Live birth chances in women with extremely low-serum anti-mullerian hormone levels

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1 Human Reproduction, Vol.26, No.7 pp , 2011 Advanced Access publication on April 30, 2011 doi: /humrep/der134 ORIGINAL ARTICLE Reproductive endocrinology Live birth chances in women with extremely low-serum anti-mullerian hormone levels Andrea Weghofer 1,2, *,, Wolf Dietrich 3,, David H. Barad 2,4, and Norbert Gleicher 2,5 1 Department of Obstetrics and Gynecology, Medical University Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria 2 The Center for Human Reproduction and The Foundation for Reproductive Medicine, New York, New York, USA 3 Department of Obstetrics and Gynecology, General Hospital Mistelbach, Mistelbach, Austria 4 Departments of Epidemiology and Social Medicine and Obstetrics, Gynecology and Women s Health, Albert Einstein College of Medicine, Bronx, NY, USA 5 Department of Obstetrics, Gynecology and Reproductive Sciences Yale University School of Medicine, New Haven, CT, USA *Correspondence address. Tel: ; Fax: ; andrea.weghofer@meduniwien.ac.at Submitted on August 30, 2010; resubmitted on March 16, 2011; accepted on April 1, 2011 background: To determine whether women with extremely low-serum anti-mullerian hormone (AMH) levels (, ng/ml) still demonstrate live birth potential with assisted reproduction and whether such potential is age dependent. methods: Between January 2006 and October 2009, 128 consecutive infertility patients with AMH 0.4 ng/ml were retrospectively evaluated for pregnancy chances and live birth rates after IVF. results: Patients presented at a mean (+SD) age of years, with mean (+SD) baseline FSH of miu/ml and mean (+SD) AMH of ng/ml. One hundred and twenty-eight women underwent a total of 254 IVF cycles. Twenty clinical pregnancies were recorded (7.9% per cycle start [95% confidence interval (CI): %]; 15.6% cumulative [CI: %]). These pregnancies resulted in 13 live births in 12 women (i.e. 11 singletons and a pair of twins) and 8 patients miscarried. Eight deliveries occurred after the first cycle (6.3% per cycle start) and four after subsequent IVF cycles (3.2%). When evaluated according to female age, 70 women 42 years presented with 16 clinical pregnancies that resulted in 10 deliveries (14.3%), while 58 patients.42 years presented with four clinical pregnancies that resulted in 2 deliveries (3.4%), representing a reduced pregnancy chance (P ¼ 0.013) and delivery rate (P ¼ 0.036) versus age 42 years. conclusions: With extremely low-serum AMH levels, moderate, but reasonable pregnancy and live birth rates are still possible. Extremely low AMH levels do not seem to represent an appropriate marker for withholding fertility treatment. Key words: anti-mullerian hormone / dehydroepiandrosterone / diminished ovarian reserve / IVF / pregnancy Introduction The discussion as to who should and should not be offered infertility treatments has intensified in recent years. In some countries offering fertility treatment coverage as part of a national health insurance system, one increasingly hears arguments in favor of restricting IVF to women with reasonable pregnancy chances. In some countries, therefore, fertility coverage is limited by age (Bundeskanzleramt_Österreich, 2004), while in others there is debate about withholding treatments from excessively obese women (Awartani et al., 2009; Dondorp et al., 2010) or those who smoke (Lintsen et al., 2005; Freour et al., 2008; Dondorp et al., 2010). Medical experts have also suggested specific clinical criteria for withholding fertility treatment. For example, ovarian function testing has been proposed to determine treatment admittance, primarily concentrating on baseline FSH (Klinkert et al., 2004; Roberts et al., 2005). Its poor sensitivity and specificity in predicting pregnancy potential, however, does not justify treatment refusal (Klinkert et al., 2004; Sun et al., 2008; Toner, 2004). More recently, anti-mullerian hormone (AMH) has been gaining popularity as its intra-cycle stability makes it a more convenient tool (La Marca et al., 2006; La Marca et al., 2007). Moreover, AMH appears superior to FSH in predicting oocyte yield and pregnancy