ORIGINAL STUDY
BRCA Mutation Carriers Do Not Have Compromised Ovarian Reserve Rachel Michaelson-Cohen, MD,*Þþ Pnina Mor, PhD,*Þ Naama Srebnik, MD,* Uziel Beller, MD,*Þ Ephrat Levy-Lahad, MD,*Þ and Talia Eldar-Geva, MD, PhDÞ
Controversy exists about the impact of BRCA1/2 mutations on female fertility. Previous studies are small or based on indirect parameters (eg, self-reported infertility), which depend on additional factors unrelated to true fertility potential. Most of the previous studies did not use strict fertility markers. Objective: The aim of this study is to evaluate the relation between carrying a BRCA1/2 mutation and fertility using the level of anti-mu¨llerian hormone (AMH), which has been previously shown to be an accurate marker of ovarian reserve and fertility potential. Patients and Methods: Forty-one healthy BRCA1/2 mutation carriers, aged 26 to 40 years, attending a multidisciplinary breast and ovarian cancer surveillance clinic, were tested for AMH levels using a 2-site ELISA. Levels were compared with those of our general population and with well-established normograms of the general population. Results: The mean age of carriers was 33.2 years (26Y39 years; SD, 3.99 years). The mean parity of carriers was 1.97 (0Y7; SD, 1.49). All women carried at least 1 Ashkenazi Jewish founder mutation. The AMH levels for most carriers were in the reference range, 2.71 T 0.59 ng/mL (approximately 50th percentile of normograms). These levels were similar to those in the control group, in which the AMH levels were 2.02 T 0.12 ng/mL (P = 0.27). Conclusions: The AMH levels of healthy BRCA1/2 mutation carriers are similar to those of noncarrier women matched for age; therefore, their ovarian reserve is comparable. This is the only study, to the best of our knowledge, that directly examines ovarian reserve in a relatively large group of carriers with an accurate marker. The results of this study may possibly give reassurance to female carriers concerning fertility potential. Key Words: BRCA1/2 mutation carriers, Ovarian reserve, Anti-mu¨llerian hormone Received August 7, 2013, and in revised form November 4, 2013. Accepted for publication November 4, 2013. (Int J Gynecol Cancer 2014;24: 233Y237)
*Noga BRCA Carrier Surveillance Clinic, †Department of Gynecology, and ‡Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel. Address correspondence and reprint requests to Rachel Michaelson-Cohen, MD, Department Gynecology and Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University of Jerusalem, PO Box 3235 Jerusalem 91031, Israel. E-mail: [email protected]. This study was supported by a generous yearly gifting, over 5 years, to the Noga Carrier Clinic at the Shaare Zedek Medical Center by Jack Klein, in memory of his dear wife, Elisa Klein. The authors declare no conflicts of interest. Copyright * 2014 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000058 International Journal of Gynecological Cancer
and BRCA2 both function as ‘‘genomic careB RCA1 takers’’ important for genomic integrity and prevention of
instability that leads to malignancy. They are primarily involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. They are also involved in the repair and maintenance of chromosome telomeres.1,2 However, they have additional multiple functions, and not all functions have been discovered. Mutations in either gene are associated with breast and ovarian cancer susceptibility, and these mutations are inherited in an autosomal dominant fashion.3 There is controversy about the impact of these mutations on female fertility. Because maintenance of telomere integrity is fundamental to reproductive fitness, BRCA mutation carriers may have a higher risk of fertility-related problems; however,
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there is no clinical evidence of this. In addition to telomere length, factors that affect cell cycle and division, or DNA repair, may also affect the reproductive life span. Furthermore, because infertility is associated with breast and ovarian cancer risks, it was hypothesized that BRCA mutations may be associated with primary ovarian insufficiency.4 In 2010, an analysis of the results from the prospective controlled COST-LESS study was published. The COST-LESS study examined the use of letrozole (aromatase inhibitor) with gonadotropins for ovarian stimulation in patients with breast cancer undergoing fertility preservation treatments. The analysis showed higher rates (odds ratio, 28.7; 95% confidence interval, 1.8Y447; P = 0.016) of low ovarian reserve in carriers of BRCA1 mutations (defined as retrieval of 4 or fewer oocytes in women younger than 38 years) compared with that in noncarriers.4 Many other surveys have failed to show a decrease in fertility among carriers. The effect of BRCA mutations on female fertility was analyzed in 96 BRCA1/2 mutation carriers with ovarian cancer, compared with 164 noncarriers with ovarian cancer and 331 noncarriers without ovarian cancer. Fertility was defined as the number of pregnancies during the reproductive period (excluding the time on contraception). There were no significant differences between the carrier and noncarrier cases and controls with regards to fertility after adjusting for covariates. However, the authors comment that factors pertaining to the study design may have affected the results.5 In another questionnaire-based case-control study performed on 2254 BRCA mutation carriers and 764 noncarrier family members, there was no difference in the self-reported rate of fertility problems or of taking a fertility medication between carriers and noncarriers. There was also no difference in parity between the 2 groups,6 suggesting that there is no effect of these mutations on fertility. However, the authors expected that if fertility differences were to exist, they would be expressed toward the limits of reproductive life, and larger studies with age-stratified analyses would be necessary for demonstration of this theoretical difference. In an additional questionnaire-based case-control study conducted in 2008 on a group of