Serum anti-mullerian hormone levels across different ethnic groups: a cross-sectional study

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1 DOI: / Fertility and assisted reproduction Serum anti-mullerian hormone levels across different ethnic groups: a cross-sectional study P Bhide, A Gudi, A Shah, R Homburg Homerton Fertility Centre, Homerton University Hospital, London, UK Correspondence: Dr P Bhide, Homerton Fertility Centre, Homerton University Hospital, Homerton Row, London E9 6SR, UK. priya. bhide@homerton.nhs.uk Accepted 13 August Published Online 7 October Objective To assess whether ethnic differences in serum anti- Mullerian hormone (AMH) exist in a population of subfertile women presenting to a fertility clinic. Design Observational cross-sectional study. Setting Homerton University Hospital Fertility Centre, London, UK. Population A total of 865 women attending the fertility clinic for their first consultation appointment between September 2012 and September Methods Serum AMH was compared amongst women from five different ethnic groups. Main outcome measures Serum AMH and ethnicity were the primary outcome variables. Results Although initial comparison showed South Asian women to have a higher serum AMH, compared with white European and Afro-Caribbean women (F = 3.817; P < 0.005), South Asian women attending the clinic were significantly younger and less likely to be smokers than women from other ethnic groups. The prevalence of polycystic ovary syndrome (PCOS) was significantly higher in South Asian and South East Asian women than in other ethnic groups. Differences in serum AMH were no longer significant after controlling for confounding factors: age, body mass index (BMI), and smoking status with (P = 0.869) and without (P = 0.215) controlling for PCOS. Conclusion The results from our study show that there was no independent association of ethnicity and serum AMH levels in an unselected population of women attending the fertility clinic. Keywords Ethnicity, infertility, serum AMH. Please cite this paper as: Bhide P, Gudi A, Shah A, Homburg R. Serum anti-mullerian hormone levels across different ethnic groups: a cross-sectional study. BJOG 2015;122: Introduction Evidence suggests that ethnic differences exist in the outcomes of assisted reproductive treatments. 1 4 The success of these treatments is multifactorial, and associated with a large number of confounding factors. Ovarian reserve/ reproductive age is one of the important predictors in the success rate of fertility treatments. 5,6 Anti-Mullerian hormone, secreted by the granulosa cells in the ovary, is used as an estimate of ovarian reserve and to predict the ovarian response to ovarian stimulation during assisted conception treatments. Differences in serum AMH levels amongst various ethnic groups have been reported in the literature. In 2009, Seifer et al. 7 reported lower serum AMH levels in black women as compared with white women, but no significant differences between white and Hispanic women, in one of the earliest studies comparing AMH in different ethnicities. Ethnic differences between Africans, Asians, and white women were confirmed by Gleicher et al. 8 in 2012, with white women showing lower levels of serum AMH than Asian women. In 2014, comparing serum AMH in a multi-ethnic population, Bleil et al. 9 showed serum AMH levels to be lower in Hispanic women compared with white women. In 2014, Iglesias et al. 10 showed Indian women to have a lower AMH than Spanish women when controlled for age. This contrasted with the Penn Ovarian Aging Study published in 2007, 11 in which black and white women had comparable AMH levels. The results from literature have been conflicting and not homogenous as regards the population and ethnic groups studied. Most studies have been performed in American populations, with very few studies performed in European populations and none reported from the UK. 1625

2 Bhide et al. As ovarian reserve remains an important predictor of the success of fertility treatments, ethnic differences in AMH may contribute to the differences in outcome of these treatments. The present study aimed to assess whether ethnic differences in serum AMH exist in a population of subfertile women presenting to a fertility clinic. The five ethnic groups compared in the study were Afro-Caribbean, South Asian (Indian subcontinent), white European, Middle Eastern, and South East Asian women. Methods This single-centre cross-sectional study was carried out in the UK at an inner London NHS fertility centre. Data were collected from participants attending the clinic from September 2012 to September 2013 over a period of 13 months. Medical records of women attending the fertility clinic for their first consultation appointment were screened for eligibility for the study. Data for ethnicity and serum AMH were collected as the primary outcome variables from these women. Data for ethnicity was collected from hospital demographic records and self-identification by the participants. Serum AMH measurement is performed as a part of routine fertility investigations and work-up for every woman attending the fertility clinic. Measurement of AMH was not restricted to a particular time of the menstrual cycle. 