Submitted on March 17, 2011; resubmitted on December 1, 2011; accepted on December 22, 2011

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1 Human Reproduction, Vol.27, No.4 pp , 2012 Advanced Access publication on January 24, 2012 doi: /humrep/der479 ORIGINAL ARTICLE Infertility Antral follicle responsiveness to follicle-stimulating hormone administration assessed by the Follicular Output RaTe (FORT) may predict in vitro fertilization-embryo transfer outcome V. Gallot 1,2,3, A.L. Berwanger da Silva 1,2,3, V. Genro 1,2,3, M. Grynberg 1,2,3, N. Frydman 1,2,3, and R. Fanchin 1,2,3, * 1 AP-HP, Centre de Médecine et Biologie de la Reproduction, Hôpital Antoine Béclère, Clamart F-92141, France 2 Univ Paris-Sud, Clamart F-92140, France 3 INSERM, U782, Clamart F-92140, France *Correspondence address. Center of Reproductive Medicine and Biology, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, Clamart, France. renato.fanchin@abc.aphp.fr. Submitted on March 17, 2011; resubmitted on December 1, 2011; accepted on December 22, 2011 background: Looking for a qualitative marker of ovarian function, we aimed to verify whether responsiveness of antral follicles to FSH administration, as reflected by the Follicular Output RaTe (FORT), is related to their reproductive competence. methods: We studied 322 IVF-ET candidates aged years who underwent controlled ovarian hyperstimulation with similar initial FSH doses. Antral follicle (3 8 mm) count (AFC) and pre-ovulatory follicle (16 22 mm) count (PFC) were performed, respectively, at the achievement of pituitary suppression (before FSH treatment) and on the day of hcg administration. The FORT was calculated by PFC 100/AFC. FORT groups were set according to tercile values: low (,42%; n ¼ 102), average (42 58%; n ¼ 123) and high (.58%; n ¼ 97). results: The average FORT was 50.6% (range, %). Clinical pregnancy rates per oocyte retrieval increased progressively from the low to the high FORT groups (33.3, 51.2 and 55.7%, respectively, P, 0.003) and such a relationship assessed by logistic regression was independent of the confounding covariates, women s ages, AFC and PFC. conclusions: The observed relationship between IVF-ET outcome and the percentage of antral follicles that effectively respond to FSH administration reaching pre-ovulatory maturation suggests that FORT may be a qualitative reflector of ovarian follicular competence. Further studies with broader inclusion criteria and more personalized protocols are needed to validate these results. Key words: follicular output rate / FORT / FSH / controlled ovarian hyperstimulation / IVF-ET Introduction Although the regulatory mechanisms determining the extent of sensitivity of individual antral follicles to FSH remain to be elucidated, adequate responsiveness to this glycoprotein presumably is characteristic of healthy and differentiated granulosa cells (Shima et al., 1987; Gougeon, 1996). Indeed, granulosa cells displaying appropriate reactivity to FSH are not only endowed with functional FSH receptors but are also able to properly execute a cascade of specialized tasks as signal transduction, steroidogenesis and cell proliferation and differentiation. This physiological context suggests that responsiveness to FSH of antral follicles may constitute a marker of their health and reproductive competence, and it leads us to hypothesize that patients endowed with a large proportion of FSH-responsive antral follicles should be more likely to become pregnant after assisted reproductive technologies. Yet, conclusive evidence of the relationship between responsiveness to FSH and reproductive competence of antral follicles is still lacking. Some investigators have attempted to address this issue by merely analyzing the strength of ovarian response to controlled & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 Follicular output rate and IVF-ET outcome 1067 ovarian hyperstimulation (COH) (Sharma et al., 2002; Carrera-Rotllan et al., 2007; Ottosen et al., 2007; Melo et al., 2009). Unfortunately, the number of pre-ovulatory follicles obtained at the end of COH is not a reliable reflection of antral follicle sensitivity to FSH, as it is greatly influenced by the number of small antral follicles available before treatment. This contingency constitutes a possible explanation for the inconstant relationship between the absolute counting of growing follicles obtained in COH and IVF-ET outcome. In an effort to objectively evaluate antral