Received August 2, 2005; revised and accepted November 8, Supported by Joint Research Board of St Bartholomew s Hospital, London.

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1 Follicular fluid levels of inhibin A, inhibin B, and activin A levels reflect changes in follicle size but are not independent markers of the oocyte s ability to fertilize Xuesong Wen, M.D., a,b,c Amanda J. Tozer, M.D., b Stephen A. Butler, Ph.D., d Celia M. Bell, Ph.D., a Suzanne M. Docherty, Ph.D., a and Ray K. Iles, Ph.D. a,b a Biomedical Sciences, Institute of Social and Health Research, School of Health and Social Science, Middlesex University, Enfield, United Kingdom; b The Williamson Laboratory, St. Bartholomew s and the Royal London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom; c The First Hospital of Harbin Medical University, Harbin, China; and d Institute for Health Research and Policy, London Metropolitan University, London, United Kingdom Objective: To investigate the biochemical relationship between follicular/oocyte maturity and follicular inhibins and activin levels. Design: Prospective study. Setting: Research laboratory in university hospital. Patient(s): Thirty-five women undertook IVF/ICSI program. Intervention(s): Individual follicular fluid aspirations, oocyte isolation, follicular fluid storage. Main Outcome Measure(s): Inhibin A, inhibin B, and activin A concentrations, oocyte retrieval, and fertility outcome. Result(s): Inhibin A, inhibin B, and activin A concentrations varied from 7.9 to 436 ng/ml, 9.7 to 786 ng/ml, and 1.7 to ng/ml, respectively. There was no change of inhibin A concentrations, whereas inhibin B and activin A concentrations dropped dramatically as the follicles enlarged. Total follicular content of inhibin A and activin A increased, and inhibin B remained constant. Both inhibin A and inhibin B levels were significantly higher in those follicles from which an oocyte could be recovered, but they did not differ with respect to subsequent oocyte fertilization. Conclusion(s): Inhibin A is actively produced throughout follicular growth to retain a set concentration. In contrast, inhibin B appears not to be actively produced, and the concentration drops as follicles enlarge. Activin A concentrations also decrease, but there is some extra synthesis. Higher levels of inhibin A and B are associated with oocyte presence but not with fertilization rates. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Inhibin A, inhibin B, activin A, follicular size, oocyte presence, fertilization Follicular size is associated with oocyte development in most species, and this may indicate that a specific size is necessary to initiate the molecular cascade of normal nuclear/cytoplasmic maturation (1). It has been demonstrated that activins and inhibins can enhance oocyte nuclear maturation along with developmental competence of the cumulus-oocyte complex (2 4). Observing the dynamic changes in the concentration of inhibins and activins within different-sized follicles may, therefore, indicate which of these cytokines is essential for follicular maturation and ovulation. Received August 2, 2005; revised and accepted November 8, Supported by Joint Research Board of St Bartholomew s Hospital, London. This work was presented on the conference of 23rd Joint Meeting of the British Endocrine Society, March 22 24, 2004, Brighton, United Kingdom. Reprint requests: Professor Ray Iles, Biomedical Sciences, Institute of Social and Health Research, School of Health and Social Science, Middlesex University, Enfield EN3 4SA, UK (FAX: ; ray@iles.net). To determine the dynamic changes within the follicle during oocyte development, maturation, and ovulation, numerous studies have been carried out on the role of different hormones and cytokines. In particular, inhibins and activins have been considered to exert an important endocrine role, acting as local regulators during folliculogenesis (5). However, most previous work has been based on pooled follicular fluid samples from either natural or controlled ovarian stimulation cycles (6) without taking into account the effect of size on the levels detected. This study was designed to measure the levels of inhibin A, inhibin B, and activin A in individual follicular fluid samples taken from preovulatory luteinized follicles. By comparing the concentration and total content profiles of these cytokines along with follicular size which is the current surrogate marker of follicular maturation we examined the relationship between inhibins and activin A expression together with follicular development to determine their role in oocyte presence and subsequent fertilization /06/$32.00 Fertility and Sterility Vol. 85, No. 6, June 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 1723