12,13 All AMH assays were performed using the Beckman Coulter Generation II assay, and values are presented as pmol/l. Interassay coefficients of variation for a low and high control were 10.3 and 10.0%, respectively. The previously reported instability of AMH was mainly caused by delays in processing and long storage times for samples. As a result of an onsite laboratory where the samples were delivered, spun, and stored at 20 C, and analysed within 14 days, we obtained a reasonable consistency in our results. Only women with a complete data set for the primary outcome variables were included in the study. Data was also collected for confounding variables such as age, body mass index, smoking status (current smokers), known endometriosis, and previous ovarian surgery. Smoking status was self-reported. As AMH levels are linked closely to the presence of polycystic ovary syndrome (PCOS), data for the prevalence of PCOS in the study population was also noted. PCOS was defined according to the Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group criteria. 14 Statistical analyses were performed using SPSS 19 for WIN- DOWS (IBM, Chicago, IL, USA). Serum AMH was the primary outcome variable. Tests of normality for serum AMH showed significant deviation from normality. Therefore, the serum AMH variable was log-transformed prior to analysis. Statistical tests included the analysis of variance and covariance. Initially, analysis of variance (ANOVA) was used to test for differences of AMH levels between ethnic groups. Analysis of covariance (ANCOVA) was used to test for differences of AMH levels between ethnic groups while controlling for confounding factors. Because of the possible impact of missing data for some of the confounding variables, each of these was first tested as a sole covariate in a series of ANCOVAs, before also being tested together in an overall ANCOVA. The results were consistent using both approaches. Results A total of 865 women were confirmed eligible and recruited into the study for the specified study period. Twenty-five women were from ethnic groups (e.g. South American and mixed race) with very small numbers, and these groups were deemed unsuitable for statistical analysis. Data from 840 women were analysed. Women belonged to one of five ethnic groups: Afro- Caribbean, South Asian (Indian subcontinent), white European, Middle Eastern, and South East Asian. The baseline clinical and endocrine descriptive data from the five ethnic groups is summarised in Table 1. Table 1. Clinical and endocrine parameters in different ethnic groups. Variable Afro-Caribbean (150) South Asian (214) White European (384) Middle Eastern (67) South East Asian (25) P Serum AMH (pmol/l) Log serum AMH <0.005 Age (years) <0.001 BMI <0.001 PCOS 11 (9.3%) 37 (20%) 34 (10.1%) 10 (15.6%) 6 (26.1%) <0.005 Smoking status 8 (5.6%) 3 (1.4%) 48 (12.8%) 16 (23.9%) 2 (8.3%) <0.001 Known endometriosis 7 (5.2%) 12 (5.9%) 22 (6%) 1 (1.5%) 1 (4.2%) NS Previous ovarian surgery 13 (9%) 15 (7.1%) 39 (10.4%) 11 (16.4%) 1 (4%) NS Data presented as means SDs or %. 1626

3 AMH and ethnicity Analysis of variance (ANOVA) was used to test for differences in AMH levels between ethnic groups after suitable log transformation. This showed that a significant difference existed amongst the ethnic groups [F 4,835 = 3.817; P < 0.005]. Post-hoc tests (Tukey) confirmed that South Asians had a significantly higher serum AMH than Afro- Caribbeans and white Europeans, but none of the other group comparisons were significant. The possible confounding factors were each first analysed by using a oneway ANOVA. These tests showed significant differences for age, BMI, PCOS, and smoking status amongst the five groups. Tukey s post-hoc tests showed that the Afro-Caribbean group has significantly higher BMI than all the other groups (P < 0.001). South Asians had a significantly lower age than white Europeans, Afro-Caribbeans, and South East Asians (P < 0.001), whereas the incidence of smoking was significantly higher in Middle Eastern women (P < 0.005). The incidence of PCOS was significantly higher in South Asians and South East Asians (P < 0.001). There was no significant difference for previous ovarian surgery and known endometriosis amongst the five groups. In view of significant differences in the confounding variables amongst the five groups, ANCOVA was used to test for differences of AMH levels between ethnic groups while controlling for any confounding effects of age, BMI, and smoking. Analyses performed with and without controlling for PCOS showed that the differences in serum AMH amongst the five ethnic groups were no longer significant (P = 0.869, when controlled for PCOS; P = 0.215, when not controlled for PCOS; Tables 2 and 3). Discussion Main findings The results from our study show no independent association between levels of serum AMH and ethnic groups in an Table 2. Multivariate analysis to assess differences in log AMH with confounding factors and controlling for PCOS. Source Type III sum Of squares df Mean square F Significance Dependent variable: LOG_AMH Corrected model * Intercept Age BMI Smoking PCOS Ethnicity Error Total Corrected total *R 2 = (Adjusted R 2 = 0.318) Table 3. Multivariate analysis to assess differences in log AMH with confounding factors without controlling for PCOS. Source Type III sum of squares df Mean square F Significance Noncent parameter Observed power* Tests of between-subjects effects (Dependent variable: LOG_AMH) Corrected model ** Intercept Age BMI Smoke Ethnic Error Total Corrected total *Computed using alpha = 0.05 **R 2 = (Adjusted R 2 = 0.210) 1627