follicle responsiveness to exogenous FSH, we used the Follicular Output RaTe (FORT) (Genro et al., 2011). This index is assessed by the ratio between the number of pre-ovulatory follicles obtained in response to FSH administration and the pre-existing pool of small antral follicles. Therefore, the present investigation aimed at testing the possible relationship between the FORT and the reproductive competence of antral follicles as reflected by the outcome of oocytes and embryos obtained in IVF-ET cycles. Materials and Methods Subjects We prospectively studied 322 patients, years of age, having accomplished COH for IVF-ET. All of them met the following inclusion criteria: (i) both ovaries present, without morphological abnormalities (such as cysts, endometriomas, etc.), and adequately visualized in transvaginal ultrasound scans; (ii) regular menstrual cycles lasting between 25 and 35 days; (iii) total number of antral follicles measuring 3 8 mm in diameter after pituitary desensitization (baseline),25; (4) BMI ranging from 17 to 29 kg/m 2. Exclusion criteria were: (i) current or past diseases affecting ovaries or gonadotrophin or sex steroid secretion, clearance or excretion; (ii) clinical and/or biological signs of hyperandrogenism; and (iii) patients who were not expected to meet the criteria set for hcg administration, defined as 4 pre-ovulatory follicles (16 22 mm in diameter) and estradiol (E 2 ) levels per pre-ovulatory follicle.200 pg/ml, and who therefore underwent COH cancellation in the present cycle. Indications for IVF-ET were male factor (40%), tubal factor (16%), endometriosis without ovarian lesions (11%) and idiopathic (25%); in 8% of cases, the same couple presented more than one of the IVF-ET indications listed above. As the present study was merely observational and included only analysis of data from routine measurements, it did not require prior submission to our Institutional Review Board. COH and IVF-ET protocol COH and IVF-ET protocols used in the present investigation are detailed in Fig. 1 and have been described elsewhere (Genro et al., 2011). In brief, within the 3 months preceding COH, serum E 2 and FSH levels were measured on cycle day 3. Between cycle days 1 3 of a subsequent menstrual cycle, patients received a single-dose, time-release gonadotrophinreleasing hormone (GnRH) agonist, triptorelin (3 mg, i.m., Decapeptyl, Beaufour Ipsen Pharma, Paris, France). Three weeks later, complete pituitary desensitization was confirmed by the detection of low serum levels of progesterone (P 4 ), E 2 and LH (baseline), the presence of ovarian cysts was ruled out and endometrial thickness,5 mm was verified. Thereafter, recombinant FSH (recfsh) therapy (Gonal-F, Serono Pharmaceuticals, Boulogne, France) was initiated, at a dose of 300 IU/day for at least 5 days, and continued until the day of hcg (Gonadotrophine Chorionique Endo, Organon Pharmaceuticals, Saint-Denis, France, IU, i.m.) administration. From the sixth day of recfsh therapy onwards, daily FSH doses were adjusted according to E 2 levels and/or the number of growing follicles. During the last days of COH, patients had daily visits at our institution for ultrasonographic and hormonal examinations to define the proper timing for hcg administration. Administration of hcg (day of hcg administration; dhcg) was performed as soon as 4 preovulatory follicles (16 22 mm in diameter) were observed and E 2 levels per pre-ovulatory follicle were.200 pg/ml. Oocytes were retrieved 35 h after hcg administration by transvaginal ultrasound-guided aspiration and ETs were performed 2 days after oocyte retrieval. Top quality embryos were defined on Day 2 as those having no multinucleated blastomeres, four or five blastomeres and,20% anucleated fragments (Van Royen et al., 1999). Luteal phase was supported with micronized P 4, 600 mg/day, administered continuously by the vaginal route, starting on the evening of ET. Clinical pregnancy was defined as the presence of a gestational sac observed at ultrasound scan at around 7 weeks of amenorrhea; ongoing pregnancy was defined as pregnancy beyond 12 weeks of amenorrhea. Embryo implantation rate was defined as the total number of gestational sacs 100/total number of embryos transferred. FORT calculation Calculation of FORT is also explained in Fig. 1. At baseline and dhcg, ovarian ultrasound scans were performed using a MHz multifrequency transvaginal probe (Voluson 730 Expert, General Electric Figure 1 Study design.