more than 2500 BRCA mutation carriers, infertility rates were similar to the rates known in the general population. The study was conducted to determine whether the use of fertility medication or the process of in vitro fertilization (IVF) was associated with an increased risk of breast cancer in BRCA mutation carriers.7 The study included 1380 women with BRCA1 or BRCA2 mutations. Case subjects were carriers with a diagnosis of invasive breast cancer. Control subjects were women who never had breast cancer and who were also carriers of a mutation in BRCA1 or BRCA2. The reported infertility rate in the entire cohort was 16%. Only 4% of the carriers had used fertility medication, and less than 1% had undergone IVF treatment. These rates are similar to those of the general population.8 None of the previously cited studies have used strict markers of fertility potential or of ovarian reserve, such as antimu¨llerian hormone (AMH). The AMH, also called mu¨llerianinhibiting substance, belongs to the TGF-A superfamily. In male fetuses, AMH is secreted by testicular Sertoli cells,
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stimulating the degradation of the mu¨llerian duct.8 In female fetuses, AMH is secreted toward the end of pregnancy by ovarian granulosa cells, and it continues to be produced until menopause.9 The AMH is secreted from primary, secondary, and small antral follicles up to 4 to 6 mm in diameter. There is a direct relationship between the number of early-stage follicles and serum AMH levels. The AMH levels decrease with age until it ceases to be secreted at menopause.10 Accordingly, women with polycystic ovaries (PCOs), who have a large number of early-stage follicles, have higher AMH levels than normal women matched for age.11 The serum AMH level has been shown to be among the best predictors of ovarian reserve.12,13 As a single marker, AMH can sufficiently predict ovarian reserve and response to fertility treatment. In comparison with other markers that are frequently used to predict ovarian reserve, AMH is at least as good, with the practical advantage of being cycle independent. This enables ovarian reserve testing for women attending clinics at any given time regardless of their menstrual cycle. A recent large meta-analysis demonstrated that AMH and antral follicle count (AFC) each have added value to age in predicting ovarian response to IVF. In addition, as single tests, AMH and AFC both fully covered the prediction of ovarian response regardless of age.14 A recent study15 has examined the relation between impaired BRCA gene function and ovarian dysfunction. In mouse studies, the authors noted that expression of BRCA1 declined in old mice compared with that in young mice. By examining BRCA1-deficient mice, they demonstrated that diminished DSB repair in aging oocytes leads to accumulation of lethal DSBs. Three BRCA1-deficient mice had lower response to ovarian stimulation. In human oocytes, they showed that DSBs were increased and BRCA1 expression decreased with age. In a group of 15 women with BRCA1 mutations, serum AMH levels were lower than in controls. The group came to the conclusion that BRCA deficiency might deteriorate ovarian function. This pilot study aimed to evaluate the relationship between carrying a BRCA mutation and fertility, using AMH as an accurate and efficient method to estimate ovarian reserve and fertility potential, in a large group of BRCA1 and BRCA2 mutation carriers from a wide range of ages.
PATIENTS AND METHODS The study includes consecutive 41 carriers of BRCA 1/2 mutations, aged 40 years or younger, attending a multidisciplinary breast and ovarian cancer surveillance clinic at the Shaare Zedek Medical Center during 2010 to 2011. Women with only 1 ovary or who had an ovarian cystectomy or other ovarian surgeries were excluded. All women had clinical breast examination, vaginal ultrasonography, serum CA125 measurement, and magnetic resonance imaging or mammography every 6 months. None of the conditions of these women were diagnosed with ovarian, breast, or other cancer during the study period. Institutional review board approval was obtained, and all participants gave informed consent. * 2014 IGCS and ESGO
Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
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Data were collected from all women including age, parity, menstrual cycle regularity, and type of BRCA mutation. Frozen serum samples were tested for AMH using a 2-site ELISA (Diagnostic System Laboratory, TX). Assay sensitivity was 0.017 ng/mL; inter-assay and intra-assay coefficients of variation were 8% and 4.6%, respectively, for serum AMH concentration of 0.15 ng/mL and 6.7%, and 3.3% for a serum AMH concentration of 4.34 ng/mL. The AMH levels in this study group were compared with that in the group of 324 healthy controls (women with normal ovulatory cycles). These women had been previously evaluated in our laboratory. Levels were compared by t test. Results are presented as mean AMH levels with SE. We also compared the 2 groups by subdividing them by age groups. In addition, we compared the AMH levels of the study group to those in the 2 well-established AMH normograms16,17; the latter normogram is based on a multicenter study population of 3871 women, including the 324 women from our own center. The former normogram (Nelson model) was built on the basis of infertile women; however, it was validated on a cohort of 423 women with confirmed ovulation and normal pelvic ultrasound findings.16
RESULTS We describe 41 BRCA1/2 mutation carriers, younger than 40 years, attending the breast and ovarian cancer surveillance clinic during the year 2010 to 2011. Their mean age at the time of analysis was 33.2 years (26Y39 years; SD, 3.99 years). The mean parity was 1.97 (0Y7; SD, 1.49). All women were carriers of at least 1 of the 3 Ashkenazi Jewish founder mutations (185 delAG and 5382 insC in BRCA1, and 6174 delT in BRCA2). Twenty-five women were BRCA1 mutation carriers, 12 were BRCA2 mutation carriers, and 3 women were carriers of a mutation in both genes. When inquired about infertility at history taking, none of the 41 women reported such a history. Demographic and genetic parameters are presented in Table 1. The AMH levels for most of the mutation carriers were in the reference range (2.71 T 0.59 ng/mL). These levels were similar to those in the control group, in which the AMH levels were 2.02 T 0.12 ng/mL (P = 0.27). There were also no differences in AMH levels when comparing the women in the study and the control groups by subdividing them according to age groups. A 33-year-old woman in the study group had an AMH level of 23.8 ng/mL, associated with PCO syndrome. This value was included in the analysis despite its extreme high level because the control group also included some women with PCO syndrome. Serum AMH levels in both
BRCA Mutations and Ovarian Reserve
study and control groups are presented in Figure 1, presented according to various age groups. Comparing the levels to the Nelson normogram,16 the results were close to the 50 percentile (Fig. 2). Although AMH levels were slightly higher for carriers of mutations in BRCA1 versus BRCA2 (mean, 2.48 vs 1.70 ng/mL, respectively), there was no statistically significant difference.
DISCUSSION The relation between BRCA mutations and infertility is poorly described in the literature, and there are conflicting results from different studies. To date, there is no recommendation for routine evaluation of fertility in female carriers of BRCA mutations. This is the first study, to the best of our knowledge, that evaluates ovarian function in a large group of BRCA mutation carriers with a wide range of ages using a wellvalidated marker of ovarian reserve/fertility potential, namely, the serum AMH.12,13 A recent study has addressed this issue in mice; 3 BRCA1-deficient mice showed lower response to ovarian stimulation than wild-type mice. They also performed a human study, and 15 women with BRCA1 mutations had lower serum AMH levels than controls. The group came to the conclusion that BRCA deficiency might deteriorate ovarian function. The differences between this study and ours may be due to the difference in the size of the studies and the ages of the women tested. The use of AMH was first used in infertility populations, in which it has great value in predicting outcome in the setting of IVF, particularly for women in their later reproductive years. In more recent studies, AMH levels were measured in young and fertile women, including women from the general population attending our center.17 The AMH values in this study group were similar to the control group, which is our own healthy reference population that had been previously tested for AMH. By comparing AMH levels in a cohort of BRCA mutation carriers with 2 well-established AMH normograms,16,17 we have shown that mutation carriers have AMH levels comparable to the general population. We conclude that there is no substantial difference in ovarian reserve between carriers and the general population. Whether carrying a BRCA mutation increases the risk for infertility has clinical implications. With the ongoing accumulation of molecular and clinical information from studies on women carrying BRCA mutations, many questions arise regarding the recommended management methods for healthy
TABLE 1. Demographic and genetic parameters of the 41 BRCA mutation carriers Age Group, y No. Patients No. Children, Median (Range) 26Y30 31Y34 35Y36 37Y39
13 10 10 8
1 2 2 3
(0Y3) (0Y4) (0Y3) (1Y7)
BRCA1 Mutation
BRCA2 Mutation
BRCA1 + 2 Mutations
9 6 6 5
3 3 3 3
1 1 1 0
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FIGURE 1. Serum AMH levels in 41 female carriers of BRCA1/2 mutations (black bars) compared with that in control women matched by age (white bars). All bar graphs show means (SE). carriers.18 Current recommendations for mutation carriers are to complete family planning by age 35 to 40 years and then to undergo salpingo-ooporectomy to reduce the risk of breast and ovarian cancer.19,20 If mutation carriers have reduced fertility, it may be appropriate to encourage family planning at an earlier age. In addition, the effect of BRCA mutations on fertility would be an issue to discuss with young women undergoing genetic testing for these mutations. Previous studies have addressed this issue indirectly by examining parity or self-reported history of infertility.5,7 However, these parameters depend on additional factors unrelated to the true fertility potential of women. Furthermore, some of the latter studies were subjected to recall bias, because the collection of data was based on questionnaires or phone interviews.6 Nevertheless, most studies could not show reduced fertility in BRCA mutation carriers.5,7 Our results contradict the conclusions of 2 studiesVthe study by Oktay et al,4 who found an increased risk of poor ovarian response in carriers of BRCA1 mutation undergoing fertility preservation as compared with that in noncarriers, and the study by Titus et al,15 in which 15 women with BRCA1 mutations had lower serum AMH levels than that in controls. However, in the former study, the study group in this research was small, 12 women with either BRCA1 or 2 mutations, with only 8 BRCA1 carriers of pathogenic mutations. Furthermore, the women included in the study had breast cancer (ie, they were not healthy carriers). The differences between these studies and ours may be due to the difference in the size of the studies and the ages of the women tested. Our study examined 41 BRCA mutation carriers at a larger range of ages (26Y39 years). The women found to have low ovarian response among the BRCA1 mutation carriers in both previous studies were older (in the former, older than 33 years, and in the latter, mean age was 34.8). Many of the BRCA mutation carriers go through risk reducing BSO at this age, and the impact of infertility at this age is less profound. In any case, our results show that AMH levels for healthy carriers even at this age group are similar to our general population; therefore, their fertility potential seems to be similar to that of noncarrier women of the same age.
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Some authors have noted early menopause in BRCA mutation carriers. Our study was on a group of women aged 26 to 39 years, and the ovarian reserve was similar to the general population (matched for age). Therefore, from the evaluation of ovarian reserve at these ages, one can extrapolate on the ovarian function throughout the years of fertility. However, it is possible that women with these mutations have normal ovarian function until a premature sudden decline in ovarian function. It is possible that if we had a larger number of women at the upper end of the age spectrum (ie, closer to 40 years), the results would have been different. It is also possible that mutations in BRCA genes may actually have a positive effect on fertility. Common mutations in other genes in certain populations have been shown to have a survival advantage (eg, lower malaria prevalence for carriers of mutations in the A hemoglobin in endemic countries). Mutations in BRCA genes may theoretically improve conditions for success of the embryonal implantation process. Greater fertility could provide a selective advantage for BRCA1 and BRCA2 mutations. To date, molecular mechanisms of this hypothetical effect are unclear. In this study, we found neither a positive nor a negative effect of BRCA1/2 mutations on ovarian function. This is only a preliminary study on a small group of BRCA1/2 mutation carriers. A long-term, large scale study, comparing actual outcomes of fertility, is needed to support our findings. This study did not address the issue of oral contraceptive use, age of women at conception, and response of patients to fertility treatment. These parameters would provide important information. In addition, another accurate marker of ovarian reserve, AFC, should be used to support our findings. This study did not include a large enough group of mutation carriers to perform a subgroup analysis
FIGURE 2. Serum AMH levels (ng/mL) in 41 female carriers of BRCA1/2 mutations as compared with the Nelson normogram.15 * 2014 IGCS and ESGO
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International Journal of Gynecological Cancer
& Volume 24, Number 2, February 2014
comparing AMH levels in women with mutations in BRCA1 versus BRCA2. Nonetheless, this is currently the only study, to the best of our knowledge, that directly examines ovarian reserve with 1 of the best predictors known today to evaluate ovarian reserve, serum AMH level,12,13 in a relatively large group of BRCA mutation carriers with a wide range of ages. The study addresses an issue of great interest to carriers of a common gene mutation and their caregivers. If the results of this pilot study are ascertained by future larger scale studies addressing all the issues mentioned previously, they may give reassurance to female carriers of BRCA mutations concerning their fertility potential. At this point, according to our preliminary results, a woman with identified as a mutation carrier may assume her fertility potential to be similar to the general population. This may not only alleviate anxiety but also affect decision making concerning family planning.
REFERENCES 1. Lansdorp PM. Repair of telomeric DNA prior to replicative senescence. Mech Ageing Dev. 2000;118:23Y34. 2. McPherson JP, Hande MP, Poonepalli A, et al. A role for BRCA1 in chromosome end maintenance. Hum Mol Genet. 2006;15:831Y838. 3. Ford D, Easton DF, Peto J. Estimates of the gene frequency of BRCA1 and its contribution to breast and ovarian cancer incidence. Am J Hum Genet. 1995;57:1457Y1462. 4. Oktay K, Kim JY, Barad D, et al. Association of BRCA1 mutations with occult primary ovarian insufficiency: a possible explanation for the link between infertility and breast/ovarian cancer risks. J Clin Oncol. 2010;28:240Y244. 5. Moslehi R, Singh R, Lessner L, et al. Impact of BRCA mutations on female fertility and offspring sex ratio. Am J Hum Biol. 2010;22:201Y205. 6. Pal T, Keefe D, Sun P, et al. Fertility in women with BRCA mutations: a case-control study. Fertil Steril. 2010;93: 1805Y1808. 7. Kotsopoulos J, Librach CL, Lubinski J, et al. Infertility, treatment of infertility, and the risk of breast cancer among women with BRCA1 and BRCA2 mutations: a case-control study. Cancer Causes Control. 2008;19:1111Y1119. 8. Speroff L, Fritz MA. Clinical Gynecologic Endocrinology and Infertility. 8th ed. Lippicont Williams & Wilkins, Baltimore, MD; 2011.
BRCA Mutations and Ovarian Reserve
9. Rajpert-De Meyts E, Jorgensen N, Graem N, et al. Expression of anti-Mu¨llerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells. J Clin Endocrinol Metab. 1999; 84:3836Y3844. 10. van Rooij IA, Broekmans FJ, te Velde ER, et al. Serum anti-Mu¨llerian hormone levels: a novel measure of ovarian reserve. Hum Reprod. 2002;17:3065Y3071. 11. Laven JS, Mulders AG, Visser JA, et al. Anti-Mu¨llerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab. 2004;89:318Y323. 12. Broer SL, Eijkemans MJ, Scheffer GJ, et al. Anti-mu¨llerian hormone predicts menopause: a long-term follow-up study in normoovulatory women. J Clin Endocrinol Metab. 2011;96: 2532Y2539. 13. Kwee J, Schats R, McDonnell J, et al. Evaluation of anti-Mu¨llerian hormone as a test for the prediction of ovarian reserve. Fertil Steril. 2008;90:737Y743. 14. Broer SL, van Disseldorp J, Broeze KA, et al. Added value of ovarian reserve testing on patient characteristics in the prediction of ovarian response and ongoing pregnancy: an individual patient data approach. Hum Reprod Update. 2013;19:26Y36. 15. Titus S, Li F, Stobezki R, et al. Impairment of BRCA1-related DNA double-strand break repair leads to ovarian aging in mice and humans. Sci Transl Med. 2013;5:172ra21. 16. Nelson SM, Messow MC, Wallace AM, et al. Nomogram for the decline in serum antimu¨llerian hormone: a population study of 9,601 infertility patients. Fertil Steril. 2011; 95:736Y741 e1Ye3. 17. Almog B, Shehata F, Suissa S, et al. Age-related normograms of serum antimu¨llerian hormone levels in a population of infertile women: a multicenter study. Fertil Steril. 2011;95:2359Y2363, 2363 e1. 18. Eitan R, Michaelson-Cohen R, Levavi H, et al. The counseling and management of young healthy BRCA mutation carriers. Int J Gynecol Cancer. 2009;19:1156Y1159. 19. Rebbeck TR, Lynch HT, Neuhausen SL, et al. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations. N Engl J Med. 2002;346:1616Y1622. 20. Samuel JC, Ollila DW. Prophylaxis and screening options: recommendations for young women with BRCA mutations. Breast Dis. 2005;23:31Y35.
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BRCA Mutation Carriers Do Not Have Compromised Ovarian Reserve Rachel Michaelson-Cohen, MD,*Þþ Pnina Mor, PhD,*Þ Naama Srebnik, MD,* Uziel Beller, MD,*Þ Ephrat Levy-Lahad, MD,*Þ and Talia Eldar-Geva, MD, PhDÞ
Controversy exists about the impact of BRCA1/2 mutations on female fertility. Previous studies are small or based on indirect parameters (eg, self-reported infertility), which depend on additional factors unrelated to true fertility potential. Most of the previous studies did not use strict fertility markers. Objective: The aim of this study is to evaluate the relation between carrying a BRCA1/2 mutation and fertility using the level of anti-mu¨llerian hormone (AMH), which has been previously shown to be an accurate marker of ovarian reserve and fertility potential. Patients and Methods: Forty-one healthy BRCA1/2 mutation carriers, aged 26 to 40 years, attending a multidisciplinary breast and ovarian cancer surveillance clinic, were tested for AMH levels using a 2-site ELISA. Levels were compared with those of our general population and with well-established normograms of the general population. Results: The mean age of carriers was 33.2 years (26Y39 years; SD, 3.99 years). The mean parity of carriers was 1.97 (0Y7; SD, 1.49). All women carried at least 1 Ashkenazi Jewish founder mutation. The AMH levels for most carriers were in the reference range, 2.71 T 0.59 ng/mL (approximately 50th percentile of normograms). These levels were similar to those in the control group, in which the AMH levels were 2.02 T 0.12 ng/mL (P = 0.27). Conclusions: The AMH levels of healthy BRCA1/2 mutation carriers are similar to those of noncarrier women matched for age; therefore, their ovarian reserve is comparable. This is the only study, to the best of our knowledge, that directly examines ovarian reserve in a relatively large group of carriers with an accurate marker. The results of this study may possibly give reassurance to female carriers concerning fertility potential. Key Words: BRCA1/2 mutation carriers, Ovarian reserve, Anti-mu¨llerian hormone Received August 7, 2013, and in revised form November 4, 2013. Accepted for publication November 4, 2013. (Int J Gynecol Cancer 2014;24: 233Y237)
*Noga BRCA Carrier Surveillance Clinic, †Department of Gynecology, and ‡Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel. Address correspondence and reprint requests to Rachel Michaelson-Cohen, MD, Department Gynecology and Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University of Jerusalem, PO Box 3235 Jerusalem 91031, Israel. E-mail: [email protected]. This study was supported by a generous yearly gifting, over 5 years, to the Noga Carrier Clinic at the Shaare Zedek Medical Center by Jack Klein, in memory of his dear wife, Elisa Klein. The authors declare no conflicts of interest. Copyright * 2014 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000058 International Journal of Gynecological Cancer
and BRCA2 both function as ‘‘genomic careB RCA1 takers’’ important for genomic integrity and prevention of
instability that leads to malignancy. They are primarily involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. They are also involved in the repair and maintenance of chromosome telomeres.1,2 However, they have additional multiple functions, and not all functions have been discovered. Mutations in either gene are associated with breast and ovarian cancer susceptibility, and these mutations are inherited in an autosomal dominant fashion.3 There is controversy about the impact of these mutations on female fertility. Because maintenance of telomere integrity is fundamental to reproductive fitness, BRCA mutation carriers may have a higher risk of fertility-related problems; however,
& Volume 24, Number 2, February 2014
Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
233
Michaelson-Cohen et al
International Journal of Gynecological Cancer
there is no clinical evidence of this. In addition to telomere length, factors that affect cell cycle and division, or DNA repair, may also affect the reproductive life span. Furthermore, because infertility is associated with breast and ovarian cancer risks, it was hypothesized that BRCA mutations may be associated with primary ovarian insufficiency.4 In 2010, an analysis of the results from the prospective controlled COST-LESS study was published. The COST-LESS study examined the use of letrozole (aromatase inhibitor) with gonadotropins for ovarian stimulation in patients with breast cancer undergoing fertility preservation treatments. The analysis showed higher rates (odds ratio, 28.7; 95% confidence interval, 1.8Y447; P = 0.016) of low ovarian reserve in carriers of BRCA1 mutations (defined as retrieval of 4 or fewer oocytes in women younger than 38 years) compared with that in noncarriers.4 Many other surveys have failed to show a decrease in fertility among carriers. The effect of BRCA mutations on female fertility was analyzed in 96 BRCA1/2 mutation carriers with ovarian cancer, compared with 164 noncarriers with ovarian cancer and 331 noncarriers without ovarian cancer. Fertility was defined as the number of pregnancies during the reproductive period (excluding the time on contraception). There were no significant differences between the carrier and noncarrier cases and controls with regards to fertility after adjusting for covariates. However, the authors comment that factors pertaining to the study design may have affected the results.5 In another questionnaire-based case-control study performed on 2254 BRCA mutation carriers and 764 noncarrier family members, there was no difference in the self-reported rate of fertility problems or of taking a fertility medication between carriers and noncarriers. There was also no difference in parity between the 2 groups,6 suggesting that there is no effect of these mutations on fertility. However, the authors expected that if fertility differences were to exist, they would be expressed toward the limits of reproductive life, and larger studies with age-stratified analyses would be necessary for demonstration of this theoretical difference. In an additional questionnaire-based case-control study conducted in 2008 on a group of more than 2500 BRCA mutation carriers, infertility rates were similar to the rates known in the general population. The study was conducted to determine whether the use of fertility medication or the process of in vitro fertilization (IVF) was associated with an increased risk of breast cancer in BRCA mutation carriers.7 The study included 1380 women with BRCA1 or BRCA2 mutations. Case subjects were carriers with a diagnosis of invasive breast cancer. Control subjects were women who never had breast cancer and who were also carriers of a mutation in BRCA1 or BRCA2. The reported infertility rate in the entire cohort was 16%. Only 4% of the carriers had used fertility medication, and less than 1% had undergone IVF treatment. These rates are similar to those of the general population.8 None of the previously cited studies have used strict markers of fertility potential or of ovarian reserve, such as antimu¨llerian hormone (AMH). The AMH, also called mu¨llerianinhibiting substance, belongs to the TGF-A superfamily. In male fetuses, AMH is secreted by testicular Sertoli cells,
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stimulating the degradation of the mu¨llerian duct.8 In female fetuses, AMH is secreted toward the end of pregnancy by ovarian granulosa cells, and it continues to be produced until menopause.9 The AMH is secreted from primary, secondary, and small antral follicles up to 4 to 6 mm in diameter. There is a direct relationship between the number of early-stage follicles and serum AMH levels. The AMH levels decrease with age until it ceases to be secreted at menopause.10 Accordingly, women with polycystic ovaries (PCOs), who have a large number of early-stage follicles, have higher AMH levels than normal women matched for age.11 The serum AMH level has been shown to be among the best predictors of ovarian reserve.12,13 As a single marker, AMH can sufficiently predict ovarian reserve and response to fertility treatment. In comparison with other markers that are frequently used to predict ovarian reserve, AMH is at least as good, with the practical advantage of being cycle independent. This enables ovarian reserve testing for women attending clinics at any given time regardless of their menstrual cycle. A recent large meta-analysis demonstrated that AMH and antral follicle count (AFC) each have added value to age in predicting ovarian response to IVF. In addition, as single tests, AMH and AFC both fully covered the prediction of ovarian response regardless of age.14 A recent study15 has examined the relation between impaired BRCA gene function and ovarian dysfunction. In mouse studies, the authors noted that expression of BRCA1 declined in old mice compared with that in young mice. By examining BRCA1-deficient mice, they demonstrated that diminished DSB repair in aging oocytes leads to accumulation of lethal DSBs. Three BRCA1-deficient mice had lower response to ovarian stimulation. In human oocytes, they showed that DSBs were increased and BRCA1 expression decreased with age. In a group of 15 women with BRCA1 mutations, serum AMH levels were lower than in controls. The group came to the conclusion that BRCA deficiency might deteriorate ovarian function. This pilot study aimed to evaluate the relationship between carrying a BRCA mutation and fertility, using AMH as an accurate and efficient method to estimate ovarian reserve and fertility potential, in a large group of BRCA1 and BRCA2 mutation carriers from a wide range of ages.
