The association between anti-müllerian hormone and IVF pregnancy outcomes is influenced by age

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1 Reproductive BioMedicine Online (2010) 21, ARTICLE The association between anti-müllerian hormone and IVF pregnancy outcomes is influenced by age Jeff G Wang *, Nataki C Douglas, Gary S Nakhuda, Janet M Choi, Susanna J Park, Melvin H Thornton, Michael M Guarnaccia, Mark V Sauer Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, College of Physicians and Surgeons, Columbia University, New York, NY, USA * Corresponding author. address: jw781@columbia.edu (JG Wang). Jeff Wang, MD, is an assistant professor of clinical obstetrics and gynaecology in the Division of Reproductive Endocrinology and Infertility at Columbia University. His current interests include clinical outcomes research in reproductive medicine. Abstract The conflicting results from studies on the predictive capabilities of serum anti-müllerian hormone (AMH) for IVF pregnancy outcomes may be attributed to small sample sizes and disparities in the age of the study populations. The relationship between AMH and IVF pregnancy outcomes was clarified with retrospective cross-tabulation analyses (n = 1558) stratified by age to control for its confounding effects. Serum AMH concentrations were divided into tertiles (0.29, , 1.21 ng/ml) and ages into four groups (<34, 34 37, 38 41, 42 years). For women <34, having serum AMH in the lowest tertile did not reduce the chance of IVF pregnancy/live birth compared with those with higher AMH concentrations. For women 34 41, a significant positive relationship existed between serum AMH and pregnancy rates. For women 42, serum AMH concentrations 0.29 ng/ml were associated with a 3% chance of pregnancy, and women with AMH 1.21 ng/ml had the same pregnancy rate as women with concentrations ng/ml. In conclusion, AMH has limited predictive value for IVF outcomes in the two extremes of female reproductive age; however, for women between 34 and 41, higher serum AMH concentrations are associated with significantly greater chances of pregnancy (P < 0.01). RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: age, AMH, IVF, MIS, pregnancy Introduction Securing accurate prognostic tests for predicting pregnancy success is essential for counselling patients regarding their individual chances from assisted reproduction technologies and to obviate embarking on expensive medical care if minimal benefit is expected. Determinants of success in assisted reproduction are complex and involve a myriad of interactive host factors ranging from the quantity and the quality of the individual gametes, the receptivity of the uterus and maternal medical conditions that may interfere with implantation /$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 758 JG Wang et al. Presently, numerous quantitative ovarian reserve markers such as serum concentrations of FSH, oestradiol, inhibin B and anti-müllerian hormone (AMH), as well as the antral follicle count and ovarian volume measured by ultrasonography, have been shown to predict ovarian responsiveness to gonadotrophin stimulation and the number of oocytes retrieved with reasonable accuracy (Broekmans et al., 2006; McIlveen et al., 2007; Sun et al., 2008). However, clinically available predictors for oocyte quality are still lacking. In their absence, it is not surprising the quantitative ovarian reserve markers alone have limited predictive value for pregnancy and live birth rates, the ultimate outcomes of interest (Broekmans et al., 2006). Within the confines of this incomplete information, many quantitative ovarian reserve markers have been tested for their associations with pregnancy rates with variable results. Specifically, AMH has demonstrated considerable predictive capability in several studies (Barad et al., 2009; Ebner et al., 2006; Eldar-Geva et al., 2005; Kwee et al., 2008; Wunder et al., 2008). However, the results have not been consistent across all studies (Broer et al., 2009; Fanchin et al., 2007; Ficicioglu et al., 2006; Gnoth et al., 2008; Hazout et al., 2004; McIlveen et al., 2007; Penarrubia et al., 2005; Smeenk et al., 2007). Nonetheless, small sample size with inadequate power, disparities in age distribution of the study populations and differences in study design possibly account for many of the inconsistencies. In an attempt to resolve these conflicting findings, a simple model in this study is developed using a large sample size to analyse IVF clinical pregnancy and live birth rates utilizing only AMH as the predictor for quantitative ovarian reserve and patient age as the best available proxy for oocyte quality. It is postulated that a significant relationship exists between AMH and pregnancy outcomes, but its clinical significance may differ by patient age group. Materials and methods From January 