potential in women undergoing assisted reproduction techniques These authors contributed equally to the preparation of the manuscript. & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 1906 Weghofer et al. (ART) (Nakhuda et al., 2007; Nardo et al., 2007; Barad et al., 2009; Singer et al., 2009). So far, only one ongoing pregnancy has been reported after ART in a patient with extremely low AMH levels (,0.1 ng/ml) (Fraisse et al., 2008). Tocci et al., 2009 described a live birth in a 34-year old woman with AMH concentrations,0.5 ng/ml. Nelson et al., in contrast, could not establish pregnancies in 26 women up to age 44 years with AMH concentrations,0.15 ng/ml after different treatment approaches, performed in the course of a multicenter trial (Nelson et al., 2009). In the here reported study, we attempt to determine specific pregnancy chances in a larger cohort with AMH levels 0.4 ng/ml and whether pregnancy chances change with female age. Materials and Methods Between January 2006 and October 2009, 140 consecutive patients with AMH levels 0.4 ng/ml at initial evaluation presented to the Center for Human Reproduction (New York, NY, USA). All 140 women were referred because of infertility and underwent work up for fertility treatment at our center. Those 128 women who started an IVF cycle during the study period (January 2006 to December 2009) were included. Follow-up for pregnancy outcomes was completed in October Serum AMH levels were measured at initial presentation and prior to all following treatment cycles. In women who underwent more than one cycle, AMH levels at initial presentation and prior to their following cycles are, therefore, reported. The AMH cut-off level of 0.4 ng/ml was chosen, since Gnoth et al. (2008) recently reported this cut-off as a good predictor of poor response to controlled ovarian hyperstimulation. Serum AMH levels were measured using a Diagnostics System Laboratories (DSL) assay (Beckman Coulter, Inc., Brea, CA, USA). The assay for serum AMH involved an enzymatically amplified two-site immunoassay: the DSL active MIS/AMH enzyme-linked immunosorbent assay. According to the manufacturer s manual, the sensitivity of the assay was mg/l with intra-assay variation,15% (Freour et al., 2007). Interand intra-assay variations were,15% in our laboratory. Following our center s routine treatment protocol, all women with diminished ovarian reserve, i.e. abnormally high age-specific baseline FSH and/or abnormally low age-specific AMH levels, were supplemented with 25 mg of micronized dehydroepiandrosterone (DHEA) three times daily for at least 6 weeks prior to ART, as previously reported (Barad and Gleicher, 2005; Gleicher and Barad, 2006; Barad et al., 2007a,b). All women who underwent IVF received microdose agonist stimulation with a daily gonadotrophin dosage of 600 IU (with 3:1 FSH over hmg preponderance) (Gleicher and Barad, 2006). Ovulation induction and oocyte retrieval were performed in all women who presented with at least one follicle of 17 mm diameter. The diagnosis of a clinical pregnancy was established by the presence of a gestational sac on vaginal ultrasound circa 5 weeks after oocyte retrieval. Statistical analysis was undertaken using the Statistical Package for the Social Sciences 17.0 (SPSS, Chicago, IL, USA). Baseline characteristics of patients were compared using t-tests. Outcome parameters are presented as proportions. A P-value,0.05 was considered statistically significant. Continuous values are presented as mean + SD and as median; outcome parameters are presented as mean + SD with 95% confidence intervals (CI). Subgroup analyses were performed based on age 42 and. 42 years since Klipstein et al. (2005) reported substantially lower pregnancy rates in women above age 42 years. Logistic regression of pregnancy against AMH as a continuous variable was used to evaluate an association between different AMH levels and pregnancy potential. The data presented here only involved retrospective review of medical records. Patients at our Center sign at initial consultation an informed consent which allows for such reviews if the patient s medical record remains confidential and her identity protected. These conditions were met in