4 Bhide et al. unselected population of women attending the fertility clinic. Although initial comparison showed South Asian women to have a higher serum AMH, South Asian women attending the clinic were significantly younger and had a lower incidence of smoking. The prevalence of PCOS was significantly higher in South Asian and South East Asian women, compared with other ethnic groups. After controlling for confounding factors, differences in serum AMH were then found to be no longer significant. Strengths and limitations The study population comprised an unselected population of women attending the fertility clinic for various diagnoses and treatments. There were significant differences in the baseline characteristics of these women in terms of demographic and clinical parameters. Using a wide-ranging unselected population of subfertile women would improve the generalisability of the results. Fertility treatment within the UK and funded by the National Health Service is restricted by limits on age and BMI. Our data were not restricted to these limits as our study population was selected before the start of treatment, and also included women ineligible for NHS funding. Although the typical UK population comprises only 14.6% of ethnic minorities, 15 our sample had 54% of ethnic minorities because the study was conducted in a tertiary referral clinic in inner city London. This may be considered a major strength of the study, as it allows wellbalanced and adequate sample sizes in each ethnic group studied. The study population comprised subfertile women attending the fertility clinic. Hence caution should be exercised when extrapolating these results to the general population; however, serum AMH measurements are most often requested in the setting of subfertility, and the findings are valid for this very group. Serum AMH levels may be affected by genetic and environmental confounding factors. Some of these factors could not be assessed and hence their effect remains unknown. Also, the study population did not have the extremes of range for age and body mass index, as these women may have been less likely to be referred for fertility assessment and treatment. Another limitation of the study may be considered to be its retrospective design, which may introduce an element of selection bias as all screened women were not included in the study. This is unlikely to be a major influencer of the results, however, as the proportion of ethnic groups in the included and excluded groups did not significantly differ (P = 0.65). The assessment of serum AMH was performed at a single point in time and women were not followed-up to assess the changes to serum AMH over time. Interpretation The results from our study do not agree with publications reporting a difference in serum AMH amongst various ethnic groups The published data on ethnic differences is conflicting. Although previous studies indicate differences in serum AMH amongst ethnic groups, these differences are variable. Seifer et al. 7 report no significant differences in serum AMH between Hispanic and white women, in contrast to Bleil et al. 9, who show that Hispanic women have a lower AMH than white women. These observed differences in results may arise from the heterogeneous nature of the populations studied and varying environmental conditions. Serum AMH levels may be influenced by genetic and environmental factors Genetic factors may be reflective of true biological differences, whereas environmental factors would be variable in different study populations and settings. Most of the previous studies have been carried out in the USA, with very few from Europe and none reported from the UK. In the study by Iglesias et al. 10 the two populations compared were in different environmental settings, which may have affected the results of the study. Interestingly, studies performed to compare IVF outcomes in different ethnic groups have shown the ovarian response, number of oocytes retrieved, and fertilisation rates to be similar in different ethnic groups. 1 This is in agreement with our results that show a similar ovarian reserve in all ethnic groups. Ovarian reserve and serum AMH are affected by certain conditions, such as polycystic ovarian syndrome. These have an increased prevalence in certain ethnicities, 21 and this is in agreement with our results. Previous studies performed in the general community setting have not taken these factors into account, 7,9 whereas those performed in an IVF setting have excluded this subset of women. 10 Our study has considered the prevalence of PCOS in the study population, as it is closely linked to serum levels of AMH, and has analysed the data with and without controlling for the AMH. In spite of its association with serum AMH, when considered with other confounding variables, PCOS has not affected AMH levels across different ethnicities. Conclusion The results of this study indicate that there is no independent effect of ethnicity on serum AMH levels in an unselected population of women attending a fertility clinic. As AMH may be considered a marker of ovarian reserve, women pursuing fertility treatment may be assured that ovarian reserve and hence ovarian response to treatment in different ethnic groups is likely to be similar. Although treatment outcomes may be different in these groups, these are unlikely to result from differences in ovarian reserve. 1628