2005 to March 2009, 2712 non-donor, fresh IVF cycles performed at the Centre for Women s Reproductive Care at Columbia University with available AMH concentrations were retrospectively identified. The study population consisted of 1558 different couples with the mean female age of 38 ± 4 years (range 22 45). The primary causes of infertility among entered couples included 44% diminished ovarian reserve, 40% male factor infertility, 10% tubal factor, 8% unexplained and 4% endometriosis. Of the 1558 women who initiated the first cycle, 691 (44%) returned for a second cycle. Given this high percentage of second repeat cycles, the first two IVF attempts were included in the study, resulting in a total of 2249 cycles selected for the analysis. The 463 further repeated cycles were excluded from the study to minimize selection bias. Serum AMH was drawn within 3 months prior to ovarian stimulation, independent of menstrual cycle day. Assays were performed at the Centre using a two-site immunoassay from Diagnostic Systems Laboratories (Webster, Texas). Ovarian stimulation was carried out using standard IVF protocols. Cycles were cancelled if there were less than three dominant follicles 8 mm at the completion of ovarian stimulation. Intracytoplasmic sperm injection was performed in 56% of the cases either due to previously diagnosed male factor or poor semen sample obtained on the day of oocyte retrieval. Clinical pregnancies were defined by the presence of at least one intrauterine fetal heartbeat 4 5 weeks after oocyte retrieval. For the cross-tabulation analyses, the female patients were stratified by their age and serum AMH concentrations (Table 1). Specifically, the AMH concentrations were divided into low, middle and high tertiles (0.29, and 1.21 ng/ml) and the female age into four groups (<34, 34 37, and >42 years). Clinical pregnancy rates in the initial analysis were defined per initiated cycle, including cycles cancelled due to poor response, to provide realistic prognostic information for patients before initiating IVF cycles. In the second analysis, clinical pregnancy rates were defined per oocyte retrieval in order to investigate more accurately the association between quantitative ovarian reserve and oocyte quality. In addition, the associations between serum AMH concentrations and live birth rates per embryo transfer in different age groups were also analysed. For each age group, clinical pregnancy, cycle cancellation and live birth rates were compared across AMH tertiles using logistic regression analysis. The results are presented as mean ± SEM. A P-value <0.05 was considered significant. Statistical analyses were performed using Statistical Package for Social Sciences version 15.0 (SPSS, USA). This study was exempt from the Columbia University institutional review due to its retrospective nature. Results For women younger than 34 years initiating IVF treatment, having an AMH concentration in the highest tertile (1.21 ng/ml) increased the likelihood of clinical pregnancy Table 1 Patient demographics and IVF cycle parameters. Age groups (years) < Number of patients Number of cycles AMH (ng/ml) 2.43 ± ± ± ± 0.06 No. of oocytes retrieved 13.3 ± ± ± ± 0.3 No. of embryos transferred 2.12 ± ± ± ± 0.11 AMH = anti-müllerian hormone.

3 Age, AMH and pregnancy outcomes 759 by 25% compared with that of women with AMH in the lowest tertile (55 ± 3% versus 30 ± 6%, P < 0.02). The clinical pregnancy rate for women with AMH concentrations in the mid-tertile was 46 ± 4% which did not differ statistically from those of the higher or the lower tertiles (Figure 1A). The risk of cycle cancellation for women with AMH 0.29 ng/ml was 40 ± 7%, significantly higher than those of women in the middle and the high tertiles (9 ± 2%, P < and 8 ± 2%, P < 0.001, respectively) (Figure 1B). However, once cycles cancelled due to poor response to ovarian stimulation were excluded, the clinical pregnancy rates per retrieval were equivalent across all three AMH tertiles (50 ± 9% versus 50 ± 5% versus 59 ± 3%) (Figure 1C). In addition, no significant differences were found in the live birth rates per embryo transfer across the three AMH tertiles (Figure 1D). For women 42 years and older initiating IVF treatment, having AMH concentrations in the highest tertile was associated with the same clinical pregnancy rate as that of the middle tertile (18 ± 5% versus 14 ± 2%, respectively). However, having AMH concentrations in the lowest tertile was associated with only a 3 ± 1% clinical pregnancy rate, significantly different from those of the middle (P < 0.004) and the high (P < 0.001) tertiles (Figure 1A). The cycle cancellation risk for women with AMH 0.29 ng/ml was 54 ± 3%, significantly higher than those of the middle (17 ± 2%, P < 0.001) and the high (8 ± 3%, P < 0.001) tertiles (Figure 1B). Clinical pregnancy rate adjusted