3 1068 Gallot et al. Medical Systems, Paris, France) to evaluate the number and sizes of antral follicles. We carefully determined, at baseline, the number of all follicles measuring 3 8 mm in diameter (antral follicle count, AFC) and, on dhcg, the number of all follicles measuring mm in diameter (preovulatory follicle count, PFC), in both ovaries. The FORT was calculated by the ratio between PFC on dhcg 100/ AFC at baseline. The choice of considering only mm follicles for the calculation of FORT was used in a previous investigation of our group (Genro et al., 2011) and represented a methodological attempt for discriminating, among the cohort of small antral follicles, those that were the most FSH-responsive. Definition of FORT and other ancillary study groups To make the interpretation of the possible relationship between follicle responsiveness to COH and IVF-ET outcome easier, we decided to sort our population into three distinct FORT groups. The three FORT groups were arbitrarily chosen according to whether FORT values were under the 33th percentile (,42%, low FORT group; n ¼ 102), between the 33th and the 67th percentile (42 58%, average FORT group; n ¼ 123), or above the 67th percentile (.58%, high FORT group; n ¼ 97) of distribution. To compare the predictability of FORT with that of other pre-ivf factors that might influence outcome, we established three other sets of three different groups according to ages, AFC and PFC values. For them, we used a similar methodology as for FORT (terciles of data distribution). Therefore, for women s age: young group (,33 years, n ¼ 89), middle group (33 37 years, n ¼ 151) and older group (.37 years, n ¼ 82); for AFC: low AFC group (,14 antral follicles, n ¼ 103), average AFC group (14 17 antral follicles, n ¼ 116) and high AFC group (.17 antral follicles, n ¼ 103); and for PFC: low PFC group (,6 pre-ovulatory follicles, n ¼ 77), average PCF group (6 8 pre-ovulatory follicles, n ¼ 140) and high PFC group (.8 pre-ovulatory follicles, n ¼ 105). Hormonal measurements Serum P 4,E 2, LH and FSH levels were determined by an automated multianalysis system using a chemiluminescence technique (Advia-Centaur, Bayer Diagnostics, Puteaux, France). For P 4, the lower detection limit was 0.10 ng/ml, linearity up to 60 ng/ml and intra- and inter-assay coefficients of variation (CVs) were 8 and 9%, respectively. For E 2, the lower detection limit was 30 pg/ml, linearity up to 1000 pg/ml and intra- and inter-assay CVs were 8 and 9%, respectively. For LH and FSH, the lower detection limit was 0.1 miu/ml and intra- and inter-assay CVs were 3 and 5%, respectively. Statistics The measure of central tendency used was the mean and the measure of variability was the standard error for parametrical data. Medians and minimum maximum values were used when normality of data distribution could not be ascertained. The relationship between two continuous variables was assessed by correlation when they were independent from each other and by simple regression when there was a dependency relationship. To determine if coefficients of correlation were significantly different from zero, Pearson s test was used if variables were parametric and Spearman s test if variables were non-parametric. Comparisons of continuous variables from the low, average and high FORT, AFC and PFC groups were performed using analysis of variance. Categorical variables in each set of three groups were compared using the two-sided Pearson x 2 test. The relationship between pregnancy rate and FORT was adjusted for possible covariates, ages, AFC and PFC, by running a binary regression model, including dummy variables obtained from terciles. A P-value of,0.05 was considered statistically significant. Results Overall population, COH characteristics and IVF-ET results At the time of inclusion, women were aged years and presented BMI values at kg/m 2. On cycle day 3, serum E 2 and FSH levels were pg/ml and miu/ml, respectively. At baseline, AFC was at follicles and serum P 4,E 2 and LH levels were at ng/ml, pg/ml and miu/ml, respectively. On dhcg, PFC was at follicles and serum P 4 and E 2 levels were ng/ml and pg/ml, respectively. COH lasted days and required a total dose of IU of recombinant FSH. Overall, FORT was 50.6% (range, %). An average of oocytes were retrieved, embryos were obtained and embryos were transferred into the uterus. From the 322 patients having received hcg and having reached oocyte retrieval, 301 underwent ET. Overall, clinical pregnancy and ongoing pregnancy rates per retrieval and embryo implantation rates were, respectively, 46.9, 37.3, and 32.0%. With regard to the prevalence of FORT groups according to AFC before recfsh administration, we observed that in the low AFC group, 19, 40 and 41% of patients belonged to the low, average and high FORT groups, respectively; in the average AFC group, 35, 36 and 29% of patients belonged to the low, average and high FORT groups, respectively; and in the high AFC group, 41, 39 and 20% of patients belonged to the low, average and high FORT groups, respectively. Population, COH characteristics and IVF-ET results in the FORT, AFC and PFC groups Population and COH characteristics in all FORT groups are summarized in Table I. As shown, the three FORT groups were comparable in terms of women s ages, BMIs, indications for IVF-ET, serum FSH and E 2 levels on cycle day 3, total recfsh dose used for COH, duration of recfsh therapy and serum P 4 levels on dhcg. Conversely, AFC values showed a stepwise decrease from the low to the high FORT groups (P, 0.001), paralleled by an increase in serum E 2 levels and PFC values on dhcg (P, 0.001). In line with this, FORT was correlated with AFC (r ¼ 20.27; P, 0.001), with serum E 2 levels on dhcg (r ¼ 0.27; P, 0.001) and with PFC (r ¼ 0.67; P, 0.001). With regard to the AFC and PFC groups (data not shown), women s ages, BMIs and indications for IVF-ET were similar in both sets of groups and, as expected, AFC and PFC were negatively correlated with serum Day 3 FSH and E 2 levels and positively correlated with serum E 2 and P 4 levels on dhcg. IVF-ET data and outcome in the FORT groups are summarized in Table II. As shown, the number of oocytes and embryos obtained increased progressively from the low to the high FORT groups (P, 0.001), but fertilization rates remained steady. The overall percentage of patients who underwent ET was lower in the low FORT group when compared with the remaining groups. Yet, reasons for not

4 Follicular output rate and IVF-ET outcome 1069 Table I Patient characteristics and COH data in the low, average and high FORT groups. Low FORT <42% Average FORT 42 58% High FORT >58% P-value (n 5 102) (n 5 123) (n 5 97)... Age (years) BMI (kg/m 2 ) Indications for IVF-ET Male (%) Tubal (%) Endometriosis (%) Idiopathic (%) Mixed (%) Serum FSH levels (IU/ml) a Serum estradiol levels (pg/ml) a AFC b ,0.001 Total recombinant FSH dose (IU) 3, , , Duration of recombinant FSH therapy (days) Serum estradiol levels (pg/ml) c 2, , , ,0.001 Serum progesterone levels (pg/ml) c PFC c ,0.001 FORT (%) d 35.0 ( ) 50.0 ( ) 66.7 ( ) a On cycle day 3. b At baseline (just before the start of FSH treatment). c On dhcg. d Medians (minimum maximum). Table II IVF-ET data and outcome in the low, average and high FORT groups. Low FORT <42% Average FORT 42 58% High FORT >58% P-value (n 5 102) (n 5 123) (n 5 97)... Number of oocytes retrieved ,0.001 Number of metaphase II oocytes ,0.001 Fertilization rate (%) Number of embryos obtained ,0.002 Top-morphology embryos (%) Number of embryos transferred Clinical pregnancies/oocyte retrieval ,0.004 (%) Ongoing pregnancies/oocyte retrieval ,0.003 (%) Embryo implantation rate (%) a ,0.004 a Defined as total number of gestational sacs 100/total number of embryos transferred. reaching ET (fertilization failure, 67% of cases; marked embryo fragmentation, 33% of cases) were similar among the three FORT groups. A decreased prevalence of top-morphology embryos was observed in the low when compared with the high FORT groups. In agreement with this, FORT levels were slightly yet significantly correlated (r ¼ 0.14; P, 0.02) with the percentage of top-morphology embryos. Whereas the number of embryos transferred into the uterus was similar, we noted a significant reduction in clinical and ongoing pregnancy rates per oocyte retrieval as well as in embryo implantation rates in the low when compared with the remaining FORT groups. Incidentally, as expected, AFC and PFC were positively correlated with the number of oocytes and embryos obtained but not with fertilization or top-morphology embryo rates. In addition, neither the AFC nor PFC groups showed statistically significant differences in

