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Anti-Müllerian Hormone (AMH)
Anti-Mu¨llerian Hormone (AMH) Francesca Mossa, University of Sassari, Sassari, Italy James J Ireland, Michigan State University, East Lansing, MI, United States © 2018 Elsevier Inc. All rights reserved.
Introduction What is AMH and Its Source in Females AMH is a dimeric glycoprotein and a member of the transforming growth factor b (TGFb) superfamily of growth and differentiation factors. It is produced by Sertoli cells of the testes in the male and by granulosa cells of healthy, growing ovarian follicles in females (Visser et al., 2006).
AMH Mechanism of Action Like other TGFb family members, AMH signals through a receptor complex consisting of two type I and two type II receptors. AMH binding causes type II receptor mediated phosphorylation and activation of the type I receptor, which in turn activates the SMAD pathway. Receptor-activated SMADs then interact with SMAD4 and translocate to the nucleus to modify target gene expression. The type II AMH receptor (AMHRII) is specific for AMH, whereas there are three type I AMH receptors (ACVR1, BMPR1A, and BMPR1B) that are shared with bone morphogenetic proteins (BMPs), which are also members of the TGFb family. AMHRII mRNA is expressed in granulosa cells of small growing follicles. Type I AMH receptors mRNA is expressed in oocytes, granulosa, and theca cells at different stages of follicular development (reviewed in Visser et al., 2006).
AMH Functions Mammals, such as human beings, cattle, swine, and sheep are born with a finite number of morphologically healthy follicles and oocytes in the ovaries (the ovarian reserve) that dwindle rapidly during aging and are never replenished. Follicular recruitment starts soon after birth and is continuous throughout life. In female AMH null mice, follicles are recruited at a faster rate, resulting in an exhausted pool of primordial follicles at a younger age compared to wild-type controls. Also, AMH inhibits primordial follicle assembly and decreases the initial primordial follicular pool size in a rat ovarian organ culture. Similarly, AMH inhibited the initiation of follicle growth in cultured pieces of human ovaries (reviewed in Dewailly et al., 2014) and recently, AMH was shown to inhibit follicular activation and attenuate the growth of primary follicles in bovine fetal ovarian cortical pieces in vitro (Yang et al., 2017). These findings indicate that AMH acts as a brake on follicular development in humans, rodents and in cattle, inhibiting primordial follicular growth from the ovarian reserve and avoiding its premature exhaustion. AMH is also believed to reduce the responsiveness of preantral and small antral follicles to FSH, modulating follicular development. In cultured human granulosa cells, AMH inhibited FSH-induced aromatase mRNA and protein expression and estradiol production and AMH inhibited FSH effects on follicular growth in women and mice (reviewed in Dewailly et al., 2014).
Regulation of AMH Secretion AMH secretion changes as the ovarian follicle develops; AMH expression starts in primary follicles, reaches its highest level in preantral and small antral follicles, whereas it then decreases as the selected, FSH-dependent follicle progresses toward the preovulatory stage and is absent in atretic follicles. This pattern of expression was first assessed in rodents and subsequently in women, cattle and sheep. Nevertheless cumulus cells of preovulatory follicles continue to express AMH in women and mice (reviewed by Monniaux et al., 2012). AMH production is stimulated by the oocyte: evidence indicates that BMPs, such as BMP4, BMP6, and BMP15 originating from oocyte and theca cells, stimulate AMH mRNA expression by granulosa cells. On the contrary, FSH appears to have an inhibitory effect on AMH secretion, albeit still poorly understood. FSH decreased AMH mRNA expression in rat and cattle, but not in sheep (reviewed by Monniaux et al., 2012); however, FSH enhanced AMH gene expression and protein secretion in bovine granulosa cells in vitro at low concentrations, but inhibited them at higher concentrations (Scheetz et al., 2012). In summary, the regulation of AMH secretion by FSH is complex and the mechanism that regulates AMH secretion is unclear.
