Medecine de la Reproduction, H^opital Antoine Beclere, Clamart, France; b Universite Paris-Sud, Clamart, France; and c Institut National de la Sante et de la Recherche Medicale, U782, Clamart, France
Objective: To verify whether, during the luteal–follicular transition, increased inhibin B production by abnormally large follicles modifies the expected relationship between the number of follicles and peripheral inhibin B and FSH levels. Design: Prospective study. Setting: Teaching hospital, France. Patient(s): A total of 192 normo-ovulatory women. Intervention(s): Serum inhibin B and FSH levels and numbers and sizes of antral follicles (3–12 mm) were measured on cycle day 3. Main Outcome Measure(s): The strength of hormonal–follicular correlations was assessed in two groups formed according to the presence (large follicle group; n ¼ 73) or absence (small follicle group; n ¼ 119) of one follicle measuring >7 mm. Result(s): Serum inhibin B and FSH levels were correlated with antral follicle counts (r ¼ 0.32 and r ¼ 0.44, respectively). These relationships were significantly weaker in the large follicle group (r ¼ 0.24 and r ¼ 0.28, respectively) than in the small follicle group (r ¼ 0.51 and r ¼ 0.55, respectively). Conclusion(s): Antral follicle size influences serum inhibin B and FSH levels and alters their expected relationship with the number of antral follicles on day 3. These results contribute to clarifying the controversial role of serum inhibin B and FSH levels in the prediction of ovarian follicular status. (Fertil Steril 2010;93:894–9. 2010 by American Society for Reproductive Medicine.) Key Words: Inhibin B, FSH, follicle count, ovarian reserve, menstrual cycle
The progressive loss of female fertility potential with age is essentially due to the quantitative and qualitative attrition of ovarian follicles, a process that accelerates during the forth decade of life. Indeed, from the early 30s to the early 40s, women are expected to exhaust three quarter of their follicular reserve (1). This issue assumes a pivotal importance in reproductive medicine, given that ovarian aging is the single most important factor in determining the effectiveness of treatments, in particular for women who delay the age of first childbearing. Given that the adult ovary is a set of follicles at different stages of growth and whose ability to proReceived August 1, 2008; revised October 8, 2008; accepted October 17, 2008; published online December 4, 2008. M.G. has nothing to disclose. E.F. has nothing to disclose. J.B.S. has nothing to disclose. P.K. has nothing to disclose. R. Frydman has nothing to disclose. R. Fanchin has nothing to disclose. Reprint requests: Michael Grynberg, M.D., Department of Obstetrics and ^ pital Antoine Be cle re, 157 Gynecology and Reproductive Medicine, Ho rue de la Porte de Trivaux, 92141 Clamart, France (FAX: þ33 1 45 37 49 80; E-mail: [email protected]).
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duce hormones is heterogeneous, the clinical assessment of the extent of follicle loss by the exclusive analysis of peripheral hormonal levels often is problematic. This physiologic contingency constitutes an attractive explanation for the debated reliability of some hormonal predictors of ovarian follicular status, such as serum inhibin B (2–4), E2 (5), and FSH (4, 6, 7) measurements performed on cycle day 3, which are essentially based on the activity of cyclic antral follicles. Untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women (8, 9) and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase, is likely to modify the expected relationship between hormonal parameters and the number of antral follicles. Because, by definition, outsized antral follicles contain more granulosa cells, their ability to produce inhibin B (10, 11), and in some severe cases E2 (12), is possibly increased as compared with timely selected,
Fertility and Sterility Vol. 93, No. 3, February 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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small follicles. This, in turn, significantly attenuates FSH secretion (13) and dissociates the profile of these hormones from the underlying ovarian reserve of antral follicles. Hence, to test this hypothesis, we compared the strengths of the relationships between serum inhibin B, E2, and FSH levels and the number of antral follicles in normo-ovulatory women displaying or not advanced follicle growth on cycle day 3. MATERIALS AND METHODS Subjects One hundred ninety-two infertile women, aged 19–42 years, undergoing routine exploration during an unstimulated cycle that preceded assisted reproductive technologies at our center were studied prospectively. All patients met the following inclusion criteria: [1] regular, ovulatory menstrual cycles every 25–35 days, [2] both ovaries present, [3] no clinical signs of hyperandrogenism, [4] no current or past diseases affecting ovaries or gonadotropin or sex steroid secretion, clearance, or excretion, [5] no current hormone therapy, [6] adequate visualization of ovaries at transvaginal ultrasound scans, and [7] total number of small antral follicles (3–12 mm in diameter) between 1 and 24 follicles, including both ovaries. All patients signed an informed consent form for this analysis. Because the present study was merely observational and included only data from routine measurements, it did not require previous submission to our institutional review board. Hormonal Measurements and Ultrasound Scans On cycle day 3, each woman underwent blood sampling by venipuncture for serum inhibin B, E2, and FSH measurement and a transvaginal ovarian ultrasound scan for follicle measurement. Serum inhibin B levels were determined using a doubleantibody ELISA (Serotec, Varilhes, France) as described previously (10). For inhibin B, functional sensitivity was 15 pg/ mL, and intra-assay and interassay coefficients of variation (CV) were <6% and <9%, respectively. Serum levels of E2 and FSH were determined using an automated multianalysis system with chemiluminescence detection (ACS-180; Bayer Diagnostics, Puteaux, France). For E2, functional sensitivity was 15 pg/mL, and intra-assay and interassay CV were 8% and 9%, respectively. For FSH, functional sensitivity was 0.1 mIU/mL, and intra-assay and interassay CV were 3% and 5%, respectively. Ultrasound scans were performed using a 3.7–9.3 MHz multifrequency transvaginal probe (RIC5-9H; General Electric Medical Systems, Paris, France) by a single operator who was blinded to the results of hormone assays. The objective of ultrasound examination was to evaluate the number and size of small antral follicles. Follicles measuring 3–12 mm in mean diameter (mean of two orthogonal diameters) in both ovaries were considered. To optimize the reliability of ovarian follicular assessment, the ultrasound scanner was equipFertility and Sterility
ped with a tissue harmonic imaging system (14), which allowed improved image resolution and adequate recognition of follicular borders. Intra-analysis CV for follicular and ovarian measurements were <5%, and their lower limit of detection was 0.1 mm. In an effort to evaluate the bulk of granulosa cells in both ovaries, we calculated the mean follicle diameter (cumulative follicle diameter divided by the number of follicles measuring 3–12 mm in diameter in both ovaries) and the largest follicle diameter. Definition of Groups To study the possible influence of antral follicle size on the relationship between antral follicle count and serum inhibin B, E2, and FSH levels, participants were arbitrarily sorted into two groups. The first group (small follicle group; n ¼ 119) comprised women having all follicles %7 mm in diameter; the second group (large follicle group; n ¼ 73) comprised those presenting at least one follicle measuring >7 mm. The choice of 7 mm as a cutoff to discriminate cases with early follicle development was arbitrary. It corresponded to the 95% percentile of follicle size distribution of the 2,433 follicles included in the present series, and it was based both on our own experience of typical follicle sizes and that of others (15, 16), according to which it is unusual to observe >7-mm follicles on cycle day 3 in normally cycling women. In an additional analysis, participants were sorted into four different groups according to both follicle size and follicle count. Women with <12 follicles and no follicle >7 mm in diameter were included in the few-small group (n ¼ 48); those with <12 follicles and at least one follicle >7 mm in diameter were included in the few-large group (n ¼ 38); women with R12 follicles and no follicle >7 mm in diameter were included in the many-small (n ¼ 71) group; and those with R12 follicles and at least one follicle >7 mm in diameter (n ¼ 35) were included in the many-large group. The choice of 12 follicles as a quantitative cutoff was also arbitrary and corresponded to the 50th percentile of follicle counts in the present series and elsewhere (16). Statistical Analysis The measures of central tendency and of variability used were the mean and SE when data distribution was normal, and median and range when normality could not be ascertained. Differences between small and large follicle groups were evaluated with the Student’s t or the Mann-Whitney tests, when appropriate. Differences among the few-small, few-large, many-small, and many-large groups were evaluated with analysis of variance or the Kruskall-Wallis test, when appropriate. Relationships between two different continuous variables were assessed by correlation. The Fisher r to z test was used to determine whether the coefficient of correlation (r) was significantly different from zero. Strengths of correlations were compared using comparison of correlation coefficients. A P value of < .05 was considered statistically significant. 895
TABLE 1 Patient and follicle characteristics in the small and large follicle groups. Variable
Small follicle group (n [ 119)
Large follicle group (n [ 73)
P value
33.8 0.4 13.3 0.5 7 (0–20) 4 (0–16) 0 (0–0) 57.8 2.3 4.3 0.1 6 (3–7) 64.6 1.9 32.8 2.2 8.2 0.4
34.6 0.4 11.6 0.6 5 (0–18) 3 (0–10) 1 (1–3) 56.8 2.7 5.1 0.1 8 (8–12) 77.9 3.6 45.7 3.2 7.1 0.3
NS < .03 < .001 < .04 < .001 NS < .001 < .001 < .001 < .001 < .05
Ages (y) No. of follicles 3–12 mm No. of follicles 3–4 mm No. of follicles 5–6 mm No. of follicles >7 mm Total follicle diameter (mm)a Mean follicle diameter (mm)b Largest follicle diameter (mm) Serum inhibin B level (pg/mL) Serum E2 level (pg/mL) Serum FSH level (mIU/mL)
Note: Values are mean SE or median (range). NS ¼ nonsignificant. a Sum of diameters of follicles measuring 3–12 mm. b Total follicle diameter divided by no. of follicles 3–12 mm. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
RESULTS Patient and Follicle Characteristics Overall, at the time of the investigation, patients were 34.1 0.3 years of age and had a median menstrual cycle length of 27.8 0.2 days. Their ovaries contained 12.7 0.4 antral follicles, with a mean diameter of 4.6 0.1 mm; the cumulative size of these follicles reached 57.4 1.8 mm. Thirty-eight percent of patients (n ¼ 73) displayed at least one follicle measuring >7 mm in diameter.
Hormonal–Follicular Relationships According to Follicle Size Overall, the number of 3–12-mm follicles was significantly correlated with serum inhibin B (r ¼ 0.32, P<.001) and FSH (r ¼ 0.44, P<.001) levels but failed to correlate with serum E2 levels (r ¼ 0.08, not significant). The size of the largest antral follicle and the number of follicles exceeding 7 mm were correlated with serum levels of inhibin B (r ¼ 0.35, P<.001 and r ¼ 0.22, P<.003, respectively), E2 (r ¼ 0.31, P<.001 and r ¼ 0.20, P<.004, respectively), and FSH (r ¼ 0.25, P<.001 and r ¼ 0.16, P<.03, respectively).
Patient and follicle characteristics in the small and large follicle groups are summarized in Table 1. Although the number of antral follicles was significantly smaller in the large as compared with the small follicle group, serum inhibin B levels were higher and serum FSH levels were lower in the large as compared with the small antral follicle group. In addition, serum E2 levels were significantly higher in the large than in the small follicle group.
The strength of hormonal–follicular relationships in the small and large follicle groups is displayed in Table 2. As shown, the intensity of the relationships (r values) between the number of 3–12-mm follicles and serum inhibin B and FSH levels varied according to the presence or the absence
TABLE 2 Strength of hormonal–follicular relationships in the small and large follicle groups. Correlation coefficients (r) Variable
Small follicle group (n [ 119) Large follicle group (n [ 73) P value
No. of follicles (3–12 mm) serum inhibin B levels No. of follicles (3–12 mm) serum E2 levels No. of follicles (3–12 mm) serum FSH levels
0.51
0.24
< .04
0.07
0.01
NS
0.55
0.28
< .03
Note: NS ¼ nonsignificant. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
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TABLE 3 Hormonal–follicular relationships in the few-small, few-large, many-small, and many-largea groups. P value
Variable Age (y) Menstrual cycle length (d) No. of follicles (3–12 mm) Total follicle diameter (mm) Largest follicle diameter (mm) Serum inhibin B level (pg/mL) Serum E2 level (pg/mL) Serum FSH level (mIU/mL)
I Few-small (n [ 48)
II Few-large (n [ 38)
III Many-small (n [ 71)
IV Many-large (n [ 35)
(I vs. II)
(I vs. III)
(I vs. IV)
(II vs. III)
(II vs. IV)
(III vs. IV)
34.6 0.6 27.4 0.4
34.9 0.6 26.8 0.5
33.2 0.4 28.2 0.3
34.2 0.6 28.5 0.4
NS NS
< .05 NS
NS < .05
< .03 < .01
NS < .005
NS NS
8.5 (1–11)
8.0 (2–11)
17.0 (12–24)
16.0 (12–24)
NS
< .001
< .001
< .001
< .001
NS
33.4 1.8
39.5 1.9
74.3 2.1
75.6 2.7
NS
< .001
< .001
< .001
< .001
NS
6 (3–7)
9 (8–12)
6 (4–7)
8 (8–12)
< .001
NS
< .001
< .001
< .004
< .001
54.6 3.2
69.9 4.1
71.4 2.0
86.6 5.6
< .004
< .001
< .001
NS
< .003
< .003
33.2 3.9 10.5 0.9
46.0 4.9 7.7 0.4
32.7 2.6 6.7 0.2
45.5 4.2 6.5 0.3
< .03 < .001
NS < .001
< .04 < .001
< .01 NS
NS NS
< .02 NS
Note: Values are mean SE or median (range). NS ¼ nonsignificant. a Few-small group (I): <12 follicles and no follicle >7 mm in diameter; few-large group (II): <12 follicles and at least one follicle R8 mm in diameter; many-small group (III): R12 follicles and no follicle >7 mm in diameter; many-large group (IV): R12 follicles and at least one follicle R8 mm in diameter. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
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of a follicle measuring >7 mm in diameter. It was significantly stronger in the small as compared with the large follicle groups for inhibin B (P<.04) and for FSH (P<.03). Incidentally, serum FSH levels were more strongly correlated (P<.003) with serum inhibin B (r ¼ 0.46, P<.001) than with serum E2 (r ¼ 0.16, P<.02) levels and, as expected, menstrual cycle lengths were negatively correlated with the size of the largest follicle (r ¼ 0.16, P<.03). Hormonal–Follicular Relationships According to Follicle Size and Follicle Count Hormonal–follicular relationships in the few-small, fewlarge, many-small, and many-large groups are summarized in Table 3. As shown, in keeping with data presented above, women included in the few-large group had shorter menstrual cycle duration but astonishingly comparable inhibin B and FSH levels as those included in the many-small group, whereas patients included in the few-small group had, as expected, lower serum inhibin B and higher serum FSH levels than those of the many-small group. In addition, serum E2 levels tended to be higher in women presenting at least one >7-mm follicle, irrespective of follicle counts. DISCUSSION The present investigation was designed to test the hypothesis that advanced follicle development during the luteal–follicular transition may alter the expected relationship between serum inhibin B and FSH levels and the number of antral follicles on cycle day 3. We hypothesized that an outsized follicle would by itself be capable of producing as much inhibin B as a number of smaller follicles, a phenomenon that could therefore dissociate antral follicle count from the corresponding peripheral inhibin B and FSH levels. From a clinical standpoint, this work also aimed at offering an explanation to the debated predictability of these latter hormonal markers (2, 6, 7). This issue assumes a particular importance, given that untimely and/or advanced follicle growth during the luteal–follicular transition is often observed in ovarian-aged patients (8, 9), a population that specifically requires careful and reliable evaluation of their ovarian follicle status. Our data showed that women presenting at least one overdeveloped follicle also had fewer antral follicles and tended to present shorter menstrual cycles than those who did not. This confirms and expands the reported association between untimely and/or accelerated antral follicle growth and ovarian aging (8, 9). Besides, in women showing at least one overdeveloped follicle, the expected relationship between serum inhibin B and FSH levels and antral follicle count was markedly weaker as compared with those having only small follicles. This indicates that, in patients presenting a reduced number of antral follicles, the activity of outsized follicles may increase inhibin B and reduce FSH levels. The correlation between the size of the largest antral follicle and serum inhibin B levels (positive) and serum FSH levels (negative) supports this contention and is in keeping with previous 898
