IVF-ICSI outcome in relation to the antral follicle count. Study population and amount of treatments.

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1 Antral follicle counts are strongly associated with live-birth rates after assisted reproduction, with superior treatment outcome in women with polycystic ovaries Jan Holte, M.D., Ph.D., a,b Thomas Brodin, M.D., a,b Lars Berglund, M.Sc., Ph.D., c Nermin Hadziosmanovic, M.Sc., c Matts Olovsson, M.D., Ph.D., a and Torbj orn Bergh, M.D., Ph.D. b a Department of Women s and Children s Health, Uppsala University, b Carl von Linne Clinic, Uppsala Science Park, and c Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden Objective: To evaluate the association of antral follicle count (AFC) with in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) outcome in a large unselected cohort of patients covering the entire range of AFC. Design: Prospective observational study. Setting: University-affiliated private infertility center. Patient(s): 2,092 women undergoing 4,308 IVF-ICSI cycles. Intervention(s): AFC analyzed for associations with treatment outcome and statistically adjusted for repeated treatments and age. Main Outcome Measure(s): Pregnancy rate, live-birth rate, and stimulation outcome parameters. Result(s): The AFC was log-normally distributed. Pregnancy rates and live-birth rates were positively associated with AFC in a log-linear way, leveling out above AFC 30. Treatment outcome was superior among women with polycystic ovaries, independent from ovulatory status. The findings were significant also after adjustment for age and number of oocytes retrieved. Conclusion(s): Pregnancy and live-birth rates are log-linearly related to AFC. Polycystic ovaries, most often excluded from studies on ovarian reserve, fit as one extreme in the spectrum of AFC; a low count constitutes the other extreme, with the lowest ovarian reserve and poor treatment outcome. The findings remained statistically significant also after adjustment for the number of oocytes retrieved, suggesting this measure of ovarian reserve comprises information on oocyte quality and not only quantity. (Fertil Steril Ò 2011;96: Ó2011 by American Society for Reproductive Medicine.) Key Words: AFC, antral follicle count, infertility, IVF, ovarian reserve, PCO A woman s age is the main determinant of the chance to deliver a healthy child in natural procreation as well as in assisted reproduction. Apart from chronologic age, it has long been suggested that more specific information on the ovarian age, often referred to as the ovarian reserve, may improve the prediction of pregnancy chances. Ovarian reserve is largely genetically determined, the number of oocytes at birth varies between individuals, as does their depletion rate (1). Ovarian reserve may also be affected by adnexal surgery, chemotherapy, pelvic radiotherapy, and smoking (1, 2). Consequently, although ovarian reserve is covariant with age, it varies between women at menarche and even more so at later ages (1). Measures of ovarian reserve include analyses of basal or stimulated gonadotropins levels, antim ullerian hormone (AMH) levels, and sonographic estimates of ovarian volume or the number of antral follicles, the antral follicle count (AFC) (3). Most studies on ovarian reserve have been designed to predict poor response at ovarian stimulation, including the risk of treatment cycle cancellation (4 9), and have established the role of measures of ovarian reserve as an aid in Received April 14, 2011; revised June 7, 2011; accepted June 27, J.H. has nothing to disclose. T.B. has nothing to disclose. L.B. has nothing to disclose. N.H. has nothing to disclose. M.O. has nothing to disclose. T.B. has nothing to disclose. Reprint requests: Thomas Brodin, M.D., Department of Women s and Children s Health, Akademiska sjukhuset, , Uppsala, Sweden ( thomas.brodin@kbh.uu.se). individualization of ovarian stimulation protocols to predict ovarian response (3). Although estimates of ovarian reserve would theoretically also qualify as a prognostic means for pregnancy and delivery, with few exceptions (10 14) the majority of studies have failed to convincingly establish such associations, which has led to a skeptical point of view on the clinical usefulness of ovarian