2 MATERIALS AND METHODS Subjects Two hundred follicular fluid samples were collected from 35 women undergoing controlled ovarian stimulation for IVF/ intracytoplasmic sperm injection (ICSI) at the Fertility Centre, Saint Bartholomew s Hospital, London. The ages of patients varied from 29 to 38 years old and the follicular size from 10.2 to 35 mm. All couples were undergoing treatment for tubal or male factor infertility. Serum concentration of LH and FSH were determined on day 3 of the treatment cycle. Body mass index (BMI), the years of infertility, and the number of treatment cycles were also recorded. Couples whose infertility was due to endometriosis, polycystic ovarian syndrome, and other ovulatory disorders were excluded. All women underwent a long GnRH agonist stimulation protocol commencing in the midluteal phase for pituitary down-regulation. Multiple follicular development was stimulated with subcutaneous injections of recombinant FSH (Puregon; Organon, Cambridge, UK), highly purified urinary FSH (Metrodin; Serono, Welwyn Garden City, UK), or hmg (Menogon containing both FSH and LH; Ferring, Datchet, UK) ranging from 10 to 14 days and monitored by transvaginal ultrasound measurements. The dose ranged from 1,250 to 4,875 IU (median 2,300, interquartile range 1, ,075). Both dose and the duration of ovarian stimulation were dependent on the serum E 2 level and the ultrasound scan result; there were no significant differences between patients. Follicular fluid sample collection was 36 hours after SC administration of hcg (10,000 IU; Profasi; Serono) and when at least three follicles in each patient had reached 18 mm in diameter, as measured by ultrasound scan. Out of the 35 patients, 7 were treated for male factor infertility. The embryologists assessed all sperm parameters. If the indication for treatment was male factor, those requiring ICSI included cases where the preparation of sperm yielded 1 million motile sperm/ml or 90% abnormal forms as defined by the WHO criteria. None of the patients had surgically retrieved sperm or had immotile sperm injected at the time of ICSI. Those requiring IVF included those whose sperm preparation yielded 6 million sperm/ml (but 1million sperm/ml). For the standard IVF protocol, oocytes were inseminated 40 hours after hcg administration then checked the following day for normal fertilization, i.e., the presence of two pronuclei (2PN). For cases of ICSI, cumulus cells were digested 1 hour after oocyte retrieval and maturity was evaluated. The chosen sperm were injected into the oocyte at metaphase II and the presence of 2PN checked the next day. Written consent was obtained from each patient, and the study was approved by the East London and The City Health Authority Research Ethics Committee (study number P/98/ 222). Follicular Fluid Aspiration Individual follicles were randomly selected prior to aspiration and measured in two dimensions by transvaginal ultrasound (model EUB-525; Hitachi, Northants, UK) to obtain a mean diameter. All measurements and aspirations were performed by the same operator. Following identification of a suitable follicle, the follicle was gently pierced using a double lumen needle and aspirated, allowing the follicle to collapse slowly around the needle. Each follicle was emptied completely to ensure the maximum oocyte recovery rate. The follicle was then flushed with 4.5 ml heparinized saline (3 1.5-mL automated flushes) and aspirated again. If an oocyte was not obtained, three further flushes were carried out to obtain it, but flushings were not added to samples. Heavily blood stained aspirates were discarded, and a further follicle was measured and aspirated if appropriate. A detailed record was kept of the volume of fluid aspirated per individual follicle prior to washing. Following examination of the follicular fluid from the individual follicles by the embryologist, the fluid was placed into a sterile tube. The follicular fluids were taken immediately to the Williamson Laboratory. Each tube was centrifuged at 200g for 10 minutes and the follicular fluid supernatant frozen at 20 C for later analysis. Immunoassays Inhibin A, inhibin B, and activin A were measured by enzyme-linked immunoabsorbent assay (Oxford Bio- Innovation, Oxford, UK). For the inhibin A assay, the intraand interassay coefficients of variation were 10%, the limit of detection was 10 pg/ml, and the assay had 0.012% cross-reactivity with inhibin B and 0.002% cross-reactivity with activin A. For the inhibin B assay, the intra- and interassay coefficients of variation were 10%, the limit of detection was 16 pg/ml, and its cross-reactivity was 0.001% with activins and 1% with inhibin A. For the activin A assay, the intra- and interassay coefficients of variation were 10%, the limit of detection was 78 pg/ml, and its cross-reactivity with inhibin A and inhibin B was 0.001%. Follicular fluid samples were diluted 1:100 in phosphatebuffered solution (PBS) containing 6% (w/v) fetal bovine serum (FBS) for inhibin A and inhibin B assays. The samples were diluted 1:10 in PBS containing 6% FBS for activin A assays. Statistical Analysis Scatter plots were used to show the relationship of follicle diameter with the concentrations and total content of each cytokine using the Origin 6.1 software package (OriginLab Corp., Northampton, MA). The Mann-Whitney U test was used to compare median values of concentrations and total content for inhibin A, inhibin B, and activin A with respect to oocyte retrieval and subsequent fertilization. These were performed 1724 Wen et al. Inhibin and activin levels in follicular fluid Vol. 85, No. 6, June 2006

3 TABLE 1 General patient information. No. patients 35 Age (y) BMI (kg/m 2 ) 24 (21 25) FSH (U/L) 6.1 (4.5 8) LH (U/L) 5.5 ( ) Duration of subfertility (years) 3.5 (2.8 6) Cycle number 1 (1 2) with the StatsDirect software package (StatsDirect, Cheshire, UK). P.05 was taken as significant. RESULTS There was no statistically significant difference with respect to age, BMI level, duration of subfertility, cycle number, or serum concentration of LH and FSH among the 35 patients who were recruited to this study (Table 1). Follicular diameter demonstrated a polynomial correlation to aspirated follicular fluid volume (Fig. 1): volume follicular diameter follicular diameter 2 (P.0001). There could have been high pressure leakage of follicular fluid around the needle insertion point from the large follicles simultaneous with aspiration. However, although there was a very large variation in the amount of follicular fluid recovered from large follicles, there was no clear bias toward lower recovered volume with respect to the predicted (regression) volumes of such follicles. The relationships between cytokine levels and follicular diameter were examined by scatter plot analysis (Figs. 2 4). Inhibin A concentration remained constant with follicular enlargement, and there was no relationship between follicular size and concentrations (r 0.08; Fig. 2A). However, when corrected for follicular volume, total follicular content of inhibin A increased with follicular diameter (r 0.5), and there was significant difference between different follicles (P.0001; Fig. 2B). Inhibin B concentration dropped sharply with follicular diameter (r 0.7; P.0001; Fig. 3A). However, total content increased only marginally with follicular size (r 0.24; P.01; Fig. 3B). Activin A concentration decreased with follicular enlargement (r 0.4; Fig. 4A), but total content had the opposite profile when concentration was corrected for follicular volume (r 0.56; Fig. 4B). Both concentration and total content changes were statistically significant (P.0001). To allow for the variation in levels of these cytokines with follicle development, we compared inhibin A concentrations (because this remained constant whereas follicular size increased), inhibin B total follicular content FIGURE 1 Scatter plot to show the correlation between follicular size (mm) and aspirated follicular volume (ml). Polynomial regression and 95% confidence interval around the regression is shown (P.0001). Fertility and Sterility 1725

4 FIGURE 2 Scatter plots to show the relationship of follicular diameter (mm) with concentration (ng/ml) (A) and total content (ng) (B) of inhibin A in individual follicles. There was no significant difference between inhibin A concentrations and follicular diameter (r 0.08; P.24). Total content of inhibin A correlated with follicular diameter (r 0.5; P.0001). DISCUSSION There are several markers that have been used to evaluate maturation in the selection of oocytes for assisted reproduction. These include transvaginal ultrasound scans for follicular diameter, serum E 2 concentration, and cycle day 3 serum inhibin B concentrations. However, when these criteria are met, the fertilization and pregnancy rates are still not dramatically higher, and lately this has been shown to plateau (7). Studies have been undertaken to identify improved markers for follicular/oocyte maturation to increase fertilization and subsequent pregnancy rates. Since the recognition of inhibins as nonsteroidal regulators of FSH in 1932, and the subsequent discovery of activin and follistatin (8 10), these cytokines in particular have been studied for their roles in follicular development, maturation, and ovulation. FIGURE 3 Scatter plots to show the relationship of follicular diameter with concentration (ng/ml) (A) and total content (ng) (B) of inhibin B in individual follicles. Inhibin B concentration correlated with follicular size (r 0.7; P.0001). (because this remained constant), and activin A concentrations (because this changed less than the total content as the follicles enlarged) with whether the follicle contains an oocyte and its subsequent fertilization outcome (Table 2). Inhibin levels were higher in follicles from which an oocyte was obtained. Inhibin A concentrations were almost three times higher (9.8 to 28.9 ng/ml; P.0001), and inhibin B total content was one-third higher (203.5 to ng; P.0005). Activin A concentrations, however, did not differ (17.4 to 17.5 ng/ml; P.83). There was no significant difference in the levels of all three cytokines with respect to fertilization (Table 2) Wen et al. Inhibin and activin levels in follicular fluid Vol. 85, No. 6, June 2006