5 1070 Gallot et al. terms of pregnancy or embryo implantation rates. As with the FORT groups, age groups were also associated with IVF-ET outcome. Clinical parameters influencing IVF-ET outcome Patient characteristics and COH data in pregnant and non-pregnant patients are shown in Table III. In line with the results described above, FORT was significantly higher in women who achieved a clinical pregnancy when compared with those who did not ( versus %, respectively, P, 0.001), whereas both groups of patients showed comparable AFC ( versus antral follicles, respectively) and PFC ( versus preovulatory follicles, respectively). In addition, no other pre-ivf clinical parameter (BMI, Day 3 E 2 and FSH levels, E 2 and P 4 levels on dhcg, total recfsh dose, duration of recfsh therapy), except women s age ( versus years, respectively, P, 0.004), reached statistical significance as different between patients having or not become clinically pregnant. As shown in Table IV, binary logistic regression analysis demonstrated that the relationship Table III Patient characteristics and COH data in pregnant and non-pregnant patients. Clinical No P-value pregnancy pregnancy (n 5 151) (n 5 171)... Ages (years) ,0.004 BMI (kg/m 2 ) Serum FSH levels (mui/ml) a Serum E 2 levels (pg/ml) a AFC b Total recfsh dose 3, , (IU) Duration of recfsh therapy (days) PFC c FORT (%) ,0.001 Serum E 2 levels 2, , (pg/ml) c No. of oocytes ,0.05 retrieved No. of metaphase II ,0.001 oocytes Fertilization rate (%) No. of embryos ,0.03 obtained Top-morphology ,0.001 embryos (%) No. of embryos transferred a On cycle day 3. b AFC (3 8 mm) at baseline (just before the start of FSH treatment). c PFC on dhcg. Table IV Binary logistic regression analysis of factors potentially associated with the occurrence of clinical pregnancy. B SE Wald df P-value Exp (B)... Age PFC a FORT b a Pre-ovulatory follicle count (on dhcg). b Follicular output rate defined as pre-ovulatory follicle (16 22 mm) count 100/ small antral follicle (3 8 mm) count. Logistic regression parameters: B is the estimated logit coefficient; SE the standard error of the coefficient; Wald is given by the formula [B/SE] 2 ; df the degrees of freedom; P the significance level of the coefficient; and Exp(B) the odds ratio of the individual coefficient. between FORT and pregnancy outcome was independent from and robust to the influence of confounding covariates as women s ages, AFC and PFC. Discussion The central finding of the present investigation is the remarkable, covariate-independent, relationship between FORT and IVF-ET outcome. Irrespective of age and absolute pre-coh AFC and post-coh PFC, patients endowed with a larger proportion of FSH-responsive antral follicles were more prone to become pregnant after IVF-ET. This supports the hypothesis that scant responsiveness of antral follicles to exogenous FSH reveals some degree of follicle/ oocyte dysfunction (Shima et al., 1987; Gougeon, 1996) and places the FORT as a promising qualitative marker of antral follicles. In line with this, previous investigators have observed that the proportion of atretic selectable follicles during the first days of the follicular phase in the menstrual cycle exceeds 40% (Gougeon and Testart, 1990), a percentage that comes close to the mean FORT values observed in our infertile population (50.6%). Also, the present results offer a reasonable explanation for the reported weak predictability of absolute AFC (Broer et al., 2009) and invite us to reconsider criteria for COH cancellation based on the output of follicle response to exogenous FSH (FORT) rather than the absolute counting of follicles recruited by the treatment. It is noteworthy that FORT was not significantly influenced by usual parameters of ovarian aging such as women s ages (in contrast with PFC) and basal E 2 and FSH levels. This suggests that, with ovarian aging, antral follicles do not lose notably their aptitude to respond to FSH, which corroborates previous indirect data (Hanoch et al., 1998). Whether the maintenance of follicular responsiveness to FSH with aging reveals a compensating mechanism for preserving ovulatory folliculogenesis in older women remains to be elucidated. In line with this hypothesis, anti-müllerian hormone (AMH), a putative inhibitor of antral follicle sensitivity to FSH (Durlinger et al., 2001), with serum levels which are strongly and positively related to AFC (Fanchin et al., 2003) and which decrease progressively as the ovary ages (De Vet et al., 2002), has been negatively correlated with FORT (Genro et al., 2011). Incidentally, the improved FORT results in patients having reduced AFC observed in the present series is in agreement