reserve measurements (3). It has also been questioned whether the available methods of measuring ovarian reserve can estimate oocyte quality in addition to oocyte quantity (11, 15). Women with polycystic ovaries, with the overall greatest number of antral follicles (16 19), are usually excluded from ovarian reserve studies although they constitute a large subgroup in assisted reproductive technology (ART) populations. Using larger study populations and including all patients, thus covering the entire range of ovarian reserve, we found that two markers of ovarian reserve (basal gonadotropin levels and mean menstrual cycle length) may indeed be associated with pregnancy and delivery rates and thus may be of value for prognostic counseling before treatment (20, 21). Our prospective study investigated the association of AFC and treatment outcome in all patients eligible for sonographic scans before IVF-ICSI treatment. Our hypothesis was that AFC is associated with pregnancy and live-birth rates, and that polycystic ovaries form one end of the spectrum of ovarian reserve. We also investigated whether AFC is merely a quantitative measure of ovarian reserve or if it also reflects a qualitative component. The preliminary 594 Fertility and Sterility â Vol. 96, No. 3, September /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 TABLE 1 IVF-ICSI outcome in relation to the antral follicle count. Study population and amount of treatments. Age (y) at OPU, mean SD Patients, N 2,092 Total no. of treatments, N 4,308 Treatments with ET, N 3,701 Mean no. of treatments, N 2.06 Treatments per patient, N (%) >5 936 (44.7) 571 (27.3) 323 (15.4) 208 (10) 54 (2.6) IVF-ICSI outcome in relation to the antral follicle count. Type of stimulation, type of treatment and transfer type. Stimulation type Treatment type Transfer type Long GnRH-agonist down regulation (%) (91.7) IVF% (53.4) Single embryo (%) (50.3) Antagonist protocol (%) (8.3) ICSI% (35.7) Double embryo (%) (49.7) rfsh (%) (84.7) Combined IVF and ICSI% (10.9) hmg (%) (15.3) Note: ET ¼ embryo transfer; GnRH ¼ gonadotropin-releasing hormone; hmg ¼ human menopausal gonadotropin; ICSI ¼ intracytoplasmic sperm injection; IVF ¼ in vitro fertilization; OPU ¼ ovum pickup; rfsh ¼ recombinant follicle-stimulating hormone; SD = standard deviation. findings were reported as an abstract at the ESHRE meeting in Copenhagen 2005 (22). MATERIALS AND METHODS For demographic data, see Table 1. Before their first treatment, all patients were scanned with two-dimensional ultrasound for AFC by one of two investigators (JH, TBe), both with more than 10 years of experience of ovarian scanning. Patients were scanned regardless of the day in their cycle by means of a 7.5 MHz vaginal probe, either with a Pie Medical Scanner 150 or 240 (Pie Medical) or Sonoace X6 (Medison Co., Ltd.). The AFC was recorded as the sum of all follicles of 2 to 10 mm in size. Patients were enrolled regardless of the amount of antral follicles and regardless of the cause and duration of infertility. Treatments from January 1999 to October 2009 were included in the analyses. Frozen and thawed cycles were not included. The diagnosis of ovulation and anovulation among women with polycystic ovaries (16) was restricted to the combination of 12 or more antral follicles per ovary and the presence or absence of clinical signs of anovulation (amenorrhea or irregular cycles >35 days). Serum levels of androgens were not analyzed, nor were any clinical signs of hyperandrogenism recorded. The procedure of ovarian stimulation has been described elsewhere (20). Cycles were canceled in case of a threatening ovarian hyperstimulation syndrome (OHSS) and a failed coasting (>4 days of coasting with an inadequate fall in estradiol levels and/or a symptomatic patient), or if ovarian stimulation was suboptimal (predominantly poor response; fewer than three follicles maturing). Embryos were transferred on day 2 after ovum pickup (OPU). Luteal phase support was given vaginally for 2 weeks after embryo transfer (ET) as 1,200 mg of progesterone vagitories (Apoteket AB) or as 180 mg of gel (Merck Serono) daily. Embryos were scored in a 10-degree scale according to the integrated morphology cleavage (IMC) embryo score (23). Pregnancy was defined as the visualization of a gestational sac with vaginal ultrasound in gestational week >7. Delivery of at least one living child was used in the definition of live-birth rates. All data were prospectively collected with the intention to evaluate impact on treatment outcome. In Sweden, at the time of study initiation, an institutional review board approval was not needed for studies of this kind and was therefore not required. Statistical Methods The AFC was log-transformed (natural logarithm) due to a skewed distribution. Logarithmic transformation adjusts a lognormal distribution to a Gaussian distribution, enabling the use of parametric statistical tests. As 55.3% of the women underwent more than one treatment cycle, we have analyzed our data with generalized estimating equation (GEE) models (24) that fully account for any dependence between treatments for the same woman and appropriately addresses error estimates in the context of correlated observations. The main outcome variables were pregnancy and live birth per started stimulation, OPU and ET. To aid in clinical use, AFC was subsequently stratified into four groups, defined by cutoffs corresponding to pregnancy rates 15%, 25%, and 35%, and the grouped variable was used as a continuous variable in the analyses. Odds ratios (OR) for each AFC stratum with 95% confidence intervals (CIs) and P values were presented. All models were adjusted for age (continuous variable). Given doses of follicle-stimulating hormone/human menopausal gonadotropin (FSH/hMG) at ovarian stimulation, the number of oocytes, and other treatment variables were analyzed using GEE models for dichotomous or for continuous outcome variables, where the predictor was the four group AFC categorization. These results are presented as mean or percentage CIs per AFC group and as P values unadjusted and adjusted for age. Pregnancy rates and live-birth rates per started cycle were compared between women with and without polycystic ovaries, unadjusted and adjusted for age and body mass index (BMI). The statistical package SAS (v. 9.1; SAS Institute) was used for all calculations. P<.05 was considered statistically significant. RESULTS The AFC ranged from 3 to 80. The mean overall AFC (standard deviation) was 19.2 (11.7). The pregnancy rate and live-birth rate per started treatment cycle were positively associated with the AFC in a log-linear way (Fig. 1). The pregnancy rate intervals 0% Fertility and Sterility â 595

3 FIGURE 1 Pregnancy rate (%) per started stimulation (solid line) relative to log transformed antral follicle count (AFC), with confidence interval (dotted lines). Pregnancy rates increased (log ) linearly and leveled out above the AFC 30. The corresponding graph for the live-birth rate described the same pattern. AF ¼ antral follicles. Density (red) denotes the distribution after log transformation. The AFC numbers given along the x-axis are back-transformed for ease of interpretation. N ¼ 4,308 IVF-ICSI treatment cycles. to 15%, 16% to 25%, 26% to 35%, and >35% corresponded to the AFC strata 0 5, 6 11, 12 23, and >23, respectively (see Fig. 1). Above an AFC 30 (approximately 25% of all treatments) there was no further increase in pregnancy and live-birth rates. Treatment outcomes according to the four AFC strata are shown in Table 2. The four groups had different pregnancy rates and livebirth rates. There were statistically significant associations between AFC strata and the amount of retrieved oocytes and given doses of FSH/hMG (inverse relationship). There was no interaction between age and stratified AFC on pregnancy and live-birth rates. Figure 2 illustrates live-birth rates/opu at different age groups within each AFC-stratum (for ease of interpretation, age was here subgrouped into three age groups; <34 years, 34 to 38 years, and >38 years). Comparing the two extremes of AFC, women with AFC >23 had at least a 50 percent higher chance of a live birth after treatment than those with AFC <5, when age was taken into account. The OR for live birth/opu relative to stratified AFC was 1.48 ( ) unadjusted and 1.30 ( ) after age adjustment, implying a 30% increased chance of delivering a child for each AFC stratum upward from >5, given the same age. The influence of stratified AFC on pregnancy and live-birth rates/ OPU