this case, allowing for expedited approval by the Institutional Review Board. Results Table I summarizes patient characteristics. The mean age for all 128 women was years [median: 41.6 years]; mean AMH levels were ng/ml [median: 0.2 ng/ml]. In women with repeated AMH measurements owing to multiple cycles, maximum AMH levels of 0.4 ng/ml were recorded. AMH values in subsequent cycles also showed mean levels of ng/ml [median: 0.3 ng/ ml]. Univariate regression analysis did not reveal a significant association between AMH levels of between,0.1 and 0.4 ng/ml and pregnancy potential or live birth. The study group underwent a total of 254 fresh IVF cycles during the study period, resulting in retrieval of oocytes [median: 2.0]. Fertilization failure or embryonic arrest occurred in 62 cycles. Only one embryo was transferred in 59 cycles (39%), two embryos were transferred in 60 cycles (40%), three in 25 cycles (17%) and four embryos were transferred in only six cycles (4%). Owing to a lack of ovarian response (no follicle development), 42 cycles (17%) Table I Patient characteristics and cycle parameters in 128 women with extremely low anti-mullerian hormone (AMH) levels (< ng/ml) who underwent 254 ART cycles. All patients (n 5 128/254) a Age 42 years (n 5 70/145) a >Age 42 years (n 5 58/109) a... Female age (years) [41.6] [39.0] [44.0] AMH (ng/ml) [0.2] [0.3] [0.2] Baseline FSH (miu/ml) [13.1] [13.2] [13.1] Number of ART cycle starts per patient [2.0] [2.0] [2.0] Total gonadotrophin dosage (IU) [6900] [6600] [7650] Cycle cancellation before retrieval n (%) 42 (17%) 27 (19%) 15 (14%) Oocytes retrieved per cycle start [2.0] [3.0] [2.0] Values are mean + SD; values in brackets are medians. a Patients/ART cycles.

3 Low anti-mullerian hormone and live birth rates 1907 Table II Pregnancy outcomes in 128 IVF patients with extremely low AMH levels ( ng/ml). All patients 95% CI Age 42 years 95% CI >Age 42 years 95% CI P (n 5 128/254) a (n 5 70/145) a (n 5 58/109) a... Clinical pregnancies 20 (7.9%) [4.9% 11.9%] 16 (11.0%) [6.4% 17.3%] 4 (3.7%) [1.0% 9.1%] per cycle Clinical pregnancies 20 (15.6%) [9.8% 23.1%] 16 (22.9%) [13.7% 34.5%] 4 (6.9%) [1.9% 16.7%] per patient Deliveries after 1st IVF 8 (6.3%) [2.7% 11.9%] 7 (10.0%) [4.1% 19.5%] 1 (1.7%) [0.04% 9.2%] cycle Deliveries per patient 12 (9.4%) [4.9% 15.8%] 10 (14.3%) [7.1% 24.7%] 2 (3.4%) [0.4% 11.9%] a Patients/ART cycles. were cancelled. When analyzed according to age (n ¼ years versus n ¼ years), no differences were found in baseline and cycle characteristics between the two groups. Table II presents pregnancy outcomes and delivery rates of 128 women after a total number of 254 IVF cycles: twenty clinical pregnancies were recorded (7.9% per cycle start [95% CI: %]; 15.6% cumulative [CI: %]). These pregnancies resulted in 13 healthy live births in 12 women (i.e. 11 singletons and a pair of twins), eight patients miscarried (40% [ %]). Eight deliveries occurred after first (6.3%) and four after consecutive IVF cycles (3.2%). Those eight women who delivered after their first IVF cycle required a median time of 3 months to conceive (time from first consultation to pregnancy test), while the four patients who conceived in subsequent cycles did so after a median time of 23 months. When evaluated according to female age, 70 women 42 years presented with 16 clinical pregnancies (22.9% [CI: %]) that resulted in 10 deliveries (14.3% [CI: %]), while 58 patients above age 42 years presented with four clinical pregnancies (6.9% [CI: %]) that resulted in two deliveries (3.4%; [CI: %]), representing a reduced pregnancy chance and delivery rate versus age 42 years (P ¼ 0.013; P ¼ 0.036, respectively). Discussion The here presented data demonstrate moderate, but still reasonable pregnancy chances and live birth rates in women with extremely low AMH levels. To the best of our knowledge, this represents the largest cohort of women with extremely compromised AMH levels who conceived and delivered in the course of ART. At many IVF centers such patients are currently counseled regarding their substantially reduced pregnancy chances with autologous oocytes, and are often advised to proceed to using egg donation (Scott, 2004). Our data concur with