by per Figure 1 (A) The relationship between IVF clinical pregnancy rates per initiated cycle and serum anti-müllerian hormone (AMH) tertiles stratified by age. For women aged <34 years, the only significant difference in pregnancy rate was between that of women in the highest and the lowest tertiles (P < 0.02). For women aged years, the clinical pregnancy rates demonstrated a significant positive linear relationship with serum AMH concentrations (P < 0.01). This relationship was also observed for women (P < 0.01). For women aged 42 years with AMH 0.29 ng/ml, the clinical pregnancy rate was significantly lower than those of the middle and higher quartiles (P < and P < respectively). The clinical pregnancy rates for women in the middle and highest tertiles were not significantly different. (B) Cycle cancellation risks for women with AMH concentrations in the lowest tertile were significantly higher than those of women whose AMH concentrations were in the higher tertiles for all age groups (all P < 0.01). (C) Clinical pregnancy rates per retrieval after excluding the cycles that were cancelled due to poor response during ovarian stimulation did not differ significantly across all three AMH tertiles for women aged <34 years. For women aged years, the only significant difference in clinical pregnancy rate was between those of the highest and the lowest tertiles (P < 0.005). For women aged years, the clinical pregnancy rate in the highest tertile was significantly greater than those of the middle (P < 0.02) and lowest tertiles (P < 0.001). For women aged 42 years, clinical pregnancy rate in the lowest tertile was significantly lower than those of the middle (P < 0.01) and highest (P < 0.01) tertiles. However, women having AMH in the highest tertile did not have a greater chance of pregnancy compared with those of women in the middle tertile. (D) The live birth rates per embryo transfer did not differ significantly across all three AMH tertiles for women aged <34 years. For women aged years, the live birth rate in women with AMH in the lowest tertile was significantly lower than those of the middle (P < 0.05) and highest tertiles (P < 0.05). For women aged years, the live birth rates for women in the low, middle and high AMH tertiles, respectively, were not statistically significantly different. For women aged 42 years, significantly more live births were observed in the highest AMH compared with the lowest. *P < 0.05 compared with the highest tertile; P < 0.05 compared with the middle tertile.

4 760 JG Wang et al. Fig 1. (continued) retrieval was 6 ± 2% for women in the lowest AMH tertile, which was significantly less than those of the middle (P < 0.01) and the high (P < 0.01) tertiles. Again, having AMH concentrations in the highest tertile did not confer a greater clinical pregnancy rate per retrieval compared with those in the middle tertile (20 ± 5% versus 17 ± 3%) (Figure 1C). There were no live births observed in women with AMH concentrations in the lowest tertile which was significantly different from that of women in the highest tertile (10 ± 4%, P < 0.05) (Figure 1D). For women in the intermediate age group 34 37, a significantly positive linear association was noted between AMH concentrations and clinical pregnancy rate (Figure 1A). The clinical pregnancy rates per initiated cycle were 21 ± 4%, 37 ± 3% and 47 ± 3% for the low, middle and high AMH tertiles. All pair-wise comparisons of clinical pregnancy rates across the three AMH groups were individually statistically significant (P < 0.01) even with adjustments made for the multiple comparisons (Figure 1A). Similarly the significant linear association was also noted for women aged with clinical pregnancy rates per initiated cycle of 14 ± 2%, 26 ± 2% and 38 ± 3% (P < 0.01) for the low, middle and high AMH tertiles (Figure 1A). Having AMH concentrations 0.29 ng/ml was associated with a 34 ± 4% and 43 ± 3% risk of cycle cancellation in the and the age groups, respectively. The cancellation risks were significantly greater than those of the higher tertiles (P < 0.01) in the respective age groups (Figure 1B). In terms of clinical pregnancy rate per retrieval for women 34 37, having AMH in the highest tertile was associated with a significantly greater clinical pregnancy rate only when compared with that of the lowest tertile (50 ± 3% versus 33 ± 5%, P < 0.005; Figure 1C). As for the live birth rates per embryo transfer in this age group, women with AMH in the lowest tertile (16 ± 5%) had significantly lower rates compared with those of the middle (29 ± 3%, P < 0.05) and the highest tertiles (30 ± 3%, P < 0.05) (Figure 1D). For women aged 38 41, having AMH in the highest tertile was associated with a significantly greater clinical pregnancy rate (40 ± 3%) than either that of the middle tertile (31 ± 2%, P < 0.02) or the lowest tertile (24 ± 3%, P < 0.001) (Figure 1C). However, there were no statistically significant differences in the live birth rates amongst the three AMH tertiles in this age group (14 ± 3% versus 16 ± 2% versus 20 ± 3%) (Figure 1D). Discussion A significant positive relationship exists between IVF clinical pregnancy rate and the quantitative ovarian reserve as measured by the serum AMH; however, the strength of the association is modulated by patient age. The variable relationship may explain the discrepant results amongst the previous investigations as the age distribution of the study populations varied (Barad et al., 2009; Broer et al., 2009; Ebner et al., 2006; Eldar-Geva et al., 2005; Ficicioglu et al., 2006; Gnoth et al., 2008; Hazout et al., 2004; Kwee et al., 2008; McIlveen et al., 2007; Penarrubia et al., 2005; Smeenk et al., 2007; Wunder et al., 2008). In addition, most of the previous investigations had sample sizes around patients. This results in inadequate statistical power to detect absolute pregnancy rate differences that were less than 20 30%. As demonstrated in the present study, the magnitude of the greatest clinical pregnancy rate difference between the highest and the lowest tertiles was

5 Age, AMH and pregnancy outcomes 761 only 26%, which suggests that the prior studies may have been underpowered for comparing pregnancy outcomes. The success of assisted reproduction technologies is jointly determined by the interaction between oocyte quality and quantity. The ovarian ageing process results qualitatively from the decline in the proportion of developmentally competent oocytes as well as quantitatively from the diminishing number of oocytes available for selection and recruitment. With a greater proportion of biologically competent oocytes, the number of oocytes needed to achieve pregnancy is expected to be less. However, with ovarian ageing, the diminishing proportion of normal oocytes needs to be compensated for quantitatively by increasing the number of available oocytes through controlled ovarian hyperstimulation. Hence, a greater number of oocytes retrieved per IVF cycle confers a higher chance of obtaining the requisite competent oocytes to achieve pregnancy each cycle in women experiencing early and middle stages of oocyte quality decline. Nonetheless, with the most advanced ovarian ageing, even the excess oocytes obtained through ovarian stimulation cannot sufficiently compensate for the paucity of normal gametes. A generalized AMH threshold value for predicting pregnancy outcomes does not exist because oocyte quality is not accounted for by quantitative ovarian reserve markers alone. As demonstrated in this study, the clinical pregnancy rate is 50% per retrieval for women younger than 34 years even with very low AMH and this rate does not differ from those of women with higher AMH concentrations in the same age group. Similarly, the live birth rates remain the same across all AMH concentrations for these women. These observations suggest that low quantitative ovarian reserve is not associated with lower oocyte quality in young patients and the prognosis remains excellent for these patients despite very low AMH concentrations. For women 42 years or older, the combination of poor quality oocyte and low quantitative reserve culminates in extremely poor prognosis. Women with AMH 0.29 ng/ml in this age group face a 97% chance of failure per initiated IVF cycle, a 54% chance of cycle cancellation during stimulation due to poor response and only a 6% chance of clinical pregnancy per cycle even if they meet the requirement to undergo oocyte retrieval. In addition, no live births were observed in women from this age group with AMH 0.29 ng/ml. Moreover, the equivalent clinical pregnancy and live birth rates between women in the middle and the highest AMH tertiles suggest a higher quantitative ovarian reserve in this age group does not imply better-quality oocytes. Hence, women older than 42 should not be falsely reassured that they have the same prognosis as that of younger women, even with high AMH concentrations. In conclusion, this simple age-stratified analysis demonstrated the variable relationships between quantitative ovarian reserve and IVF pregnancy outcomes. For women between the ages of 34 and 41, the positive linear relationship between AMH and pregnancy outcomes demonstrates that the extent of oocyte quality decline can be partially and effectively compensated by utilizing the excess oocyte reserve. Hence, the clinical application of basal AMH testing as a predictor of IVF outcomes may be most appropriate for women in this age group. In the two extremes of female reproductive age, the chance of pregnancy is predominantly determined by oocyte quality and therefore diminishes the predictive value of quantitative ovarian reserve. 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