was studied in a GEE model with adjustment for age and the number of retrieved oocytes. The AFC strata as well as the amount of oocytes and age remained highly statistically significant for both pregnancy rates (P<.0001) and live-birth rates (P<.001; no interaction between AFC strata and age or between AFC strata and amount of oocytes). The live birth/opu odds ratio for stratified AFC adjusted for age and the amount of oocytes was 1.23 ( ), indicative of an increased chance of delivering a child of 23% per AFC stratum upward, when both age and oocyte quantity are taken into account. The corresponding figure for pregnancy/opu was 1.25 ( ). These figures imply that AFC provides also qualitative information on ovarian reserve. Polycystic ovaries were found in 634 women. Of them, 519 were ovulatory (undergoing 931 cycles), and 115 were anovulatory (304 cycles). Pregnancy and live-birth rates (both P<.0001) were higher among women with polycystic ovaries compared with women with AFC <24, with differences remaining highly statistically significant after adjustment for age and BMI. There was no difference in pregnancy rates (P¼.76) or live-birth rates (P¼.95) between ovulatory and anovulatory women with polycystic ovaries, although their mean AFC (standard deviation) differed ( and , respectively; P<.0001). DISCUSSION This study demonstrates strong associations between ovarian reserve, measured by AFC, and assisted reproduction outcome. Both pregnancy and live-birth rates were strongly associated with AFC in a log-linear manner, a relationship not previously shown. Women with an AFC above 30, comprising women with polycystic ovaries, showed the highest pregnancy rates. Our findings also suggest that ovarian reserve measured by AFC is not only a quantitative measure but also reflects an age-independent qualitative component. Previous studies on measures of ovarian reserve mainly focused on predicting ovarian response to FSH/hMG stimulation and established that a low AFC indicates a risk of poor response and a risk for cycle cancellation (25 27). Our results confirm those previous findings and also demonstrate that the association between the number of retrieved oocytes and the total amount of given FSH/ hmg dose (inversely) with AFC both are continuous, that is, AFC may predict also normal and high response. These findings are in line with previous results for AMH and ovarian response in terms of oocyte yield (11). Both the number of oocytes and the administered total dose of FSH/hMG to reach mature follicles have previously been proven to be associated with pregnancy chance and have been suggested as dynamic markers of ovarian reserve (28). The log-linear relationship between treatment outcome and AFC (see Fig. 1) provides important clinical information. Pregnancy rates increased from 15% to 25% with an increase merely from 5 to 11 antral follicles, whereas a similar increase in AFC in the highest range was not associated with any improvement in prognosis. That is, differences in the low range of AFC have more impact on treatment outcome than similar numeric differences in the higher AFC range. Also, AMH, closely related to AFC (29, 30), has been shown to be log-normally distributed with a similar relationship to live-birth rates as demonstrated here for AFC (11). It has been argued that estimates of ovarian reserve merely provide quantitative information on the remaining pool of oocytes (11, 15) and do not reflect oocyte quality (31 33). Any positive influence of a well-preserved ovarian reserve would thus hinge on an increased chance of retrieving high-quality euploid oocytes from many follicles than from few. However, our results, with a statistically significant impact of AFC on live-birth rates also after adjustment for the oocyte yield at OPU, support that AFC measures of ovarian reserve also comprise a qualitative component. An interesting finding was that treatment outcome improved steadily up to an AFC of about 30 and then leveled out. These ovaries qualify for the diagnosis of being polycystic (16). Independent of their ovulatory status, these patients exhibited the best treatment 596 Holte et al. IVF outcome and AFC Vol. 96, No. 3, September 2011

4 Fertility and Sterility â 597 TABLE 2 IVF-ICSI outcome in relation to the antral follicle count stratified into four groups. Variable name No. of observations (total) AF %5 AF 6 11 AF AF >23 P value (trend) e Age-adjusted P value e N 129 (3.0%) 893 (20.7%) 2,051 (47.6%) 1,235 (28.7%) Age (stim start) 4, ( ) 37.6 ( ) 35.3 ( ) 33.5 ( ) <.0001 AFC 4, ( ) 8.4 ( ) 15.9 ( ) 33.8 ( ) Pregnancy/stim start (%) 4, ( ) ( ) ( ) ( ) <.0001 <.0001 Pregnancy/OPU (%) 4, ( ) ( ) ( ) ( ) <.0001 <.0001 Pregnancy/ET (%) 3, ( ) ( ) ( ) ( ) <.0001 <.0001 Live birth rate/stim start 4, ( ) ( ) ( ) ( ) <.0001 <.0001 (%) Live birth rate/opu (%) 4, ( ) ( ) ( ) ( ) <.0001 <.0001 Live birth rate/et (%) 3, ( ) ( ) ( ) ( ) <.0001 <.0001 FSH or hmg total dose at 4, ( ) 3684 ( ) 2475 ( ) 1587 ( ) <.0001 <.0001 stim (IU) OPU, eggs total 3,990 c 5.3 ( ) 6.8 ( ) 9.7 ( ) 11.5 ( ) <.0001 <.0001 Embryo quality (1 10) a 3, ( ) 8.6 ( ) 8.9 ( ) 9.1 ( ) <.0001 <.0001 Embryos to ET (no.) 3,699 d 1.7 ( ) 1.6 ( ) 1.5 ( ) 1.4 ( ) <.0001 <.0001 Canceled (%) 4, ( ) ( ) ( ) ( ) <.0001 <.0001 FSH cycle day 3 (IU/L) 2,722 b 10.0 ( ) 8.5 ( ) 7.2 ( ) 6.0 ( ) <.0001 <.0001 LH cycle day 3 (IU/L) 2,498 b 5.1 ( ) 4.9 ( ) 5.0 ( ) 5.9 ( ) Menstrual cycle length 4,222 b 27.0 ( ) 28.0 ( ) 28.4 ( ) 40.7 ( ) <.0001 <.0001 (days) BMI (kg/m 2 ) 4,300 b 23.2 ( ) 23.4 ( ) 23.0 ( ) 23.5 ( ) Note: AFC was stratified into four groups (0 5, 6 11, 12 23, and >23 antral follicles) based on pregnancy rate intervals (0 15%, 16 25%, 26 35%, and >35%) as illustrated in Figure 1. Mean values 95% confidence intervals. N ¼ 4,308 treatment cycles in 2,092 women. AFC ¼ antral follicle count; BMI ¼ body mass index (kg/m 2 ); ET ¼ embryo transfer; FSH ¼ follicle-stimulating hormone; LH ¼ luteinizing hormone; OPU ¼ ovum pickup; stim start ¼ start of stimulation. a Integrated morphology cleavage embryo score, IMC (23). b Difference in observations versus n stim start due to missing values. c Difference in observations versus n OPU due to missing values. d Difference in observations versus n ET due to missing values. e P values denote trend test based on generalized estimating equation models.

5 FIGURE 2 Live-birth rates/oocyte pickup (%) relative to different ages at oocyte pickup (<34; 34 38; >38 years) within each antral follicle count-stratum (<5, 6 11, 12 23, and >23 antral follicles). Mean values standard error of the mean. N ¼ 4,004 IVF-ICSI treatment cycles. AFC ¼ antral follicle count. outcome, also after adjusting for age and BMI, which strongly supports the concept that polycystic ovaries per se can be regarded as the ovarian type with the greatest ovarian reserve (18). It must however be emphasized that these results are limited to the ultrasound finding polycystic ovaries (ovulation). Analyzing androgens was beyond the scope of this study. Thus, we have not studied the entire polycystic ovary syndrome (PCOS) group, and our conclusions cannot be extrapolated to everyone with the diagnosis PCOS, especially those with a diagnosis based on hyperandrogenism. In a recent follow-up study, 15 to 20 years after a PCOS diagnosis, women 40 to 50 years of age exhibited a clearly better preserved ovarian reserve than age-matched controls (19), findings in line with earlier reports on the lower FSH levels in women with PCOS in their fifties compared with controls (34). Our present findings also conform well to our previous results, showing superior treatment outcome in the subgroup of assisted reproduction patients with high luteinizing hormone (LH) and low FSH levels basally, a hormonal pattern suggesting women with polycystic ovaries may form a substantial part of that group (21). We also reported that long menstrual cycles within the ovulatory range are associated with higher pregnancy and live-birth rates (20). According to the present findings (Table 2), long cycles are often associated with a high AFC. Recent data also show that AMH levels are higher in women with polycystic ovaries than in controls (35, 36). Together, these findings suggest that the polycystic ovary constitutes one extreme in a (log) Gaussian distribution of AFC. The AFC relies on the technical qualities of the ultrasound machine and the skill of the ultrasonographer. The risk of an interobserver variation is increased when AFC is high (37). Given our results, a variation in the higher AFC range is likely to be of minor significance, because the treatment outcome leveled out in that range. To minimize a possible interobserver variability, study inclusion was restricted to patients scanned only by two experienced investigators in our treatment center. To reduce the effect of intracycle variation, a standardization of AFC scanning to cycle days 2 to 4 has been suggested (38). In our study, patients were examined regardless of the cycle day. Due to the great number of included patients, the risk of a systematic underestimation or overestimation of the AFC (e.g., due to a dominant follicle or a corpus luteum) distorting the results is probably negligible. Ovarian stimulations were conducted with individual gonadotropin doses. We are aware that this might have influenced the results, as the doses were decided partly according to the patients AFC. However, giving all patients the same dose would not have been clinically reasonable and would probably also have caused an attenuation of the associations of AFC and treatment outcome. Our results show that AFC in a large unselected assisted reproduction population is normally distributed after log transformation. Measures of ovarian response are related to AFC over the entire AFC range. Pregnancy and live-birth rates are closely and loglinearly related to AFC up to AFC 30. Women with polycystic ovaries, regardless of ovulatory status, present the best outcome, and we hypothesize that this ovarian type forms one end of a (log) Gaussian distribution of AFC, representing the greatest ovarian reserve. We also suggest that ovarian reserve, expressed as AFC, besides being a quantitative measure also comprises an ageindependent qualitative component. REFERENCES 1. te Velde ER, Pearson PL. The variability of female reproductive ageing. Hum Reprod Update 2002;8: Maltaris T, Seufert R, Fischl F, Schaffrath M, Pollow K, Koelbl H, et al. The effect of cancer treatment on female fertility and strategies for preserving fertility. Eur J Obstet Gynecol Reprod Biol 2007;130: Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12: Loumaye E, Billion JM, Mine JM, Psalti I, Pensis M, Thomas K. Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril 1990;53: Tanbo T, Dale PO, Lunde O, Norman N, Abyholm T. Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated follicle-stimulating hormone levels. Fertil Steril 1992;57: Lass A, Skull J, McVeigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in-vitro fertilization can predict poor response. Hum Reprod 1997;12: van der Stege JG, van der Linden PJ. Useful predictors of ovarian stimulation response in women undergoing in vitro fertilization. Gynecol Obstet Invest 2001;52: Erdem M, Erdem A, Gursoy R, Biberoglu K. Comparison of basal and clomiphene citrate induced FSH and inhibin B, ovarian volume and antral follicle counts as ovarian reserve tests and predictors of poor ovarian response in IVF. J Assist Reprod Genet 2004;21: Hendriks DJ, Broekmans FJ, Bancsi LF, de Jong FH, Looman CW, Te Velde ER. Repeated clomiphene citrate challenge testing in the prediction of outcome in IVF: a comparison with basal markers for ovarian reserve. Hum Reprod 2005;20: Scott RT Jr, Elkind-Hirsch KE, Styne-Gross A, Miller KA, Frattarelli JL. The predictive value for in vitro fertility delivery rates is greatly impacted by the method used to select the threshold between normal and elevated basal follicle-stimulating hormone. Fertil Steril 2008;89: Nelson SM, Yates RW, Fleming R. Serum antim ullerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles implications for individualization of therapy. Hum Reprod 2007;22: Frazier LM, Grainger DA, Schieve LA, Toner JP. Follicle-stimulating hormone and estradiol levels independently predict the success of assisted reproductive technology treatment. Fertil Steril 2004;82: Holte et al. IVF outcome and AFC Vol. 96, No. 3, September 2011

6 13. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT Jr. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001;76: Penarrubia J, Peralta S, Fabregues F, Carmona F, Casamitjana R, Balasch J. Day-5 inhibin B serum concentrations and antral follicle count as predictors of ovarian response and live birth in assisted reproduction cycles stimulated with gonadotropin after pituitary suppression. Fertil Steril 2010;94: Haadsma ML, Groen H, Fidler V, Seinen LH, Broekmans FJ, Heineman MJ, et al. The predictive value of ovarian reserve tests for miscarriage in a population of subfertile ovulatory women. Hum Reprod 2009;24: Balen AH, Laven JS, Tan SL, Dewailly D. Ultrasound assessment of the polycystic ovary: international consensus definitions. Hum Reprod Update 2003;9: The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19: Nikolaou D, Gilling-Smith C. Early ovarian ageing: are women with polycystic ovaries protected? Hum Reprod 2004;19: Hudecova M, Holte J, Olovsson M, Sundstrom Poromaa I. Long-term follow-up of patients with polycystic ovary syndrome: reproductive outcome and ovarian reserve. Hum Reprod 2009;24: Brodin T, Bergh T, Berglund L, Hadziosmanovic N, Holte J. Menstrual cycle length is an ageindependent marker of female fertility: results from 6271 treatment cycles of in vitro fertilization. Fertil Steril 2008;90: Brodin T, Bergh T, Berglund L, Hadziosmanovic N, Holte J. High basal LH levels in combination with low basal FSH levels are associated with high success rates at assisted reproduction. Hum Reprod 2009;24: Holte J, Hadziosmanovic N, Bergh T, Berglund L. Ovarian reserve is directly proportional to antral follicle counts over the entire range, from oligofollicular to polycystic ovaries: results of a prospective study in 2568 IVF/ICSI cycles. Abstract. In: ESHRE. Copenhagen, Holte J, Berglund L, Milton K, Garello C, Gennarelli G, Revelli A, et al. Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval. Hum Reprod 2007;22: Liang K-Y, Zeger SL. Longitudinal data analysis using generalized linear models. Biometrika 1986;73: Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002;77: Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ. Antral follicle count in the prediction of poor ovarian response and pregnancy after in vitro fertilization: a meta-analysis and comparison with basal follicle-stimulating hormone level. Fertil Steril 2005;83: Hendriks DJ, Kwee J, Mol BW, te Velde ER, Broekmans FJ. Ultrasonography as a tool for the prediction of outcome in IVF patients: a comparative meta-analysis of ovarian volume and antral follicle count. Fertil Steril 2007;87: Saldeen P, Kallen K, Sundstrom P. The probability of successful IVF outcome after poor ovarian response. Acta Obstet Gynecol Scand 2007;86: Gruijters MJ, Visser JA, Durlinger AL, Themmen AP. Anti-m ullerian hormone and its role in ovarian function. Mol Cell Endocrinol 2003;211: van Rooij IA, Broekmans FJ, te Velde ER, Fauser BC, Bancsi LF, de Jong FH, et al. Serum anti-m ullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002;17: Smeenk JM, Sweep FC, Zielhuis GA, Kremer JA, Thomas CM, Braat DD. Antim ullerian hormone predicts ovarian responsiveness, but not embryo quality or pregnancy, after in vitro fertilization or intracyoplasmic sperm injection. Fertil Steril 2007;87: Hsu A, Arny M, Knee AB, Bell C, Cook E, Novak AL, et al. Antral follicle count in clinical practice: analyzing clinical relevance. Fertil Steril 2011;95: Melo MA, Garrido N, Alvarez C, Bellver J, Meseguer M, Pellicer A, et al. Antral follicle count (AFC) can be used in the prediction of ovarian response but cannot predict the oocyte/embryo quality or the in vitro fertilization outcome in an egg donation program. Fertil Steril 2009;91: Dahlgren E, Johansson S, Lindstedt G, Knutsson F, OdenA, JansonPO, etal. Womenwithpolycystic ovary syndrome wedge resected in 1956 to 1965: a long-term follow-up focusing on natural history and circulating hormones. Fertil Steril 1992;57: Cook CL, Siow Y, Brenner AG, Fallat ME. Relationship between serum m ullerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil Steril 2002;77: La Marca A, Broekmans FJ, Volpe A, Fauser BC, Macklon NS. Anti-m ullerian hormone (AMH): what do we still need to know? Hum Reprod 2009;24: Scheffer GJ, Broekmans FJ, Bancsi LF, Habbema JD, Looman CW, Te Velde ER. Quantitative transvaginal two- and three-dimensional sonography of the ovaries: reproducibility of antral follicle counts. Ultrasound Obstet Gynecol 2002;20: Broekmans FJ, de Ziegler D, Howles CM, Gougeon A, Trew G, Olivennes F. The antral follicle count: practical recommendations for better standardization. Fertil Steril 2010;94: Fertility and Sterility â 599