those of Nelson et al. who report reduced but not negligible pregnancy potential in women with AMH levels,0.7 ng/ml (Nelson et al., 2009). Age-dependent decline in fertility potential is generally attributed to quantitative and qualitative changes in follicle pools. The older a woman, the fewer follicles remain, oocytes are of a lower quality and aneuploidy increases, leading to rising miscarriage rates (Kumbak et al., 2009). The observed miscarriage rate of 40% in this patient cohort with median age of 41.6 years and severely diminished ovarian reserve has, therefore, to be considered quite low. Women with diminished ovarian reserve usually demonstrate the highest miscarriage rates amongst infertility patients (Levi et al., 2001). If AMH levels correspond to follicle numbers, but not to oocyte quality and chromosomal integrity, as stated by Guerif et al. and others (Lie Fong et al., 2008; Guerif et al., 2009), younger women are likely to experience better pregnancy rates than their older counterparts with equally low AMH. The significantly higher clinical pregnancy and delivery rates were observed in younger women ( 42 years), support this. Aging, however, represents a continuum: whatever age the cut-off is set to for comparing pregnancy and delivery rates between younger and older women, it always, to a degree, will be arbitrary. All women in this study received DHEA supplementation. As previously reported, we and others demonstrated improved pregnancy chances with DHEA (Gleicher et al., 2010a; Wiser et al., 2010) in women with diminished ovarian reserve. Here presented findings can, therefore, not automatically be assumed to apply to all women with low AMH. The DHEA studies, indeed, also demonstrated an age-related effect in favor of younger age (Gleicher et al., 2010b). As increasing numbers of women postpone pregnancy, those seeking fertility treatments are increasing in average age ( gov/art/). These older women challenge reproductive endocrinologists to offer thoughtful counsel. Predictive AMH data are of considerable potential importance in such a process. Thirteen live births in 128 women with AMH levels,0.4 ng/ml, especially in young women, however, seriously questions the value of extremely low AMH concentrations in helping to determine when to refuse IVF treatments. AMH likely represents the most specific tool for assessing pregnancy and delivery chances (Wang et al., 2010). Since women with extremely low AMH levels still experience pregnancy chances, currently available tools may be able to determine compromised pregnancy chances, they appear, however, unsuitable to accurately predict the absence of realistic pregnancy or live birth potential (Nelson et al., 2009; Gleicher et al., 2010c), which has been further supported by the data presented in this study. In conclusion, here presented data demonstrate moderate but still reasonable pregnancy chances and live birth rates with extremely lowserum AMH levels (, ng/ml). Though these post hoc observations need further confirmation in other (not treated with DHEA) patients, extremely low AMH levels do not seem to represent an appropriate marker for withholding fertility treatment.

4 1908 Weghofer et al. Authors roles A.W. and W.D. contributed equally to the preparation of this manuscript. A.W. involved in study design, execution, analysis, manuscript drafting and critical discussion, W.D. in execution, analysis, manuscript drafting and critical discussion, D.B. in study design, analysis and critical discussion and N.G. in study design, manuscript drafting and critical discussion. Conflict of interest D.H.B. and N.G. have received research grant support, travel funds and speaker honoraria from various pharmaceutical and medical device companies, none, however, related to here presented topic. Both authors also are listed as inventors on already awarded and still pending US patents, claiming beneficial effects on diminished ovarian reserve and embryo ploidy from DHEA supplementation. Funding Foundation for Reproductive Medicine and intramural grants from the Center for Human Reproduction. References Awartani KA, Nahas S, Al Hassan SH, Al Deery MA, Coskun S. 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