6 Follicular output rate and IVF-ET outcome 1071 with this. Therefore, the poor ovarian response to COH commonly observed in aged women is probably attributable to a pre-existing depletion of the follicular pool rather than a per-follicle loss of sensitivity to FSH. Yet, the significance of the present results should take into account some limitations of the FORT index, as discussed elsewhere (Genro et al., 2011). In brief, FORT could not be assessed, by design, in patients having discontinued FSH treatment before hcg administration (due to a markedly insufficient follicle recruitment), since their PFC could not be established. Further studies are needed to verify if FORT still is instrumental when the number of average-sized follicles at the mid-follicular phase of COH is used instead of the mm follicles on dhcg. Such a development might reincorporate patients vowed to cancellation into the FORT calculation. In addition, FORT implies that 3 8 mm follicles before COH respond coordinately to FSH, which is not always the case (Fanchin et al., 2005). To overcome this limitation, the individual tracking of the development of each follicle in response to FSH would be required, which is practically unrealistic. Instead, two alternative methodological measures were taken. First, to reduce follicle size discrepancies and coordinate as far as possible follicle growth during COH, endogenous FSH was beforehand profoundly suppressed by a GnRH agonist. Second, to recruit as many follicles as possible despite their pretreatment sizes, relatively high initial FSH doses were used. Finally, to prevent the possible influence of any unsuitable extension of COH duration on PFC and, therefore, the FORT calculation, strict follicle monitoring and ovulation triggering policies were respected. Indeed, all participants received hcg as early as when 4 follicles reached pre-ovulatory maturation (and E 2 levels per pre-ovulatory follicle were.200 pg/ ml) and only those who undoubtedly would not satisfy this criteria were cancelled. Furthermore, FORT assumed that, on dhcg, only mm follicles effectively responded to FSH, while it is conceivable that smaller follicles also presented some degree of FSH responsiveness. However, as very small follicles, which were not countable by ultrasound at baseline, may have also initiated their FSH-driven maturation after the start of COH and reached intermediate sizes on dhcg, the inclusion of average-sized follicles on dhcg into the calculation of FORT could confuse its interpretation. Moreover, additional studies focusing on other follicle sizes will be helpful to fine-tune alternative relevant cutoffs for the calculation of this new parameter. Together, these FORT characteristics bring out its great flexibility and spur us to further develop this new concept. Future evaluation of alternative ways of calculating the FORT, in particular using different numerators and including patients treated with weaker and possibly more discriminating exogenous FSH signals, will undoubtedly contribute to broaden its clinical applications. In conclusion, the present findings indicate that antral follicle responsiveness to FSH, as far as it is measurable by FORT, is positively related to IVF-ET outcome in normo-cycling women devoid of polycystic ovaries. This places the FORT as a promising qualitative marker of ovarian function. In agreement with our own previous report (Genro et al., 2011), FORT constitutes an objective approach to assessing the actual response of follicles to exogenous FSH, which is not influenced by the size of the pre-existing cohort of antral follicles. This innovative index invites us to revisit the usual criteria of COH cycle cancellation based on the absolute number of growing follicles and shed a new light on the long-lasting debate on the definition of poor responders to COH (Garcia et al., 1981; DeCherney et al., 1985; Benadiva et al., 1988; Hugues and Cédrin Durnerin 1998; Loutradis et al., 2008). In addition, the usefulness of FORT to predict IVF-ET outcome needs to be validated in studies adopting more personalized protocols for COH and hcg administration and broader inclusion criteria. Moreover, further studies possibly focusing on other parameters supposed to be involved in follicle responsiveness to FSH, such as FSH receptor polymorphisms (Perez Mayorga et al., 2000) and granulosa cell function (Adriaenssens et al., 2010), are required to expand the present observations. Authors roles V.G. and A.L.B.S.: collecting data, interpreting data, and writing the paper. V. 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