5 AMH and ethnicity Disclosure of interests The authors have no conflicts of interest to declare. Contribution to authorship Study concept and design, PB and RH; data collection, PB; data interpretation, PB, RH, AG, AS; drafting the article, PB and RH; critical review and final approval, PB, RH, AG, AS. Details of ethics approval Ethics committee approval was not required for this study. The process of data extraction was consistent with the data protection rules. The study was approved by the Research and Development office of the Homerton University Hospital NHS Foundation Trust, London, UK (R&D no. FE 1307, 4 April 2014). Funding The authors have no funding to declare. Acknowledgement The authors would like to thank Ms K Grayson for statistical advice and help. & References 1 Jayaprakasan K, Pandian D, Hopkisson J, Campbell BK, Maalouf WE. Effect of ethnicity on live birth rates after in vitro fertilisation or intracytoplasmic sperm injection treatment. BJOG 2014;121: Sharara FI, McClamrock HD. Differences in in vitro fertilization (IVF) outcome between white and black women in an inner-city, university-based IVF program. Fertil Steril 2000;73: Seifer DB, Zackula R, Grainger DA. Trends of racial disparities in assisted reproductive technology outcomes in black women compared with white women: Society for Assisted Reproductive Technology 1999 and 2000 vs Fertil Steril 2010;93: Purcell K, Schembri M, Frazier LM, Rall MJ, Shen S, Croughan M, et al. Asian ethnicity is associated with reduced pregnancy outcomes after assisted reproductive technology. Fertil Steril 2007;87: Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, Zamora J, Coomarasamy A. Association between the number of eggs and live birth in IVF treatment: an analysis of treatment cycle. Hum Reprod 2011;26: Iliodromiti S, Kelsey TW, Wu O, Anderson RA, Nelson SM. The predictive accuracy of anti-mullerian hormone for live birth after assisted conception: a systematic review and meta-analysis of the literature. Hum Reprod Update 2014;20: Seifer DB, Golub ET, Lambert-Messerlian G, Benning L, Anastos K, Watts DH, et al. Variations in serum m ullerian inhibiting substance between white, black, and Hispanic women. Fertil Steril 2009;92: Gleicher N, Kim A, Weghofer A, Barad DH. Differences in ovarian aging patterns between races are associated with ovarian genotypes and sub-genotypes of the FMR1 gene. Reprod Biol Endocrinol 2012;10:77. 9 Bleil ME, Gregorich SE, Adler NE, Sternfeld B, Rosen MP, Cedars MI. Race/ethnic disparities in reproductive age: an examination of ovarian reserve estimates across four race/ethnic groups of healthy, regularly cycling women. Fertil Steril 2014;101: Iglesias C, Banker M, Mahajan N, Herrero L, Meseguer M, Garcia- Velasco JA. Ethnicity as a determinant of ovarian reserve: differences in ovarian aging between Spanish and Indian women. Fertil Steril 2014;102: Freeman EW, Gracia CR, Sammel MD, Lin H, Lim LC, Strauss JF 3rd. Association of anti-mullerian hormone levels with obesity in late reproductive-age women. Fertil Steril 2007;87: La Marca A, Stabile G, Artenisio AC, Volpe A. Serum anti-mullerian hormone throughout the human menstrual cycle. Hum Reprod 2006;21: Streuli I, Fraisse T, Pillet C, Ibecheole V, Bischof P, de Ziegler D. Serum antimullerian hormone levels remain stable throughout the menstrual cycle and after oral or vaginal administration of synthetic sex steroids. Fertil Steril 2008;90: Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81: ONS Census: key statistics for local authorities in England and Wales. In: Statistics OFN, editor. England and Wales. UK: Office for National Statistics; Schuh-Huerta SM, Johnson NA, Rosen MP, Sternfeld B, Cedars MI, Reijo Pera RA. Genetic variants and environmental factors associated with hormonal markers of ovarian reserve in Caucasian and African American women. Hum Reprod 2012;27: Ben-Shlomo I, Vitt UA, Hsueh AJW. Perspective: the ovarian kaleidoscope database-ii. Functional genomic analysis of an organspecific database. Endocrinology 2002;143: Schuh-Huerta SM, Johnson NA, Rosen MP, Sternfeld B, Cedars MI, Reijo Pera RA. Genetic markers of ovarian follicle number and menopause in women of multiple ethnicities. Hum Genet 2012;131: Gleicher N, Weghofer A, Oktay K, Barad D. Relevance of triple CGG repeats in the FMR1 gene to ovarian reserve. Reprod Biomed Online 2009;19: Tal R, Seifer DB. Potential mechanisms for racial and ethnic differences in antim ullerian hormone and ovarian reserve. Int J Endocrinol 2013;2013: Rodin DA, Bano G, Bland JM, Taylor K, Nussey SS. Polycystic ovaries and associated metabolic abnormalities in Indian subcontinent Asian women. Clin Endocrinol (Oxf) 1998;49: