Anti-Müllerian hormone as a predictor of pregnancy following IVF

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1 Reproductive BioMedicine Online (2013) 26, ARTICLE Anti-Müllerian hormone as a predictor of pregnancy following IVF Priya Bhide a, *, Anil Gudi a, Amit Shah a, Peter Timms a, Kate Grayson b, Roy Homburg a a Homerton University Hospital, Homerton Row, London E9 6SR, UK; b Statistics by Design, Camberley, Surrey GU17 0HB, UK * Corresponding author. address: priya.bhide@homerton.nhs.uk (P Bhide). Dr Priya Bhide, MD, MRCOG works as an associate specialist in fertility and assisted conception at the Homerton Fertility Centre, London, UK. She is currently involved in research projects relating to ovarian reserve, anti- Müllerian hormone and polycystic ovarian syndrome. Abstract This single-centre retrospective observational study was performed at a university IVF centre. The aim was to examine the predictive power of AMH concentrations for clinical pregnancy rate (CPR) and establish a cut-off concentration of AMH below which no pregnancies were achieved. Data from 820 women with one treatment cycle each were analysed. There was a significant difference in CPR (24.4% and 40.0%; P < 0.01) between the lowest and highest quartiles of AMH. This study failed to establish a cut-off concentration of AMH below which there were no clinical pregnancies as several pregnancies were achieved despite an AMH less than 1 pmol/l. Log AMH showed a strong positive correlation with number of oocytes retrieved (r = 0.522; P < 0.001). Log AMH and overall CPR were weakly correlated (r = 0.112, P < 0.001), but this was not maintained when controlled for the number of oocytes. Age was a stronger independent predictor of CPR than AMH. In conclusion, although an excellent marker of ovarian response, AMH is only a weak predictor of clinical pregnancy. With AMH below the third percentile, CPR was 15%. However AMH is very useful for patient counselling and assessment when used in conjunction with age. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: AMH, clinical pregnancy rate, IVF Introduction Anti-Müllerian hormone (AMH) is widely used in assisted conception clinics as a marker of ovarian response prior to starting IVF/intracytoplasmic sperm injection (ICSI) treatment. It is used alongside several other markers of quantitative ovarian response such as age, serum FSH, and antral follicle count to predict the ovarian response. A clear positive association between AMH concentrations and oocyte yield following ovarian stimulation has been shown in previous studies (Eldar-Geva et al., 2005; Elgindy et al., 2008; Fanchin et al., 2003a,b; Fiçicioglu et al., 2006; Hazout et al., 2004; Jayaprakasan et al., 2010; Jee et al., 2008; Kwee et al., 2008; La Marca et al., 2007, 2011; Lekamge et al., 2007; Lie Fong et al., 2008; McIlveen et al., 2007; Muttukrishna et al., 2004, 2005; Nakhuda et al., 2007; /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

2 248 P Bhide et al. Nelson et al., 2007; Seifer et al., 2002; Silberstein et al., 2006; Van Rooij et al., 2002; Wunder et al., 2008). This has allowed individualization of treatment protocols to optimize oocyte yield and avoid or predict extremes of response (Nelson et al., 2007). The success of assisted conception treatments is, however, determined by both oocyte quantity and quality. Although there is an agreement on the correlation between AMH concentrations and ovarian response, the correlation between AMH and oocyte or embryo quality has been unclear. A positive correlation between AMH and live birth rate has been shown (Gleicher et al., 2010; La Marca et al., 2011; Nelson et al., 2007). This, however, was through an indirect relationship between AMH and oocyte yield. Other studies (Sahmay et al., 2012; Wang et al., 2010) have shown a limited predictive value for serum AMH in relation to clinical pregnancy rate. Clinical pregnancies have been seen at even very low values of AMH. AMH has so far not been found to be an independent predictor of live birth rate. The current study specifically aimed to examine the basal AMH in relation to clinical pregnancy rates in women undergoing IVF/ICSI treatment and attempted to determine a cut-off point of AMH below which there are no pregnancies. It also aimed to assess if there is a correlation between AMH and clinical pregnancy rates independently of the number of oocytes. This study wanted to examine the hypothesis that serum AMH concentrations indicate the prognosis for pregnancy and not merely ovarian response. Materials and methods Patients This was a single-centre, retrospective observational cohort study for which permission was received from the National Research Ethics Service (10/H0703/84). The study was performed at a tertiary referral university IVF centre. It included 820 women with a single cycle of IVF/ICSI each using their own eggs at the centre from November 2010 to December Some of these 820 women had subsequent treatment cycles in the study period. These subsequent treatment cycles were not included in the study. Only women with a complete dataset were included. Women with polycystic ovarian syndrome (Rotterdam criteria) formed a part of the study population. They were included in the study population to allow generalization of the results to a wide range of women attending the fertility clinic. Women who had an IVF/ICSI treatment without estimation of serum AMH, subsequent treatment cycles in the study period or incomplete data and those women who were lost to follow up were excluded from the study (252 patients; 24% of the original number). Data collection Data was compiled using the fertility centre database. All women had a baseline AMH concentration measurement prior to starting treatment as a part of the complete fertility workup. All AMH estimations were performed within 6 months of treatment. AMH measurement was performed independently of the time in the menstrual cycle. Although a few studies report AMH concentrations to change significantly during the cycle (Sowers et al., 2010) these are limited by small sample size. There is an evolving consensus following several other studies for non-significant intracycle variability for AMH (Da Silva et al., 2010; Hehenkamp et al., 2006; Streuli et al., 2008; Tsepelidis et al., 2007; van Disseldorp et al., 2010). This is the significant advantage of AMH compared with FSH. Ovarian stimulation and oocyte retrieval The stimulation protocol was individualized to aim for the optimal number of oocytes for each patient. The protocol and starting dose of FSH were decided based on age, AMH concentrations, ultrasound scan findings and response in previous treatment cycles if any. The standard stimulation protocol used was the long agonist protocol (Homburg, 2005). Women with polycystic ovarian syndrome, polycystic ovarian morphology, AMH concentrations >35 pmol/l or previous overresponse or ovarian hyperstimulation syndrome were started on the antagonist protocol (Homburg, 2005). The standard dose of FSH in women 35 years 150 IU, increasing in increments of 75 IU with <35 years was 150IU increasing age or low AMH. The maximum dose used was 600 IU in exceptional circumstances and extreme poor responders. Ovarian response was monitored by ultrasound scans and serum oestradiol concentrations. Oocyte retrieval was performed 35 h after human chorionic gonadotrophin administration. Fertilization and embryo transfer Insemination/ICSI was performed 40 hours after HCG administration. Fertilization was checked the following day hours after insemination/icsi. Embryos were transferred 2, 3 or 5 days later. Assessment of embryos was performed by morphological grading systems including the Gardner s scoring system (Alpha Scientists, 2011; Gardner et al., 2000). The day of transfer was determined by number of embryos available and morphological appearances of embryos. The number of embryos transferred was either 1, 2 or 3, depending on age, attempt, embryo quality and patient choice. Outcome measures A pregnancy test was performed 14 days after embryo transfer. If positive an early pregnancy scan was performed 3 weeks later and a clinical pregnancy defined as at least one intrauterine sac containing a fetus with a heartbeat. The primary outcome measure was clinical pregnancy rate (CPR). The secondary outcome measure was ovarian response (number of oocytes retrieved). AMH assay The AMH assay used was the enzyme-linked immunosorbent assay by DSL (DSL Active MIS/AMH ELISA; Diagnostic Systems Laboratories, Webster, TX, USA) until March In April 2011, it was replaced by the Beckman Coulter generation II assay (AMH Gen II A79765 ELISA kit; Diagnostic Systems Laboratories). The initial results to March 2011

3 AMH and pregnancy rate following IVF 249 were converted to pmol/l (pmol/l = lg/l 7.143) and multiplied by to compensate for the 40% positive bias of the Beckman Coulter generation II assays in comparison to the generation I assays. All values were finally equivalent to Beckman Coulter generation II assays and presented as pmol/l. Inter-assay coefficients of variation for a low and high control for the DSL assay were 10.9 and 11.1, respectively, and for the Beckman Coulter assay were 12.0 and 11.2, respectively. Statistical analysis The data were analysed using a mixture of correlation, cross-tabulation and logistic regression analysis. For the cross-tabulation analyses, the patients were stratified by their age and serum AMH concentrations. The AMH concentrations were divided into quartiles and age into four groups. Clinical pregnancy rates were defined as pregnant (clinical pregnancy) or not pregnant (negative pregnancy tests, biochemical pregnancies, ectopic pregnancy). For the correlation and regression analyses, the AMH variable used was log transformed. Statistical analyses were performed using the Statistical Package for Social Sciences for Windows version 19 (IBM Corporation, USA). Results AMH values for 820 patients undergoing IVF/ICSI treatment with their own oocytes were analysed. Mean ± SD AMH was ± pmol/l (range pmol/l; median pmol/l). The values for the 25th, 50th and 75th quartiles were 10.28, and pmol/l, respectively. The data did not follow a normal distribution. The spread was very large, as shown by the large standard deviation, and showed a high positive skew caused by the few very high values. The analysis was not controlled for women with polycystic ovarian syndrome as these women were already placed on the appropriate antagonist protocol with low-dose FSH which optimized ovarian response. In 820 cycles, AMH concentrations in the four quartiles were compared for CPR. Both CPR and AMH quartile groups were categorical variables, so the most suitable statistical test to use was chi-squared. There was a statistically significant difference in the CPR in the four AMH quartile groups. There was a significantly lower pregnancy rate of 24.4% in Table 1 Clinical pregnancy rate according to quartiles of AMH. AMH quartile (pmol/l) Not pregnant Pregnant < (75.6) 50 (24.4) a (66.3) 69 (33.7) (67.8) 66 (32.2) > (60.0) 82 (40.0) b Total 553 (67.4) 267 (32.6) Values are n (%). n = 205 for each AMH group; N = 820. Chi-squared test , 3 df; P < a CPR was lower than expected in this AMH group. b CPR was higher than expected in this AMH group. the lowest quartile below an AMH of pmol/l and significantly higher rate at 40% in the highest quartile above pmol/l with an intermediate CPR between AMH values of pmol/l (chi-squared = , 3 df; P < 0.01; Table 1). This study attempted to determine a cut off for AMH below which there were no clinical pregnancies. This attempt failed as there were clinical pregnancies at AMH concentrations as low as 0.5 pmol/l, 0.88 pmol/l and 1.6 pmol/l (below the third percentile). Of the 27 women with an AMH at or below the third percentile (2.1 pmol/l), there were four clinical pregnancies (15%). In view of a non-normal distribution, the AMH values were transformed to a log AMH value and the resulting log function distribution was then confirmed to be close to a normal distribution. A weak correlation as calculated by Spearman s correlation was seen between log AMH and CPR (r s = 0.112, n = 820; P < 0.001). The data analysis for 820 cycles showed a strong positive correlation between log AMH and number of oocytes retrieved (r = 0.522, n = 820; P < 0.001; Figure 1). This study indicated, as shown above, that CPR had a weak positive correlation to AMH. However as AMH and number of oocytes were also strongly and positively correlated, this study examined whether this increase in CPR with increased AMH concentrations was purely due to the increased number of oocytes. A correlation of AMH and CPR while partialling out (or controlling for) any effect of number of oocytes failed to show a positive correlation between AMH and CPR independently of the number of oocytes (r = 0.063, n = 820). This showed AMH to have a positive correlation with clinical