Alterations in Circulating AMH Concentrations From Birth to Puberty In healthy women, peripheral AMH concentrations change throughout life. A marked postnatal rise in AMH concentrations was reported in girls, with higher AMH at 3 months of age compared to those at birth, followed by a decrease at 12 months. This peak in early infancy was interpreted as an ovarian response to prevent FSH-induced follicle growth at a time when further differentiation of follicles would be inappropriate (Hagen et al., 2010).
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Fig. 1 Circulating AMH concentrations (ng/mL) in dairy (n ¼ 9) and cross bred beef (n ¼ 13) female calves. Results of ANOVA indicated that mean AMH concentrations were similar between dairy and beef calves (P ¼ 0.11), but varied with time (P < 0.01) (Mossa et al., 2017).
Several studies report that serum AMH levels rise steadily during childhood with greatest levels at around 15–18 years, but are stable during early adulthood (18–25 years). This pattern has been assessed in Caucasian and Chinese women, and may suggest that ethnicity does not have an impact on age-related AMH variations in women (Hagen et al., 2010). Nevertheless, the mechanisms responsible for the increase in AMH concentrations during childhood in girls remain to be elucidated. We conducted a study to illustrate the variations of AMH from birth to puberty in Holstein female calves (Mossa et al., 2017). Results depicted in Fig. 1 show that AMH concentrations increase during the first 2 months of age, decrease at 5 and are stable at 8– 9 months of age, around the time of first ovulation. We also described a similar pattern in beef calves (Mossa et al., 2017). In Maine-Anjou beef heifers, plasma AMH concentrations increased rapidly between 1 and 3 months of age, remained high at 6 months of age then declined slowly until 12 months of age, which corresponds to the age of ovulation for this breed (Monniaux et al., 2012). These findings are supported by results from others indicating that 3- to 4-month old calves have greater AMH levels compared to young adult heifers in both Holstein (14–16 months) and Bos indicus Nelore (18–24 months) cattle (reviewed in Mossa et al., 2017). In summary, AMH concentrations increase in the first months of life and decrease before puberty in cattle, like women, but the timing of such fluctuations may vary among breeds and genetic groups. Prepubertal heifers experience waves of antral follicular growth like adult cattle and number of follicles increases from 2 to 14 weeks of age (Evans et al., 1994). Thus, it is plausible that the variations in AMH concentrations observed before puberty are reflective of changes in growth patterns of small antral follicles. Another possible explanation is that prepubertal variations in circulating AMH concentrations are due to changes in the ability of granulosa cells to secrete AMH.
Alterations in Circulating AMH Concentrations during Estrous and Menstrual Cycles In women, AMH peripheral concentrations remain relatively constant during the menstrual cycle (La Marca et al., 2009). For example, in a study conducted on young (aged 19–35 years) normo-ovulatory women, the fluctuations of serum AMH levels were not significant either when aligned to day of menstrual cycle or to day of ovulation. Also, another study reported that AMH concentrations were not different within individual young women (aged 24–40) across a number of up to four consecutive menstrual cycles, ranging between 5.7 and 6.0 ng/mL (van Disseldorp et al., 2010). The limited variation in AMH peripheral concentration during the menstrual cycle indicates that AMH serum levels are not affected by dominant follicle growth during the late follicular phase of the normal menstrual cycle despite the finding that AMH expression declines in large follicles. These findings taken together imply that a single measurement of AMH is predictive of circulating AMH concentration during the menstrual cycle. The static nature of AMH concentrations enhances its clinical usefulness for example to assess response to ovarian stimulation protocols during ART compared with other currently available clinical markers of reproductive function, such as inhibin B, estradiol (E2), and FSH, which are all menstrual cycle dependent. Evidence is also accumulating that AMH concentration varies minimally during estrous cycles in cattle. For example, we reported that a single AMH measurement in young adult beef heifers was highly correlated (r ¼ 0.97) with the average for multiple AMH measurements during different days of the same or multiple estrous cycles (Ireland et al., 2011). In Holstein cows, AMH varied
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minimally during the same estrous cycle and on different days of two estrous cycles (reviewed in Mossa et al., 2017). Taken together these findings illustrate the static nature of AMH during the estrous cycle and its repeatability across multiple estrous cycles in cattle. Conversely, in mice serum AMH levels were increased at estrus and gradually declined to basal levels during the following days of the cycle (Visser et al., 2006). Such differences between mice compared with women and cattle may be explained by species differences in the dynamics of follicular growth and associated AMH secretion during reproductive cycles.