5 FIGURE 4 Scatter plots to show the relationship of follicular diameter with concentration (ng/ml) (A) and total content (ng) (B) of activin A in individual follicles. Concentration decreased (r 0.4; P.0001) although total content increased (r 0.56; P.0001). Although previous studies have confirmed that there are different patterns of inhibins and activins in serum (11 13), there is still no consensus as to the profiles of these cytokines in follicular fluid (6, 14 16). Some found that inhibin A increased and inhibin B decreased with follicular growth (14); others reported that inhibin A increased significantly with the follicular size. Neither inhibin B nor activin A varied with size or maturity (17). In a rare natural cycle study, Magoffin and Jakimiuk (6) found that inhibin B increased in follicular fluid from follicles of 4 to 14 mm and decreased when follicular size was larger than 14 mm. Some authors have also described biochemical associations between the oocyte and the follicles in which they were contained and have noted that inhibin B is predominant in preovulatory follicles (12, 16). It was also observed that inhibin B appears to play a more important paracrine role in developing follicles and a greater regulatory role with FSH secretion than inhibin A (6). In order to rectify past methodologic inconsistencies and problems with study sample size, we have studied 200 preovulatory luteinized follicles, without pooling, to examine the relationship of follicle size and fertilization outcome to inhibin and activin levels. In follicles larger than 10 mm, inhibin and activin levels do alter: Inhibin A is actively produced and maintains a set follicular fluid concentration (Fig. 2) within the enlarging follicle. However, inhibin B is not actively produced, so its effective concentration decreases although its total content remains constant (Fig. 3). Activin A is actively produced, but its follicular fluid concentration decreases, indicating that production does not match the dilution effects of size enlargement (Fig. 4). These findings would support a hypothesis that inhibin B is the dominant inhibin of preantral follicles and is overtaken by inhibin A as the follicle enlarges, resulting in inhibin A dominance during the preovulatory phase. This would fit the changing pattern of inhibin A and B in serum during the menstrual cycle: Inhibin B predominates in the early follicular phase, decreasing and giving way to rising inhibin A levels before ovulation and during the luteal phase (11, 12). Activin A is produced by the granulosa cells and the pattern of its subunits mrna expression changes during folliculogenesis (18). However, its physiologic role for a changing function in different follicles is still unclear. A recent study showed that activin from secondary follicles may cause small preantral follicles to remain dormant (19); a few other reports also suggested that activin A inhibits follicle growth whereas inhibin promotes follicle development (20 22). It is most likely that follicles with higher concentration of activin A would undergo atresia. Our results have provided further evidence for the role of activin A during follicular development, in that activin A concentration was higher in smaller immature follicles and decreased as follicular diameter increased. Interestingly, the absolute amounts of activin A present in follicular fluid increased and correlated with follicular diameter. This would suggest active secretion of activin A but at a rate of production that does not increase sufficiently to compensate for the dilution effects of a much faster increase in follicular volume. Physiologically, this would fit with the dynamics of a growing sphere, in which the rate of increase in peripheral surface area does not match the rate of increase in volume. Thus, if activin A is produced by cells at the periphery of the follicle, at a set rate, the total amount produced would increase in a linear relationship to the number of cells required to fill the follicular surface area (and by proxy the follicular diameter) but will not positively correlate with functions of volume, such as concentration, because this is increasing at a much faster rate. Fertility and Sterility 1727

6 TABLE 2 Mann-Whitney U test to compare the median levels of inhibin A concentrations, inhibin B total content, and activin A concentrations with respect to an oocyte recovery and its subsequent fertilization outcome. Activin A concentration (ng/ml), median (25th 75th centile) Inhibin B total content (ng), median (25th 75th centile) Inhibin A concentration (ng/ml), median (25th 75th centile) Group Oocyte (n 130) ( ) ( ) ( ) No oocyte (n 50) ( ) ( ) ( ) P value Fertilized (n 65) ( ) ( ) ( ) Unfertilized (n 54) ( ) ( ) (8.1 34) P value With respect to fertility outcome, follicular fluid levels of the inhibins correlated with the presence of a recoverable oocyte. However, there were no significant differences between the levels of inhibin A, inhibin B, or activin A with respect to recovered oocyte fertilization rates. These findings are consistent with one other study (14, 