pregnancy rates related to a quantitative ovarian response but a poor correlation with CPR independently of oocyte numbers. As the success of assisted conception treatment is dependent on oocyte quality and numbers, age is a variable on which embryo quality and CPR may depend. Hence a further analysis of the CPR in each of the four AMH quartiles was performed in an age-stratified manner. Four age groups (<30, 30 35, and 40) were analysed in each quartile. Analysis using the chi-squared test showed no statistically significant difference in CPR between the AMH quartiles in the <30 to 39 age groups. There was a statistically significant difference only in the 40 age group (P = 0.021; Table 2). The data analysis for 820 patients showed a negative correlation between log AMH and age (r = 0.331, n = 820; P < 0.001). The combined effect of age and log AMH on clinical pregnancy rates was assessed using a logistic regression analysis. Number of oocytes was not included as this explanatory variable is very strongly correlated to log AMH. Age is seen to be a strong predictor of CPR (Exp(B) 1.185, 95% CI ; P = 0.006). AMH is also found to be an independent predictor, although less strong of CPR (Exp(B) 0.956, 95% CI ; P = 0.027). Discussion AMH is commonly used as a marker of quantitative ovarian response in assisted conception clinics prior to embarking on IVF/ICSI treatment. Several studies have shown a positive

4 250 P Bhide et al. Correlation of log AMH and number of oocytes retrieved (r = ; n = 820; p<0.001). Figure 1 Correlation of log AMH and number of oocytes retrieved (r = 0.522, n = 820; P < 0.001). Table 2 Age (years) Age-stratified clinical pregnancy rate according to quartiles of AMH. Overall AMH quartile Chi-squared tests a 1st 2nd 3rd 4th <30 43/124 (34.7) 5/15 (33.3) 9/21 (42.9) 9/36 (25.) 20/52 (38.5) 2.449, 3, NS /270 (35.6) 17/54 (31.5) 17/59 (28.8) 28/70 (40.0) 34/87 (39.1) 2.637, 3, NS /316 (34.8) 23/82 (28.0) 36/94 (38.3) 27/82 (32.9) 24/58 (41.4) 3.387, 3, NS 40 18/110 (16.4) 5/54 (9.3) 7/31 (22.6) 2/17 (11.8) 4/8 (50.0) 9.743, 3, P = Values are n/total (%). Chi squared test significant only in >40 group: 9.743, 3 df; P = correlation between basal AMH concentrations and oocyte yield following ovarian stimulation. The earliest of these studies was published in 2002 by Seifer et al. (2002). Several studies followed which confirmed this correlation (Eldar-Geva et al., 2005; Elgindy et al., 2008; Fanchin et al., 2003a,b; Fiçicioglu et al., 2006; Hazout et al., 2004; Jayaprakasan et al., 2010; Jee et al., 2008; Kwee et al., 2008; La Marca et al., 2007, 2011; Lekamge et al., 2007; Lie Fong et al., 2008; McIlveen et al., 2007; Muttukrishna et al., 2004, 2005; Nakhuda et al., 2007; Nelson et al., 2007; Silberstein et al., 2006; Van Rooij et al., 2002; Wunder et al., 2008). Most of these studies had small patient numbers and the strength of the correlation was variable (r = ). The present study with a large cohort of 820 cycles confirms this positive correlation. It confirms that AMH is an invaluable tool for individualization of IVF/ICSI treatment protocols and for an optimal ovarian response. This study specifically aimed to assess the relationship between clinical pregnancy rates following IVF/ICSI and basal AMH concentrations. This would be more realistic in counselling patients about the outcome rather than estimating only an ovarian response. Some previous studies show an independent association between live birth rates and AMH (Gleicher et al., 2010; La Marca et al., 2011; Nelson et al., 2007). Others suggest a limited predictive value (Sahmay et al., 2012; Wang et al., 2010). The present study showed an overall weak correlation between AMH and CPR in spite of a strong correlation between AMH and ovarian response. However, the difference in clinical pregnancy rates between the lowermost and uppermost quartiles of AMH seen in the cross-tabulation analysis was very significant. The influence of age was clearly shown when this analysis was age stratified as the difference was particularly accentuated in the 40 age group. Younger women (<40 years) with an AMH in the lowermost quartile had clinical pregnancy rates not significantly different from those in the intermediate or upper quartiles. AMH concentrations are an invaluable tool in individualizing the treatment protocol allowing an optimal ovarian response and preventing/minimizing the extremes of response. However, the