PATIENTS AND METHODS The study includes consecutive 41 carriers of BRCA 1/2 mutations, aged 40 years or younger, attending a multidisciplinary breast and ovarian cancer surveillance clinic at the Shaare Zedek Medical Center during 2010 to 2011. Women with only 1 ovary or who had an ovarian cystectomy or other ovarian surgeries were excluded. All women had clinical breast examination, vaginal ultrasonography, serum CA125 measurement, and magnetic resonance imaging or mammography every 6 months. None of the conditions of these women were diagnosed with ovarian, breast, or other cancer during the study period. Institutional review board approval was obtained, and all participants gave informed consent. * 2014 IGCS and ESGO
Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
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& Volume 24, Number 2, February 2014
Data were collected from all women including age, parity, menstrual cycle regularity, and type of BRCA mutation. Frozen serum samples were tested for AMH using a 2-site ELISA (Diagnostic System Laboratory, TX). Assay sensitivity was 0.017 ng/mL; inter-assay and intra-assay coefficients of variation were 8% and 4.6%, respectively, for serum AMH concentration of 0.15 ng/mL and 6.7%, and 3.3% for a serum AMH concentration of 4.34 ng/mL. The AMH levels in this study group were compared with that in the group of 324 healthy controls (women with normal ovulatory cycles). These women had been previously evaluated in our laboratory. Levels were compared by t test. Results are presented as mean AMH levels with SE. We also compared the 2 groups by subdividing them by age groups. In addition, we compared the AMH levels of the study group to those in the 2 well-established AMH normograms16,17; the latter normogram is based on a multicenter study population of 3871 women, including the 324 women from our own center. The former normogram (Nelson model) was built on the basis of infertile women; however, it was validated on a cohort of 423 women with confirmed ovulation and normal pelvic ultrasound findings.16
RESULTS We describe 41 BRCA1/2 mutation carriers, younger than 40 years, attending the breast and ovarian cancer surveillance clinic during the year 2010 to 2011. Their mean age at the time of analysis was 33.2 years (26Y39 years; SD, 3.99 years). The mean parity was 1.97 (0Y7; SD, 1.49). All women were carriers of at least 1 of the 3 Ashkenazi Jewish founder mutations (185 delAG and 5382 insC in BRCA1, and 6174 delT in BRCA2). Twenty-five women were BRCA1 mutation carriers, 12 were BRCA2 mutation carriers, and 3 women were carriers of a mutation in both genes. When inquired about infertility at history taking, none of the 41 women reported such a history. Demographic and genetic parameters are presented in Table 1. The AMH levels for most of the mutation carriers were in the reference range (2.71 T 0.59 ng/mL). These levels were similar to those in the control group, in which the AMH levels were 2.02 T 0.12 ng/mL (P = 0.27). There were also no differences in AMH levels when comparing the women in the study and the control groups by subdividing them according to age groups. A 33-year-old woman in the study group had an AMH level of 23.8 ng/mL, associated with PCO syndrome. This value was included in the analysis despite its extreme high level because the control group also included some women with PCO syndrome. Serum AMH levels in both
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study and control groups are presented in Figure 1, presented according to various age groups. Comparing the levels to the Nelson normogram,16 the results were close to the 50 percentile (Fig. 2). Although AMH levels were slightly higher for carriers of mutations in BRCA1 versus BRCA2 (mean, 2.48 vs 1.70 ng/mL, respectively), there was no statistically significant difference.