Correlation of Circulating AMH Concentrations With Antral Follicle Count, Size of the Ovarian Reserve (All Healthy Follicles), and Aging In women, antral follicle count (AFC) is defined as the number of follicles measuring 2–10 mm in diameter and is assessed by transvaginal ultrasound. AFC is proportionally related to the size of the ovarian reserve (La Marca et al., 2009). Similar to AFC, circulating AMH is a marker of the size of the ovarian follicular reserve in humans and in mice. Several studies report that AFC and serum AMH are strongly (r ¼ 0.74) to moderately (r ¼ 0.57–0.47) positively associated. For example, when AFC and AMH were repeatedly measured during a menstrual cycle, the moderate fluctuations in serum AMH concentration paralleled the variation in number of follicles 2–8 mm in diameter (Depmann et al., 2016b). This positive correlation between AFC and AMH can be explained by the AMH production by the small antral follicles that are also detected by ultrasonography. Both AFC and AMH are often used independently or jointly to estimate the size of the ovarian reserve and to predict the ovarian response to hormonal stimulation during ART (La Marca et al., 2009). A high positive correlation was observed between the variation in AMH, AFC and histological determination of total number of morphologically healthy follicles and oocytes in ovaries of young adult cattle (Ireland et al., 2008). In beef heifers, circulating AMH concentrations were approximately six- and twofold greater in animals with high ( 25 follicles, 3 mm in diameter) or intermediate (16–24 follicles) compared with a low ( 15 follicles) AFC during follicular waves (Fig. 2, left panel). Also, the overall average AMH concentration during ovulatory follicular waves per animal was highly correlated (r ¼ 0.88; Fig. 2, right panel) with average peak AFC during the two or three waves of an estrous cycle (Ireland et al., 2008). In another study, AMH plasma concentrations were highly positively correlated with the numbers of 3–7 mm antral follicles detected by ovarian ultrasonography in primiparous dairy cattle (Rico et al., 2009). Also, a positive association was detected between the antral follicle population and circulating AMH concentrations in Murrah (Bubalus bubalus), Holstein (Bos taurus), and Gyr (Bos indicus) heifers (Mossa et al., 2017). It can be 0.50
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Fig. 2 Alterations in circulating AMH concentrations during ovulatory follicular waves in cattle. Serial ovarian ultrasonography was used to identify animals with a consistently low (15 follicles 3 mm in diameter per wave, n ¼ 4 animals), intermediate (16–24 follicles, n ¼ 8), or high (25 follicles, n ¼ 4 animals) peak AFC during ovarian follicular waves. Prostaglandin F2a then was used to synchronize estrus, and ovarian ultrasonography was used to reconfirm that animals were correctly identified and to determine day of ovulation. Blood samples were taken daily at 1100 h beginning on Day 6 of the estrous cycle and ending 1 day after ovulation. Serum samples obtained 6–8 days preceding ovulation, which corresponded with ovulatory follicular waves, and on the day of ovulation were analyzed for AMH concentrations. In the left graph, data for each animal were aligned relative to day of ovulation, and data are plotted based on results of linear regression analysis. Each point represents the mean SEM for four or eight animals. In the right graph, average AMH concentration per animal was plotted relative to the average peak number of antral follicles per wave during the estrous cycle for each animal; r ¼ correlation coefficient and n ¼ total number of animals (Ireland et al., 2008).