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data (16). In agreement with this, women combining reduced follicle count with the presence of at least one >7-mm follicle (few-large group) showed similar inhibin B and FSH levels as compared with those having adequate follicle count and no outsized follicle (many-small group), despite different ovarian follicle numbers. Furthermore, women with at least one large antral follicle also presented a slight but significant elevation in serum E2 levels as compared with those who had only %7-mm follicles. Accordingly, a correlation between serum E2 levels and the size of the largest antral follicle was shown, which confirms that the more intense is the process of follicle growth advancement, the more intense the ability of follicle cells to produce E2, as a probable result of aromatase expression (17). Although it is likely that the increase in inhibin B production by outsized follicles preceded that of E2 production (10) at least in some patients, and both phenomena concurred to reduce circulating FSH, the present data did not contribute to clarifying these issues. In conclusion, our results indicate that the presence of an overdeveloped antral follicle on cycle day 3 alters the strength of the relationship between antral follicle count and serum inhibin B and FSH levels. This phenomenon discourages the use of serum inhibin B and FSH measurements as exclusive indicators of ovarian follicular status. To improve the reliability and interpretation of these hormonal markers, careful ultrasonographic evaluation not only of follicle count but also of follicle size should be routinely performed. Clinical measures aiming at preventing the occurrence of premature follicle selection during the luteal–follicular transition, such as luteal E2 (18) or premenstrual GnRH antagonist administration (19), may be useful to improve the relationship between inhibin B and FSH and ovarian follicle status, in the case of unavailability of ovarian ultrasound scans. Finally, anti-M€ullerian hormone, a glycoprotein that is produced, presumably FSH-independently (20, 21), by the granulosa cells (22) of both cyclic and noncyclic follicles (23–25), may be less influenced by such disorders of antral follicle development during the luteal– follicular transition. Further clinical investigation is necessary to address this point. Acknowledgment: The authors thank Dr. Alain Gougeon for his kind revision of the work and advice that greatly improved the manuscript.
REFERENCES 1. Block E. Quantitative morphological investigations of the follicular system in women: variations at different ages. Acta Anat 1952;14:108–23. 2. Corson SL, Gutmann J, Batzer FR, Wallace H, Klein N, Soules MR. Inhibin-B as a test of ovarian reserve for infertile women. Hum Reprod 1999;14:2818–21. 3. Hall JE, Welt CK, Cramer DW. Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum Reprod 1999;14:409–15. 4. Creus M, Penarrubia J, Fabregues F, Vidal E, Carmona F, Casamitjana R, et al. Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum Reprod 2000;15:2341–6.
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5. Bancsi LF, Huijs AM, den Ouden CT, Broekmans FJ, Looman CW, Blankenstein MA, et al. Basal follicle-stimulating hormone levels are of limited value in predicting ongoing pregnancy rates after in vitro fertilization. Fertil Steril 2000;73:552–7. 6. van Rooij IA, de Jong E, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. High follicle-stimulating hormone levels should not necessarily lead to the exclusion of subfertile patients from treatment. Fertil Steril 2004;81:1478–85. 7. Phophong P, Ranieri DM, Khadum I, Meo F, Serhal P. Basal 17beta-estradiol did not correlate with ovarian response and in vitro fertilization treatment outcome. Fertil Steril 2000;74:1133–6. 8. Klein NA, Harper AJ, Houmard BS, Sluss PM, Soules MR. Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development? J Clin Endocrinol Metab 2002;87:5746–50. 9. Santoro N, Isaac B, Neal-Perry G, Adel T, Weingart L, Nussbaum A, et al. Impaired folliculogenesis and ovulation in older reproductive aged women. J Clin Endocrinol Metab 2003;88:5502–9. 10. Groome NP, Illingworth PJ, O’Brien M, Pai R, Rodger FE, Mather JP, et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81:1401–5. 11. Magoffin DA, Jakimiuk AJ, Inhibin A, inhibin B, activin A. in the follicular fluid of regularly cycling women. Hum Reprod 1997;2:1714–9. 12. Baird DT, Fraser IS. Blood production and ovarian secretion rates of estradiol-17 beta and estrone in women throughout the menstrual cycle. J Clin Endocrinol Metab 1974;38:1009–17. 13. Welt CK, Martin KA, Taylor AE, Lambert-Messerlian GM, Crowley WF Jr, Smith JA, et al. Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women. J Clin Endocrinol Metab 1997;82:2645–52. 14. Thomas JD, Rubin DN. Tissue harmonic imaging: why does it work? J Am Soc Echocardiogr 1998;11:803–8. 15. Pache TD, Wladimiroff JW, de Jong FH, Hop WC, Fauser BC. Growth patterns of nondominant ovarian follicles during the normal menstrual cycle. Fertil Steril 1990;54:638–42. 16. Haadsma ML, Bukman A, Groen H, Roeloffzen EM, Groenewoud ER, Heineman MJ, et al. The number of small antral follicles (2-6 mm)