23) and support the evidence that none of these cytokines has any influence on the oocyte s ability to fertilize. As far as we are aware from the current literature, this is the first study to focus on total content as well as concentrations of inhibin A, inhibin B, and activin A in individual follicles. This is also the first time that the levels of these cytokines have been correlated with the presence and fertilization of oocytes in each individual follicle. In conclusion, inhibin A is actively produced within the developing follicle in order to maintain a constant follicular concentration. In contrast, inhibin B is not actively produced, and its concentration decreases as the follicular size increases. Although inhibin levels correlate with the oocyte s presence within a follicle indicating a potential signaling pathway by which the oocyte increases granulosa inhibin expression this does not show a reciprocal relationship with respect to the oocyte s ability to fertilize. Acknowledgements: The authors appreciate the assistance from all staff in the Infertility Unit at St. Bartholomew s Hospital. We especially thank the embryologists for their help with follicular fluid collection and are grateful for the financial support for this project from Joint Research Board of Bartholomew s Hospital. REFERENCES 1. Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction 2001;121: Sadatsuki M, Tsutsumi??, Yamada R, Muramatsu M, Takitani Y. Local regulatory effects of activin-a and follistatin on meiotic maturation of rat oocytes. Biochem Biophys Res Commun 1993;196: Alak BM, Coskun S, Friedman CI, Kennard EA, Kim MH, Seifer DB. Activin-A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertil Steril 1998;70: Silva CC, Knight PG. Modulatory actions of activin-a and follistatin on the developmental competence of in vitro-matured bovine oocytes. Biol Reprod 1998;58: Woodruff TK. Regulation of cellular and system function by activin. Biochem Phaemacol 1998;55: Magoffin DA, Jakimiuk AJ. Inhibin A, inhibin B and activin A in the follicular fluid of regularly cycling women. Hum Reprod 1997;12: Annual reports and accounts. London, UK: HFEA; p Available at: 8. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, et al. Purification and characterization of an FSH-releasing protein from porcine ovarian follicular fluid. Nature 1986;321: Ueno N, Ling N, Ying SY, Esch F, Shimasaki S, Guillemin R. Isolation and partial characterization of follistatin: a single chain Mr monomeric protein that inhibits the release of follicular stimulating hormone. Proc Natl Acad Sci USA1987;84: Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9: Wen et al. Inhibin and activin levels in follicular fluid Vol. 85, No. 6, June 2006

7 11. Muttukrishna S, Fowler PA, Groome NP, Mitchell GG, Robertson WR, Knight PG. Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum Reprod 1994;9: Groome NP, Illingworth PJ, O Brien M, Pai R, Rodger FE, Mather JP, McNeilly AS. Measurement of dimeric inhibin-b throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81: Muttukrishna S, Fowler PA, George L, Groome NP, Knight PG. Changes in peripheral serum concentrations of total activin-a during the human menstrual cycle and pregnancy. J Clin Endocrinol Metab 1996;81: Lau CP, Ledger WL, Groome NP, Barlow DH, Muttukrishna S. Dimeric inhibins and activin A in human follicular fluid and oocytecumulus culture medium. Hum Reprod 1999;14: Akande AV, Asselin J, Keay SD, Cahill DJ, Muttukrishna S, Groome NP, Wardle PG. Inhibin A, inhibin B and activin A in follicular fluid of infertile women with tubal damage, unexplained infertility and emdometriosis. Am J Reprod Immunol 2000;43: Chang CL, Wang TH, Horng SG, Wu HM, Wang HS, Soong YK. The concentration of inhibin-b in follicular fluid: relation to oocyte maturation and embryo development. Hum Reprod 2002;17: Schneyer AL, Fuliwara T, Fox J, Welt CK, Adams J, Messerlian GM, Taylor AE. Dynamic changes in the intrafollicular inhibin/activin/ follistatin axis during human follicular development: relationship to circulating hormone concentrations. J Clin Endocrinol Metab 2000;85: Findlay JK, Drummond AE, Dyson AJ. Recruitment and development of the follicle: the roles of the transforming growth factor- family. Mol Cell Endocrinol 2002;191: Mizunuma H, Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K, et al. Activin from secondary follicles causes small preantral follicles to remain dormant at the resting stage. Endocrinology 1999;140: Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 1990;127: McNeilly AS, Crow W, Campbell BK. Effect of follicular fluid and inhibin immunoneutralization on FSH-induced preovulatory growth in the ewe. J Endocrinol 1991;131: Miro F, Hillier SG. Relative effects of inhibin and activin on steroid hormone synthesis in primate granulosa cells. J Clin Endocrinol Metab 1992;75: Fried G, Remaeus K, Harlin J, Krog E, Csemiczky G, Aanesen A, Tally M. Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization. J Assist Reprod Genet 2003;20: Fertility and Sterility 1729