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concluded, therefore, that AMH and AFC are positively correlated in cattle and both AFC and AMH can be used interchangeably and reliably to estimate total number of morphologically healthy follicles and oocytes in the ovaries of an individual.
Diagnostic Use of AMH to Predict Fertility, Age at Menopause and Response to ART Predict Fertility In cattle, in our recent work, AMH concentrations were determined in young adult Holstein heifers at 11–12 months of age and a variety of fertility measurements made before and after calving in the same individuals. Results showed that conception rates to first artificial insemination (AI), services per conception and days open after calving until pregnant were similar among individuals in the different AMH quartiles before calving and during the first, second and third lactations. However, the quartile (Q) of cows with the lowest AMH concentrations (Q1) as heifers tended (P < 0.10) to be lower at each lactation and had the lowest overall average for total percentage pregnant compared with cows in Q2 or Q3 but not Q4 AMH quartiles (Jimenez-Krassel et al., 2015). These findings contrast somewhat with our other study that examined fertility in dairy cows (up to eight parities) with low, intermediate or high AFC and presumably corresponding differences in AMH concentrations. In this study, dairy cows with a low AFC had a lower conception rate to first AI, greater number of AI to conceive and higher calving interval compared with cows with an intermediate or high AFC (Mossa et al., 2012). Like our finding for dairy cows with low AMH as a heifer (Jimenez-Krassel et al., 2015) overall pregnancy rate was lower for the dairy cows with a low compared with a high AFC (Mossa et al., 2012). Another study shows that dairy cows with high AMH had greater pregnancy rates and lower incidence of pregnancy loss between days 30 and 65 of gestation. Lower fertility for cattle with a relatively low AFC is further supported by evidence of lower pregnancy rates in beef heifers with a low vs. a higher AFC and by a study showing that lactating cows with a low AFC had a longer interval from calving to conception and lower pregnancy rates than cows with a high AFC (reviewed in Mossa et al., 2017). The reasons for the differences between some fertility measurements in the studies that classified cattle based on AMH or AFC are unknown but likely caused by relatively small numbers of cattle in AMH quartiles and some AFC groups coupled with mixed ages of cows in AFC groups. In addition, cross-sectional studies using older cows may be biased by previous removal (culling) of individuals from the herd with a relatively low AFC or AMH. Nevertheless, combined results (Mossa et al., 2017) imply that fertility is suboptimal in cows with low AMH concentrations as heifers and in cows with low AMH or a low AFC compared with herd mates with higher AMH concentrations or a higher AFC.
Age at Menopause In women, the size of the ovarian reserve declines with age until there are too few follicles to sustain menstrual activity and menopause (final menstrual period) is reached. Menopause occurs at a mean age of 51 years when the ovarian reserve drops to approximately 1000 follicles, but the age at which natural menopause occurs varies considerably between 40 and 60 years (te Velde & Pearson, 2002). Similarly, the rate of decline of the ovarian reserve varies significantly among individuals and the high variability of age at menopause is believed to be caused by the high variation in the number of ovarian follicles and oocytes at birth. Since AMH is a marker of the ovarian reserve, it is considered a promising marker to predict age at natural menopause. So far, six studies showed that AMH concentration is predictive of age at natural menopause (hazard ratio, 5.6–9.2) (Depmann et al., 2016), but long-term studies on large numbers of women are lacking.