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determines the outcome of endocrine ovarian reserve tests in a subfertile population. Hum Reprod 2007;22:1925–31. Erickson GF, Hsueh AJ, Quigley ME, Rebar RW, Yen SS. Functional studies of aromatase activity in human granulosa cells from normal and polycystic ovaries. J Clin Endocrinol Metab 1979;49:514–9. Fanchin R, Cunha-Filho JS, Schonauer LM, Righini C, de Ziegler D, Frydman R. Luteal estradiol administration strengthens the relationship between day 3 follicle-stimulating hormone and inhibin B levels and ovarian follicular status. Fertil Steril 2003;79:585–9. Fanchin R, Castelo Branco A, Kadoch IJ, Hosny G, Bagirova M, Frydman R. Premenstrual administration of gonadotropin-releasing hormone antagonist coordinates early antral follicle sizes and sets up the basis for an innovative concept of controlled ovarian hyperstimulation. Fertil Steril 2004;81:1554–9. Bath LE, Wallace WH, Shaw MP, Fitzpatrick C, Anderson RA. Depletion of ovarian reserve in young women after treatment for cancer in childhood: detection by anti-Mullerian hormone, inhibin B and ovarian ultrasound. Hum Reprod 2003;18:2368–74. Eldar-Geva T, Ben-Chetrit A, Spitz IM, Rabinowitz R, Markowitz E, Mimoni T, et al. Dynamic assays of inhibin B, anti-Mullerian hormone and estradiol following FSH stimulation and ovarian ultrasonography as predictors of IVF outcome. Hum Reprod 2005;20:3178–83. Vigier B, Picard JY, Tran D, Legeai L, Josso N. Production of antiM€ullerian hormone: another homology between Sertoli and granulosa cells. Endocrinology 1984;114:1315–20. Baarends WM, Uilenbroek JT, Kramer P, Hoogerbrugge JW, van Leeuwen EC, Themmen AP, et al. Anti-mullerian hormone and anti-mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 1995;136:4951–62. Durlinger AL, Visser JA, Themmen AP. Regulation of ovarian function: the role of anti-Mullerian hormone. Reproduction 2002;124:601–9. Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser JA, et al. Anti-Mullerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 2004;10:77–83.
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Assistance Publique-H^opitaux de Paris (AP-HP), Service de Gynecologie-Obstetrique et Medecine de la Reproduction, H^opital Antoine Beclere, Clamart, France; b Universite Paris-Sud, Clamart, France; and c Institut National de la Sante et de la Recherche Medicale, U782, Clamart, France
Objective: To verify whether, during the luteal–follicular transition, increased inhibin B production by abnormally large follicles modifies the expected relationship between the number of follicles and peripheral inhibin B and FSH levels. Design: Prospective study. Setting: Teaching hospital, France. Patient(s): A total of 192 normo-ovulatory women. Intervention(s): Serum inhibin B and FSH levels and numbers and sizes of antral follicles (3–12 mm) were measured on cycle day 3. Main Outcome Measure(s): The strength of hormonal–follicular correlations was assessed in two groups formed according to the presence (large follicle group; n ¼ 73) or absence (small follicle group; n ¼ 119) of one follicle measuring >7 mm. Result(s): Serum inhibin B and FSH levels were correlated with antral follicle counts (r ¼ 0.32 and r ¼ 0.44, respectively). These relationships were significantly weaker in the large follicle group (r ¼ 0.24 and r ¼ 0.28, respectively) than in the small follicle group (r ¼ 0.51 and r ¼ 0.55, respectively). Conclusion(s): Antral follicle size influences serum inhibin B and FSH levels and alters their expected relationship with the number of antral follicles on day 3. These results contribute to clarifying the controversial role of serum inhibin B and FSH levels in the prediction of ovarian follicular status. (Fertil Steril 2010;93:894–9. 2010 by American Society for Reproductive Medicine.) Key Words: Inhibin B, FSH, follicle count, ovarian reserve, menstrual cycle
The progressive loss of female fertility potential with age is essentially due to the quantitative and qualitative attrition of ovarian follicles, a process that accelerates during the forth decade of life. Indeed, from the early 30s to the early 40s, women are expected to exhaust three quarter of their follicular reserve (1). This issue assumes a pivotal importance in reproductive medicine, given that ovarian aging is the single most important factor in determining the effectiveness of treatments, in particular for women who delay the age of first childbearing. Given that the adult ovary is a set of follicles at different stages of growth and whose ability to proReceived August 1, 2008; revised October 8, 2008; accepted October 17, 2008; published online December 4, 2008. M.G. has nothing to disclose. E.F. has nothing to disclose. J.B.S. has nothing to disclose. P.K. has nothing to disclose. R. Frydman has nothing to disclose. R. Fanchin has nothing to disclose. Reprint requests: Michael Grynberg, M.D., Department of Obstetrics and ^ pital Antoine Be cle re, 157 Gynecology and Reproductive Medicine, Ho rue de la Porte de Trivaux, 92141 Clamart, France (FAX: þ33 1 45 37 49 80; E-mail: [email protected]).