5 AMH and pregnancy rate following IVF 251 association of AMH with quantitative ovarian response should not be extrapolated to clinical pregnancy rates and outcome of treatment when counselling patients. Young patients with lower AMH concentrations may be reassured about favourable pregnancy outcomes. A combination of low AMH and increasing age on the other hand may be looked upon less favourably. AMH alone should not be used to predict outcomes but in conjunction with age which is a strong independent predictor of clinical pregnancy rates. Appropriate counselling of women prior to start of treatment allows them to embark on this emotionally difficult journey with a reasonable idea about outcomes. This helps tremendously to minimize disappointment if outcomes are unsuccessful. There are enormous implications in withholding treatment to a couple based solely on AMH values and a prediction for poor response. Published studies so far have not yielded cut-off values below which treatment can be regarded as futile (Gleicher et al., 2010). The present study corroborates these findings. AMH alone should not be used as an independent indicator to refuse fertility treatment to women. Acknowledgements The authors are grateful to Merck Serono UK for providing the Beckman Coulter Generation II kits for AMH assays. They are also grateful to Ms E Timlick, senior nurse for help in compilation of the database. References Alpha Scientists in Reproductive medicine and ESHRE Special Interest Group of Embryology, The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum. Reprod. 26, Da Silva, A.L., Even, M., Grynberg, M., Gallot, V., Frydman, R., Fanchin, R., Anti-Müllerian hormone: player and marker of folliculogenesis. Gynecol. Obstet. Fertil. 38, Eldar-Geva, T., Ben-Chetrit, A., Spitz, I.M., Rabinowitz, R., Markowitz, E., Mimoni, T., Gal, M., Zylber-Haran, E., Margalioth, E.J., Dynamic assays of inhibin B, anti-müllerian hormone and estradiol following FSH stimulation and ovarian ultrasonography as predictors of IVF outcome. Hum. Reprod. 20, Elgindy, E.A., El-Haieg, D.O., El-Sebaey, A., Anti-Müllerian hormone: correlation of early follicular, ovulatory and midluteal levels with ovarian response and cycle outcome in intracytoplasmic sperm injection patients. Fertil. Steril. 89, Fanchin, R., Schonäuer, L.M., Righini, C., Frydman, N., Frydman, R., Taieb, J., 2003a. Serum anti-müllerian hormone dynamics during controlled ovarian hyperstimulation. Hum. Reprod. 18, Fanchin, R., Schonäuer, L.M., Righini, C., Guibourdenche, J., Frydman, R., Taieb, J., 2003b. Serum anti-müllerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum. Reprod. 18, Fiçicioglu, C., Kutlu, T., Baglam, E., Bakacak, Z., Early follicular antimüllerian hormone as an indicator of ovarian reserve. Fertil. Steril. 85, Gardner, D.K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W.B., Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 73, Gleicher, N., Weghofer, A., Barad, D.H., Anti-Müllerian hormone (AMH) defines, independent of age, low versus good live-birth chances in women with severely diminished ovarian reserve. Fertil. Steril. 94, Hazout, A., Bouchard, P., Seifer, D.B., Aussage, P., Junca, A.M., Cohen-Bacrie, P., Serum antimüllerian hormone/müllerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol. Fertil. Steril. 82, Hehenkamp, W.J., Looman, C.W., Themmen, A.P., de Jong, F.H., Te Velde, E.R., Broekmans, F.J., Anti-Müllerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J. Clin. Endocrinol. Metab. 91, Homburg, R., Controlled ovarian stimulation for in vitro fertilization. In: Homburg, R. (Ed.), Ovulation Induction and Controlled Ovarian Stimulation: A Practical Guide, first ed. Taylor and Francis, pp Jayaprakasan, K., Campbell, B., Hopkisson, J., Johnson, I., Raine-Fenning, N., A prospective, comparative analysis of anti-müllerian hormone, inhibin-b, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil. Steril. 93, Jee, B.C., Ku, S.Y., Suh, C.S., Kim, K.C., Lee, W.D., Kim, S.H., Serum anti-müllerian hormone and inhibin B levels at ovulation triggering day can predict the number of immature oocytes retrieved in vitro fertilization cycles. J. Korean Med. Sci. 23, Kwee, J., Schats, R., McDonnell, J., Themmen, A., de Jong, F., Lambalk, C., Evaluation of anti-müllerian hormone as a test for the prediction of ovarian reserve. Fertil. Steril. 90, La Marca, A., Giulini, S., Tirelli, A., Bertucci, E., Marsella, T., Xella, S., Volpe, A., Anti-Müllerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum. Reprod. 22, La Marca, A., Nelson, S.M., Sighinolfi, G., Manno, M., Baraldi, E., Roli, L., Xella, S., Marsella, T., Tagliasacchi, D., D Amico, R., Volpe, A., Anti-Müllerian hormone-based prediction model for a live birth in assisted reproduction. Reprod. Biomed. Online 22, Lekamge, D.N., Barry, M., Kolo, M., Lane, M., Gilchrist, R.B., Tremellen, K.P., Anti-Müllerian hormone as a predictor of IVF outcome. Reprod. Biomed. Online 14, Lie Fong, S., Baart, E.B., Martini, E., Schipper, I., Visser, J.A., Themmen, A.P., de Jong, F.H., Fauser, B.J., Laven, J.S., Anti-Müllerian hormone: a marker for oocyte quantity, oocyte quality and embryo quality? Reprod. Biomed. Online 16, McIlveen, M., Skull, J.D., Ledger, W.L., Evaluation of the utility of multiple endocrine and ultrasound measures of ovarian reserve in the prediction of cycle cancellation in a high-risk IVF population. Hum. Reprod. 22, Muttukrishna, S., Suharjono, H., McGarrigle, H., Sathanandan, M., Inhibin B and anti-müllerian hormone: markers of ovarian response in IVF/ICSI patients? BJOG 111, Muttukrishna, S., McGarrigle, H., Wakim, R., Khadum, I., Ranieri, D.M., Serhal, P., Antral follicle count, anti-müllerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG 112, Nakhuda, G.S., Sauer, M.V., Wang, J.G., Ferin, M., Lobo, R.A., Müllerian inhibiting substance is an accurate marker of

6 252 P Bhide et al. ovarian response in women of advanced reproductive age undergoing IVF. Reprod. Biomed. Online 14, Nelson, S.M., Yates, R.W., Fleming, R., Serum anti-müllerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles implications for individualization of therapy. Hum. Reprod. 22, Sahmay, S., Demirayak, G., Guralp, O., Ocal, P., Senturk, L.M., Oral, E., Irez, T., Serum anti-müllerian hormone, follicle stimulating hormone and antral follicle count measurement cannot predict pregnancy rates in IVF/ICSI cycles. J. Assist. Reprod. Genet. 29, Seifer, D.B., MacLaughlin, D.T., Christian, B.P., Feng, B., Shelden, R.M., Early follicular serum Müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil. Steril. 77, Silberstein, T., MacLaughlin, D.T., Shai, I., Trimarchi, J.R., Lambert-Messerlian, G., Seifer, D.B., Keefe, D.L., Blazar, A.S., Müllerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology. Hum. Reprod. 21, Sowers, M., McConnell, D., Gast, K., Zheng, H., Nan, B., McCarthy, J.D., Randolph, J.F., Anti-Müllerian hormone and inhibin B variability during normal menstrual cycles. Fertil. Steril. 94, Streuli, I., Fraisse, T., Pillet, C., Ibecheole, V., Bischof, P., de Ziegler, D., Serum antimüllerian hormone levels remain stable throughout the menstrual cycle and after oral or vaginal administration of synthetic sex steroids. Fertil. Steril. 90, Tsepelidis, S., Devreker, F., Demeestere, I., Flahaut, A., Gervy, Ch., Englert, Y., Stable serum levels of anti-müllerian hormone during the menstrual cycle: a prospective study in normo-ovulatory women. Hum. Reprod. 22, Van Disseldorp, J., Lambalk, C.B., Kwee, J., Looman, C.W., Eijkemans, M.J., Fauser, B.C., Broekmans, F.J., Comparison of inter- and intra-cycle variability of anti-müllerian hormone and antral follicle counts. Hum. Reprod. 25, Van Rooij, I.A., Broekmans, F.J., te Velde, E.R., Fauser, B.C., Bancsi, L.F., de Jong, F.H., Themmen, A.P., Serum anti Müllerian hormone levels: a novel measure of ovarian reserve. Hum. Reprod. 17, Wang, J.G., Douglas, N.C., Nakhuda, G.S., Choi, J.M., Park, S.J., Thornton, M.H., Guarnaccia, M.M., Sauer, M.V., The association between anti-müllerian hormone and IVF pregnancy outcomes is influenced by age. Reprod. Biomed. Online 21, Wunder, D.M., Bersinger, N.A., Yared, M., Kretschmer, R., Birkhäuser, M.H., Statistically significant changes of antimüllerian hormone and inhibin levels during the physiologic menstrual cycle in reproductive age women. Fertil. Steril. 89, Declaration: The authors report no financial or commercial conflicts of interest. Received 17 July 2012; refereed 23 November 2012; accepted 27 November 2012.