Response to ART Assisted reproductive technology (ART), which includes hormonal stimulation, AI, in vitro fertilization and surrogacy, is used in both women and cattle. Ovarian stimulation consists of the artificial increase of FSH concentration that leads to the simultaneous growth of several follicles. The oocytes can be recovered from the stimulated follicles and fertilized in vitro or they can be allowed to ovulate (superovulation) and fertilized in vivo. In women oocytes are usually collected before ovulation, whereas in cattle superovulation is commonly followed by AI and the nonsurgical recovery of the embryos. The ovarian response to hormonal stimulation varies greatly among individuals in both women and cattle; such responsiveness is negatively associated with aging and growing evidence indicates that it can be predicted by measuring serum AMH concentrations (La Marca et al., 2009). In women AMH correlates strongly with the number of oocytes retrieved following ovarian stimulation for IVF (La Marca et al., 2009), hence it now widely used to assess the size of the ovarian reserve before assisted conception. Women with high AMH are likely to respond excessively to exogenous gonadotrophins (Dewailly et al., 2014), leading to a serious condition named ovarian hyperstimulation syndrome (OHSS), thus their treatment strategy can be modified by using a less intense ovarian stimulation regime. Conversely women with a low AMH are likely to respond poorly to stimulation with consequently a low chance of pregnancy (Dewailly et al., 2014) and their expectations can be managed appropriately, reducing the financial and emotional burden on couples seeking ART treatment. Evidence is now growing to support the use of AMH as a predictor of the responsiveness to ovarian stimulatory treatment in agricultural species. In primiparous dairy cows, AMH concentrations before the superovulatory treatment were positively correlated with the number of follicles before treatment and with the numbers of large follicles and corpora lutea (CL) after treatment (Rico et al., 2009). In nonlactating Holstein cows, plasma AMH concentrations before gonadotropin treatments were highly correlated
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with the numbers of large follicles and oocytes recovered at ovum pick up (OPU), as well as with the number of large follicles at estrus and the number of embryos collected from multiple ovulation and embryo transfer protocols. Further, in Japanese Black beef cattle, AMH concentrations were positively correlated with the number of follicles, number of oocytes/embryos recovered, fertilized embryos and transferable embryos. In lactating Holstein cows, AMH concentrations for each individual cow were correlated with superovulation response (number of CL on the day of the flush), total oocytes collected and total transferable embryos. Also, when cows were classified into quartiles of circulating AMH, Q4 cows had a > 2-fold greater response to superovulation including embryo production compared with cows in other quartiles (Mossa et al., 2017). In addition, a positive correlation existed between AMH with OPU and in vitro embryo production in Holstein, beef (Korean Hanwoo) and Bos indicus (Zebu) cattle (Mossa et al., 2017).
Summary and Conclusion Evidence from studies conducted in women, cattle and rodents indicates that AMH circulating concentration (1) increases in the first months of life and then decreases until puberty; (2) is repeatable in the same or multiple estrous/menstrual cycles within individuals; (3) is positively correlated with AFC; (4) is a reliable indicator of the total number of healthy follicles and oocytes; (5) is positively associated with measures of fertility (women and cattle: response to ovarian stimulation; cattle: pregnancy rate, herd longevity); (6) is a promising candidate to predict age at menopause. Taken together, these findings support the potential utility of AMH measurement in human ART and in agricultural species as a predictor of fertility. However, uncertainty still exists concerning AMH mechanism of action and regulation of secretion. Cattle may be used as a valid animal model to further corroborate the association between AMH and fertility in mammals.