894
duce hormones is heterogeneous, the clinical assessment of the extent of follicle loss by the exclusive analysis of peripheral hormonal levels often is problematic. This physiologic contingency constitutes an attractive explanation for the debated reliability of some hormonal predictors of ovarian follicular status, such as serum inhibin B (2–4), E2 (5), and FSH (4, 6, 7) measurements performed on cycle day 3, which are essentially based on the activity of cyclic antral follicles. Untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women (8, 9) and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase, is likely to modify the expected relationship between hormonal parameters and the number of antral follicles. Because, by definition, outsized antral follicles contain more granulosa cells, their ability to produce inhibin B (10, 11), and in some severe cases E2 (12), is possibly increased as compared with timely selected,
Fertility and Sterility Vol. 93, No. 3, February 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2008.10.025
small follicles. This, in turn, significantly attenuates FSH secretion (13) and dissociates the profile of these hormones from the underlying ovarian reserve of antral follicles. Hence, to test this hypothesis, we compared the strengths of the relationships between serum inhibin B, E2, and FSH levels and the number of antral follicles in normo-ovulatory women displaying or not advanced follicle growth on cycle day 3. MATERIALS AND METHODS Subjects One hundred ninety-two infertile women, aged 19–42 years, undergoing routine exploration during an unstimulated cycle that preceded assisted reproductive technologies at our center were studied prospectively. All patients met the following inclusion criteria: [1] regular, ovulatory menstrual cycles every 25–35 days, [2] both ovaries present, [3] no clinical signs of hyperandrogenism, [4] no current or past diseases affecting ovaries or gonadotropin or sex steroid secretion, clearance, or excretion, [5] no current hormone therapy, [6] adequate visualization of ovaries at transvaginal ultrasound scans, and [7] total number of small antral follicles (3–12 mm in diameter) between 1 and 24 follicles, including both ovaries. All patients signed an informed consent form for this analysis. Because the present study was merely observational and included only data from routine measurements, it did not require previous submission to our institutional review board. Hormonal Measurements and Ultrasound Scans On cycle day 3, each woman underwent blood sampling by venipuncture for serum inhibin B, E2, and FSH measurement and a transvaginal ovarian ultrasound scan for follicle measurement. Serum inhibin B levels were determined using a doubleantibody ELISA (Serotec, Varilhes, France) as described previously (10). For inhibin B, functional sensitivity was 15 pg/ mL, and intra-assay and interassay coefficients of variation (CV) were <6% and <9%, respectively. Serum levels of E2 and FSH were determined using an automated multianalysis system with chemiluminescence detection (ACS-180; Bayer Diagnostics, Puteaux, France). For E2, functional sensitivity was 15 pg/mL, and intra-assay and interassay CV were 8% and 9%, respectively. For FSH, functional sensitivity was 0.1 mIU/mL, and intra-assay and interassay CV were 3% and 5%, respectively. Ultrasound scans were performed using a 3.7–9.3 MHz multifrequency transvaginal probe (RIC5-9H; General Electric Medical Systems, Paris, France) by a single operator who was blinded to the results of hormone assays. The objective of ultrasound examination was to evaluate the number and size of small antral follicles. Follicles measuring 3–12 mm in mean diameter (mean of two orthogonal diameters) in both ovaries were considered. To optimize the reliability of ovarian follicular assessment, the ultrasound scanner was equipFertility and Sterility
ped with a tissue harmonic imaging system (14), which allowed improved image resolution and adequate recognition of follicular borders. Intra-analysis CV for follicular and ovarian measurements were <5%, and their lower limit of detection was 0.1 mm. In an effort to evaluate the bulk of granulosa cells in both ovaries, we calculated the mean follicle diameter (cumulative follicle diameter divided by the number of follicles measuring 3–12 mm in diameter in both ovaries) and the largest follicle diameter. Definition of Groups To study the possible influence of antral follicle size on the relationship between antral follicle count and serum inhibin B, E2, and FSH levels, participants were arbitrarily sorted into two groups. The first group (small follicle group; n ¼ 119) comprised women having all follicles %7 mm in diameter; the second group (large follicle group; n ¼ 73) comprised those presenting at least one follicle measuring >7 mm. The choice of 7 mm as a cutoff to discriminate cases with early follicle development was arbitrary. It corresponded to the 95% percentile of follicle size distribution of the 2,433 follicles included in the present series, and it was based both on our own experience of typical follicle sizes and that of others (15, 16), according to which it is unusual to observe >7-mm follicles on cycle day 3 in normally cycling women. In an additional analysis, participants were sorted into four different groups according to both follicle size and follicle count. Women with <12 follicles and no follicle >7 mm in diameter were included in the few-small group (n ¼ 48); those with <12 follicles and at least one follicle >7 mm in diameter were included in the few-large group (n ¼ 38); women with R12 follicles and no follicle >7 mm in diameter were included in the many-small (n ¼ 71) group; and those with R12 follicles and at least one