DISCUSSION The relation between BRCA mutations and infertility is poorly described in the literature, and there are conflicting results from different studies. To date, there is no recommendation for routine evaluation of fertility in female carriers of BRCA mutations. This is the first study, to the best of our knowledge, that evaluates ovarian function in a large group of BRCA mutation carriers with a wide range of ages using a wellvalidated marker of ovarian reserve/fertility potential, namely, the serum AMH.12,13 A recent study has addressed this issue in mice; 3 BRCA1-deficient mice showed lower response to ovarian stimulation than wild-type mice. They also performed a human study, and 15 women with BRCA1 mutations had lower serum AMH levels than controls. The group came to the conclusion that BRCA deficiency might deteriorate ovarian function. The differences between this study and ours may be due to the difference in the size of the studies and the ages of the women tested. The use of AMH was first used in infertility populations, in which it has great value in predicting outcome in the setting of IVF, particularly for women in their later reproductive years. In more recent studies, AMH levels were measured in young and fertile women, including women from the general population attending our center.17 The AMH values in this study group were similar to the control group, which is our own healthy reference population that had been previously tested for AMH. By comparing AMH levels in a cohort of BRCA mutation carriers with 2 well-established AMH normograms,16,17 we have shown that mutation carriers have AMH levels comparable to the general population. We conclude that there is no substantial difference in ovarian reserve between carriers and the general population. Whether carrying a BRCA mutation increases the risk for infertility has clinical implications. With the ongoing accumulation of molecular and clinical information from studies on women carrying BRCA mutations, many questions arise regarding the recommended management methods for healthy
TABLE 1. Demographic and genetic parameters of the 41 BRCA mutation carriers Age Group, y No. Patients No. Children, Median (Range) 26Y30 31Y34 35Y36 37Y39
13 10 10 8
1 2 2 3
(0Y3) (0Y4) (0Y3) (1Y7)
BRCA1 Mutation
BRCA2 Mutation
BRCA1 + 2 Mutations
9 6 6 5
3 3 3 3
1 1 1 0
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FIGURE 1. Serum AMH levels in 41 female carriers of BRCA1/2 mutations (black bars) compared with that in control women matched by age (white bars). All bar graphs show means (SE). carriers.18 Current recommendations for mutation carriers are to complete family planning by age 35 to 40 years and then to undergo salpingo-ooporectomy to reduce the risk of breast and ovarian cancer.19,20 If mutation carriers have reduced fertility, it may be appropriate to encourage family planning at an earlier age. In addition, the effect of BRCA mutations on fertility would be an issue to discuss with young women undergoing genetic testing for these mutations. Previous studies have addressed this issue indirectly by examining parity or self-reported history of infertility.5,7 However, these parameters depend on additional factors unrelated to the true fertility potential of women. Furthermore, some of the latter studies were subjected to recall bias, because the collection of data was based on questionnaires or phone interviews.6 Nevertheless, most studies could not show reduced fertility in BRCA mutation carriers.5,7 Our results contradict the conclusions of 2 studiesVthe study by Oktay et al,4 who found an increased risk of poor ovarian response in carriers of BRCA1 mutation undergoing fertility preservation as compared with that in noncarriers, and the study by Titus et al,15 in which 15 women with BRCA1 mutations had lower serum AMH levels than that in controls. However, in the former study, the study group in this research was small, 12 women with either BRCA1 or 2 mutations, with only 8 BRCA1 carriers of pathogenic mutations. Furthermore, the women included in the study had breast cancer (ie, they were not healthy carriers). The differences between these studies and ours may be due to the difference in the size of the studies and the ages of the women tested. Our study examined 41 BRCA mutation carriers at a larger range of ages (26Y39 years). The women found to have low ovarian response among the BRCA1 mutation carriers in both previous studies were older (in the former, older than 33 years, and in the latter, mean age was 34.8). Many of the BRCA mutation carriers go through risk reducing BSO at this age, and the impact of infertility at this age is less profound. In any case, our results show that AMH levels for healthy carriers even at this age group are similar to our general population; therefore, their fertility potential seems to be similar to that of noncarrier women of the same age.
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Some authors have noted early menopause in BRCA mutation carriers. Our study was on a group of women aged 26 to 39 years, and the ovarian reserve was similar to the general population (matched for age). Therefore, from the evaluation of ovarian reserve at these ages, one can extrapolate on the ovarian function throughout the years of fertility. However, it is possible that women with these mutations have normal ovarian function until a premature sudden decline in ovarian function. It is possible that if we had a larger number of women at the upper end of the age spectrum (ie, closer to 40 years), the results would have been different. It is also possible that mutations in BRCA genes may actually have a positive effect on fertility. Common mutations in other genes in certain populations have been shown to have a survival advantage (eg, lower malaria prevalence for carriers of mutations in the A hemoglobin in endemic countries). Mutations in BRCA genes may theoretically improve conditions for success of the embryonal implantation process. Greater fertility could provide a selective advantage for BRCA1 and BRCA2 mutations. To date, molecular mechanisms of this hypothetical effect are unclear. In this study, we found neither a positive nor a negative effect of BRCA1/2 mutations on ovarian function. This is only a preliminary study on a small group of BRCA1/2 mutation carriers. A long-term, large scale study, comparing actual outcomes of fertility, is needed to support our findings. This study did not address the issue of oral contraceptive use, age of women at conception, and response of patients to fertility treatment. These parameters would provide important information. In addition, another accurate marker of ovarian reserve, AFC, should be used to support our findings. This study did not include a large enough group of mutation carriers to perform a subgroup analysis
FIGURE 2. Serum AMH levels (ng/mL) in 41 female carriers of BRCA1/2 mutations as compared with the Nelson normogram.15 * 2014 IGCS and ESGO
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International Journal of Gynecological Cancer
& Volume 24, Number 2, February 2014
comparing AMH levels in women with mutations in BRCA1 versus BRCA2. Nonetheless, this is currently the only study, to the best of our knowledge, that directly examines ovarian reserve with 1 of the best predictors known today to evaluate ovarian reserve, serum AMH level,12,13 in a relatively large group of BRCA mutation carriers with a wide range of ages. The study addresses an issue of great interest to carriers of a common gene mutation and their caregivers. If the results of this pilot study are ascertained by future larger scale studies addressing all the issues mentioned previously, they may give reassurance to female carriers of BRCA mutations concerning their fertility potential. At this point, according to our preliminary results, a woman with identified as a mutation carrier may assume her fertility potential to be similar to the general population. This may not only alleviate anxiety but also affect decision making concerning family planning.
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