References Depmann, M., Broer, S. L., van der Schouw, Y. T., Tehrani, F. R., Eijkemans, M. J., Mol, B. W., & Broekmans, F. J. (2016a). Can we predict age at natural menopause using ovarian reserve tests or mother’s age at menopause? A systematic literature review. Menopause, 23(2), 224–232. Depmann, M., van Disseldorp, J., Broer, S. L., Eijkemans, M. J., Laven, J. S., Visser, J. A., de Rijke, Y. B., Mol, B. W., & Broekmans, F. J. (2016b). Fluctuations in anti-Müllerian hormone levels throughout the menstrual cycle parallel fluctuations in the antral follicle count: A cohort study. Acta Obstetricia et Gynecologica Scandinavica, 95(7), 820–828. Dewailly, D., Andersen, C. Y., Balen, A., Broekmans, F., Dilaver, N., Fanchin, R., Griesinger, G., Kelsey, T. W., La Marca, A., Lambalk, C., Mason, H., Nelson, S. M., Visser, J. A., Wallace, W. H., & Anderson, R. A. (2014). The physiology and clinical utility of anti-Mullerian hormone in women. Human Reproduction Update, 20(3), 370–385. Evans, A. C., Adams, G. P., & Rawlings, N. C. (1994). Follicular and hormonal development in prepubertal heifers from 2 to 36 weeks of age. Journal of Reproduction and Fertility, 102(2), 463–470. Hagen, C. P., Aksglaede, L., Sørensen, K., Main, K. M., Boas, M., Cleemann, L., Holm, K., Gravholt, C. H., Andersson, A. M., Pedersen, A. T., Petersen, J. H., Linneberg, A., Kjaergaard, S., & Juul, A. (2010). Serum levels of anti-Müllerian hormone as a marker of ovarian function in 926 healthy females from birth to adulthood and in 172 turner syndrome patients. The Journal of Clinical Endocrinology and Metabolism, 95(11), 5003–5010. Ireland, J. J., Smith, G. W., Scheetz, D., Jimenez-Krassel, F., Folger, J. K., Ireland, J. L. H., Mossa, F., Lonergan, P., & Evans, A. C. O. (2011). Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of anti-Mullerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle. Reproduction, Fertility and Development, 23, 1–14. Ireland, J. L., Scheetz, D., Jimenez-Krassel, F., Themmen, A. P., Ward, F., Lonergan, P., Smith, G. W., Perez, G. I., Evans, A. C., & Ireland, J. J. (2008). Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle. Biology of Reproduction, 79(6), 1219–1225. Jimenez-Krassel, F., Scheetz, D. M., Neuder, L. M., Ireland, J. L., Pursley, J. R., Smith, G. W., Tempelman, R. J., Ferris, T., Roudebush, W. E., Mossa, F., Lonergan, P., Evans, A. C., & Ireland, J. J. (2015). Concentration of anti-Müllerian hormone in dairy heifers is positively associated with productive herd life. Journal of Dairy Science, 98(5), 3036–3045. La Marca, A., Sighinolfi, G., Radi, D., Argento, C., Baraldi, E., Artenisio, A. C., Stabile, G., & Volpe, A. (2009). Anti-Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Human Reproduction Update, 16(2), 113–130. Monniaux, D., Drouilhet, L., Rico, C., Estienne, A., Jarrier, P., Touzé, J. L., Sapa, J., Phocas, F., Dupont, J., Dalbiès-Tran, R., & Fabre, S. (2012). Regulation of anti-Müllerian hormone production in domestic animals. Reproduction, Fertility, and Development, 25(1), 1–16. Mossa, F., Jimenez-Krassel, F., Scheetz, D., Weber-Nielsen, M., Evans, A. C. O., & Ireland, J. J. (2017). Anti-Müllerian hormone (AMH) and fertility management in agricultural species. Reproduction, 154(1), R1–R11. Mossa, F., Walsh, S. W., Butler, S. T., Berry, D. P., Carter, F., Lonergan, P., Smith, G. W., Ireland, J. J., & Evans, A. C. (2012). Low numbers of ovarian follicles 3 mm in diameter are associated with low fertility in dairy cows. Journal of Dairy Science, 95(5), 2355–2361. Rico, C., Fabre, S., Médigue, C., di Clemente, N., Clément, F., Bontoux, M., Touzé, J. L., Dupont, M., Briant, E., Rémy, B., Beckers, J. F., & Monniaux, D. (2009). Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow. Biology of Reproduction, 80(1), 50–59. Scheetz, D., Folger, J. K., Smith, G. W., & Ireland, J. J. (2012). Granulosa cells are refractory to FSH action in individuals with a low antral follicle count. Reproduction, Fertility, and Development, 24(2), 327–336. te Velde, E. R., & Pearson, P. L. (2002). The variability of female reproductive ageing. Human Reproduction Update, 8(2), 141–154. van Disseldorp, J., Lambalk, C. B., Kwee, J., Looman, C. W., Eijkemans, M. J., Fauser, B. C., & Broekmans, F. J. (2010). Comparison of inter- and intra-cycle variability of antiMullerian hormone and antral follicle counts. Human Reproduction, 25(1), 221–227. Visser, J. A., de Jong, F. H., Laven, J. S., & Themmen, A. P. (2006). Anti-Müllerian hormone: A new marker for ovarian function. Reproduction, 131(1), 1–9. Yang, M. Y., Cushman, R. A., & Fortune, J. E. (2017). Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles. Molecular Human Reproduction, 23(5), 282–291.