follicle >7 mm in diameter (n ¼ 35) were included in the many-large group. The choice of 12 follicles as a quantitative cutoff was also arbitrary and corresponded to the 50th percentile of follicle counts in the present series and elsewhere (16). Statistical Analysis The measures of central tendency and of variability used were the mean and SE when data distribution was normal, and median and range when normality could not be ascertained. Differences between small and large follicle groups were evaluated with the Student’s t or the Mann-Whitney tests, when appropriate. Differences among the few-small, few-large, many-small, and many-large groups were evaluated with analysis of variance or the Kruskall-Wallis test, when appropriate. Relationships between two different continuous variables were assessed by correlation. The Fisher r to z test was used to determine whether the coefficient of correlation (r) was significantly different from zero. Strengths of correlations were compared using comparison of correlation coefficients. A P value of < .05 was considered statistically significant. 895
TABLE 1 Patient and follicle characteristics in the small and large follicle groups. Variable
Small follicle group (n [ 119)
Large follicle group (n [ 73)
P value
33.8 0.4 13.3 0.5 7 (0–20) 4 (0–16) 0 (0–0) 57.8 2.3 4.3 0.1 6 (3–7) 64.6 1.9 32.8 2.2 8.2 0.4
34.6 0.4 11.6 0.6 5 (0–18) 3 (0–10) 1 (1–3) 56.8 2.7 5.1 0.1 8 (8–12) 77.9 3.6 45.7 3.2 7.1 0.3
NS < .03 < .001 < .04 < .001 NS < .001 < .001 < .001 < .001 < .05
Ages (y) No. of follicles 3–12 mm No. of follicles 3–4 mm No. of follicles 5–6 mm No. of follicles >7 mm Total follicle diameter (mm)a Mean follicle diameter (mm)b Largest follicle diameter (mm) Serum inhibin B level (pg/mL) Serum E2 level (pg/mL) Serum FSH level (mIU/mL)
Note: Values are mean SE or median (range). NS ¼ nonsignificant. a Sum of diameters of follicles measuring 3–12 mm. b Total follicle diameter divided by no. of follicles 3–12 mm. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
RESULTS Patient and Follicle Characteristics Overall, at the time of the investigation, patients were 34.1 0.3 years of age and had a median menstrual cycle length of 27.8 0.2 days. Their ovaries contained 12.7 0.4 antral follicles, with a mean diameter of 4.6 0.1 mm; the cumulative size of these follicles reached 57.4 1.8 mm. Thirty-eight percent of patients (n ¼ 73) displayed at least one follicle measuring >7 mm in diameter.
Hormonal–Follicular Relationships According to Follicle Size Overall, the number of 3–12-mm follicles was significantly correlated with serum inhibin B (r ¼ 0.32, P<.001) and FSH (r ¼ 0.44, P<.001) levels but failed to correlate with serum E2 levels (r ¼ 0.08, not significant). The size of the largest antral follicle and the number of follicles exceeding 7 mm were correlated with serum levels of inhibin B (r ¼ 0.35, P<.001 and r ¼ 0.22, P<.003, respectively), E2 (r ¼ 0.31, P<.001 and r ¼ 0.20, P<.004, respectively), and FSH (r ¼ 0.25, P<.001 and r ¼ 0.16, P<.03, respectively).
Patient and follicle characteristics in the small and large follicle groups are summarized in Table 1. Although the number of antral follicles was significantly smaller in the large as compared with the small follicle group, serum inhibin B levels were higher and serum FSH levels were lower in the large as compared with the small antral follicle group. In addition, serum E2 levels were significantly higher in the large than in the small follicle group.
The strength of hormonal–follicular relationships in the small and large follicle groups is displayed in Table 2. As shown, the intensity of the relationships (r values) between the number of 3–12-mm follicles and serum inhibin B and FSH levels varied according to the presence or the absence
TABLE 2 Strength of hormonal–follicular relationships in the small and large follicle groups. Correlation coefficients (r) Variable
Small follicle group (n [ 119) Large follicle group (n [ 73) P value
No. of follicles (3–12 mm) serum inhibin B levels No. of follicles (3–12 mm) serum E2 levels No. of follicles (3–12 mm) serum FSH levels
0.51
0.24
< .04
0.07
0.01
NS
0.55
0.28
< .03
Note: NS ¼ nonsignificant. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
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TABLE 3 Hormonal–follicular relationships in the few-small, few-large, many-small, and many-largea groups. P value
Variable Age (y) Menstrual cycle length (d) No. of follicles (3–12 mm) Total follicle diameter (mm) Largest follicle diameter (mm) Serum inhibin B level (pg/mL) Serum E2 level (pg/mL) Serum FSH level (mIU/mL)
I Few-small (n [ 48)
II Few-large (n [ 38)
III Many-small (n [ 71)
IV Many-large (n [ 35)
(I vs. II)
(I vs. III)
(I vs. IV)
(II vs. III)
(II vs. IV)
(III vs. IV)
34.6 0.6 27.4 0.4
34.9 0.6 26.8 0.5
33.2 0.4 28.2 0.3
34.2 0.6 28.5 0.4
NS NS
< .05 NS
NS < .05
< .03 < .01
NS < .005
NS NS
8.5 (1–11)
8.0 (2–11)
17.0 (12–24)
16.0 (12–24)
NS
< .001
< .001
< .001
< .001
NS
33.4 1.8
39.5 1.9
74.3 2.1
75.6 2.7
NS
< .001
< .001
< .001
< .001
NS
6 (3–7)
9 (8–12)
6 (4–7)
8 (8–12)
< .001
NS
< .001
< .001
< .004
< .001
54.6 3.2
69.9 4.1
71.4 2.0
86.6 5.6
< .004
< .001
< .001
NS
< .003
< .003
33.2 3.9 10.5 0.9
46.0 4.9 7.7 0.4
32.7 2.6 6.7 0.2
45.5 4.2 6.5 0.3
< .03 < .001
NS < .001
< .04 < .001
< .01 NS
NS NS
< .02 NS
Note: Values are mean SE or median (range). NS ¼ nonsignificant. a Few-small group (I): <12 follicles and no follicle >7 mm in diameter; few-large group (II): <12 follicles and at least one follicle R8 mm in diameter; many-small group (III): R12 follicles and no follicle >7 mm in diameter; many-large group (IV): R12 follicles and at least one follicle R8 mm in diameter. Grynberg. Follicle size vs. inhibin B and FSH. Fertil Steril 2010.
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of a follicle measuring >7 mm in diameter. It was significantly stronger in the small as compared with the large follicle groups for inhibin B (P<.04) and for FSH (P<.03). Incidentally, serum FSH levels were more strongly correlated (P<.003) with serum inhibin B (r ¼ 0.46, P<.001) than with serum E2 (r ¼ 0.16, P<.02) levels and, as expected, menstrual cycle lengths were negatively correlated with the size of the largest follicle (r ¼ 0.16, P<.03). Hormonal–Follicular Relationships According to Follicle Size and Follicle Count Hormonal–follicular relationships in the few-small, fewlarge, many-small, and many-large groups are summarized in Table 3. As shown, in keeping with data presented above, women included in the few-large group had shorter menstrual cycle duration but astonishingly comparable inhibin B and FSH levels as those included in the many-small group, whereas patients included in the few-small group had, as expected, lower serum inhibin B and higher serum FSH levels than those of the many-small group. In addition, serum E2 levels tended to be higher in women presenting at least one >7-mm follicle, irrespective of follicle counts. DISCUSSION The present investigation was designed to test the hypothesis that advanced follicle development during the luteal–follicular transition may alter the expected relationship between serum inhibin B and FSH levels and the number of antral follicles on cycle day 3. We hypothesized that an outsized follicle would by itself be capable of producing as much inhibin B as a number of smaller follicles, a phenomenon that could therefore dissociate antral follicle count from the corresponding peripheral inhibin B and FSH levels. From a clinical standpoint, this work also aimed at offering an explanation to the debated predictability of these latter hormonal markers (2, 6, 7). This issue assumes a particular importance, given that untimely and/or advanced follicle growth during the luteal–follicular transition is often observed in ovarian-aged patients (8, 9), a population that specifically requires careful and reliable evaluation of their ovarian follicle status. Our data showed that women presenting at least one overdeveloped follicle also had fewer antral follicles and tended to present shorter menstrual cycles than those who did not. This confirms and expands the reported association between untimely and/or accelerated antral follicle growth and ovarian aging (8, 9). Besides, in women showing at least one overdeveloped follicle, the expected relationship between serum inhibin B and FSH levels and antral follicle count was markedly weaker as compared with those having only small follicles. This indicates that, in patients presenting a reduced number of antral follicles, the activity of outsized follicles may increase inhibin B and reduce FSH levels. The correlation between the size of the largest antral follicle and serum inhibin B levels (positive) and serum FSH levels (negative) supports this contention and is in keeping with previous 898
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data (16). In agreement with this, women combining reduced follicle count with the presence of at least one >7-mm follicle (few-large group) showed similar inhibin B and FSH levels as compared with those having adequate follicle count and no outsized follicle (many-small group), despite different ovarian follicle numbers. Furthermore, women with at least one large antral follicle also presented a slight but significant elevation in serum E2 levels as compared with those who had only %7-mm follicles. Accordingly, a correlation between serum E2 levels and the size of the largest antral follicle was shown, which confirms that the more intense is the process of follicle growth advancement, the more intense the ability of follicle cells to produce E2, as a probable result of aromatase expression (17). Although it is likely that the increase in inhibin B production by outsized follicles preceded that of E2 production (10) at least in some patients, and both phenomena concurred to reduce circulating FSH, the present data did not contribute to clarifying these issues. In conclusion, our results indicate that the presence of an overdeveloped antral follicle on cycle day 3 alters the strength of the relationship between antral follicle count and serum inhibin B and FSH levels. This phenomenon discourages the use of serum inhibin B and FSH measurements as exclusive indicators of ovarian follicular status. To improve the reliability and interpretation of these hormonal markers, careful ultrasonographic evaluation not only of follicle count but also of follicle size should be routinely performed. Clinical measures aiming at preventing the occurrence of premature follicle selection during the luteal–follicular transition, such as luteal E2 (18) or premenstrual GnRH antagonist administration (19), may be useful to improve the relationship between inhibin B and FSH and ovarian follicle status, in the case of unavailability of ovarian ultrasound scans. Finally, anti-M€ullerian hormone, a glycoprotein that is produced, presumably FSH-independently (20, 21), by the granulosa cells (22) of both cyclic and noncyclic follicles (23–25), may be less influenced by such disorders of antral follicle development during the luteal– follicular transition. Further clinical investigation is necessary to address this point. Acknowledgment: The authors thank Dr. Alain Gougeon for his kind revision of the work and advice that greatly improved the manuscript.
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