To the Editor:
The paper by Sowers et al. (1) presents valuable and carefully collected data on the variability of serum Anti-Müllerian hormone (AMH) over the menstrual cycle, with 20 women each providing daily blood samples for one complete cycle. It is unfortunate that sampling were not prolonged giving data for all or part of a second cycle which would have allowed them to estimate repeatability and obtain rigorous statistical tests of cycle effects – this should be considered for future similar studies.
Having obtained this data, the authors create 4 subgroups based on the AMH levels and then compare AMH between these groups (table 1). It is of course an elementary statistical error to perform hypothesis tests comparing groups on the variable that was used to create those groups – akin to testing whether men taller than 6ft are significantly taller than those below 6ft. This statistical howler serves to ring alarm bells for the statistically literate reader, but in itself does not undermine their conclusions. However they go on to graphically compare the variability of AMH between the low and high AMH groups (fig 1 A-D). As we would naively expect the variability in AMH to increase with the AMH level (and indeed the authors assuming this in performing their fitting on log-transformed data), it is not surprising that on the untransformed scale there is more variability in the higher AMH groups. Although it is difficult to read the graphs accurately, all 4 groups seem to be consistent with a peak-to-peak variation of a little less than two-fold. If the same data were plotted on a log scale the evidence for different behaviour between low and high AMH would, at least, be much weaker. Although their modelling framework would permit an (exploratory) hypothesis test, no such test is provided , but the graphical data would suggest that there are no significant differences between the groups.
Thus, we have to conclude that the data presented here provides no evidence for two distinct patterns of AMH profiles and is consistent with there being no variability across the cycle. Larger studies with multiple cycles per woman and rigorous, pre-specified, analyses are still required to assess whether AMH does vary across the menstrual cycle.
Dr. Stephen A. Roberts
Senior Lecturer in Medical Statistics
Health Sciences – Methodology
University of Manchester
Manchester, United Kingdom
1. Sowers M, McConnell D, Gast K, Zheng H, Nan B, McCarthy JD, Randolph JF. Anti-Müllerian hormone and inhibin B variability during normal menstrual cycles. Fertility and Sterility. 07 December 2009 (10.1016/j.fertnstert.2009.07.1674).
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.06.016
The Authors Respond:
We thank Dr. Roberts for his interest in our paper “Anti-Müllerian hormone and inhibin B variability during normal menstrual cycles” that describes variability in anti-Müllerian hormone (AMH) measured daily across the menstrual cycle in 20 women, aged 30-39 years, who reported having regular menstrual cycles in the previous year (1). In his letter to the editor (2), Dr. Roberts urges that data from more than one cycle be collected to determine between-cycle differences.
We acknowledge that multiple cycles in the same women are an optimal way of describing between-cycle patterns, although the implementation of these studies requiring daily sampling of blood and urine across months is quite challenging. It is true that a single cycle of daily AMH measures may not reflect a representative pattern of cycle length and variability in AMH measures in a given woman; however, the AMH measures may represent follicle development that takes place over several months [see McGee and Hsueh (3)] and that variability in measures of AMH may reflect the heterogeneity of follicular phase follicle-stimulating hormone, which in perimenopausal women, represents a mixture of ovulatory and anovulatory cycles.
In designing this study, we felt there were three strengths that helped assure that the data from a single cycle were informative and would minimize the likelihood of confounding from anovulatory cycles. First, the age of the healthy participants was 30-39 years of age. Second and most importantly, each woman reported a history of established menstrual cycle regularity which was sustained in the year prior to study entry; and third, AMH was measured daily across the menstrual cycle, including the follicular phase. We suggest these design strengths be considered favorably in the interpretation of these data.
AMH area under the curve (AUC) was calculated for each woman. These AUCs were then ordered from least to most and placed into quartiles as a proxy for ranks of ovarian reserve; these quartiles were then related to other measures including menstrual cycle lengths and inhibin B AUC. Dr. Roberts is correct in pointing out that the p-values associated with testing hypothesis that quartile-ordering of AMH AUC (shown as part of Table 1, column 2) are not contributory although the absolute information does verify the distinctiveness of the groups.
Further, the AMH AUC ordering is informative with respect to the inhibin B values, the lengths of the menstrual cycle, and the ages of the women, all of which informed our conclusions. Graphically, daily values for each woman across her menstrual cycle were shown to depict the AMH pattern across that cycle. The information was provided so that the reader could see the “raw data”, a frequent request when summary measures like area under the curve are used, particularly considering hormone data that has well-documented daily within-cycle variation. This graphical display did not include hypothesis testing because, as described by Dr. Roberts, this would have been preliminary.
Finally, we drew our conclusions from a synthesis not only of the AMH information but also its consistency with the data describing menstrual cycle length differences and inhibin B differences.
MaryFran Sowers, Ph.D.a,c
Huiyong Zheng, Ph.D.a
Bin Nan, Ph.D.b
John F. Randolph, Jr., M.D.c
aDepartment of Epidemiology
bDepartment of Biostatistics
cDepartment of Obstetrics and Gynecology
School of Public Health
University of Michigan
Ann Arbor, Michigan.
1. Sowers M, McConnell D, Gast K, Zheng H, Nan B, McCarthy JD, Randolph JF. Anti-Müllerian hormone and inhibin B variability during normal menstrual cycles. Fertil Steril. In press.
2. Roberts SA. Variability in Anti-Müllerian hormone levels. A comment on Sowers et al, “Anti-Müllerian hormone and inhibin B variability during normal menstrual cycles”. Fertil Steril. In press.
3. McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev 2000;21:200-14.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.06.017