Introduction What is AMH and Its Source in Females AMH is a dimeric glycoprotein and a member of the transforming growth factor b (TGFb) superfamily of growth and differentiation factors. It is produced by Sertoli cells of the testes in the male and by granulosa cells of healthy, growing ovarian follicles in females (Visser et al., 2006).
AMH Mechanism of Action Like other TGFb family members, AMH signals through a receptor complex consisting of two type I and two type II receptors. AMH binding causes type II receptor mediated phosphorylation and activation of the type I receptor, which in turn activates the SMAD pathway. Receptor-activated SMADs then interact with SMAD4 and translocate to the nucleus to modify target gene expression. The type II AMH receptor (AMHRII) is specific for AMH, whereas there are three type I AMH receptors (ACVR1, BMPR1A, and BMPR1B) that are shared with bone morphogenetic proteins (BMPs), which are also members of the TGFb family. AMHRII mRNA is expressed in granulosa cells of small growing follicles. Type I AMH receptors mRNA is expressed in oocytes, granulosa, and theca cells at different stages of follicular development (reviewed in Visser et al., 2006).
AMH Functions Mammals, such as human beings, cattle, swine, and sheep are born with a finite number of morphologically healthy follicles and oocytes in the ovaries (the ovarian reserve) that dwindle rapidly during aging and are never replenished. Follicular recruitment starts soon after birth and is continuous throughout life. In female AMH null mice, follicles are recruited at a faster rate, resulting in an exhausted pool of primordial follicles at a younger age compared to wild-type controls. Also, AMH inhibits primordial follicle assembly and decreases the initial primordial follicular pool size in a rat ovarian organ culture. Similarly, AMH inhibited the initiation of follicle growth in cultured pieces of human ovaries (reviewed in Dewailly et al., 2014) and recently, AMH was shown to inhibit follicular activation and attenuate the growth of primary follicles in bovine fetal ovarian cortical pieces in vitro (Yang et al., 2017). These findings indicate that AMH acts as a brake on follicular development in humans, rodents and in cattle, inhibiting primordial follicular growth from the ovarian reserve and avoiding its premature exhaustion. AMH is also believed to reduce the responsiveness of preantral and small antral follicles to FSH, modulating follicular development. In cultured human granulosa cells, AMH inhibited FSH-induced aromatase mRNA and protein expression and estradiol production and AMH inhibited FSH effects on follicular growth in women and mice (reviewed in Dewailly et al., 2014).
Regulation of AMH Secretion AMH secretion changes as the ovarian follicle develops; AMH expression starts in primary follicles, reaches its highest level in preantral and small antral follicles, whereas it then decreases as the selected, FSH-dependent follicle progresses toward the preovulatory stage and is absent in atretic follicles. This pattern of expression was first assessed in rodents and subsequently in women, cattle and sheep. Nevertheless cumulus cells of preovulatory follicles continue to express AMH in women and mice (reviewed by Monniaux et al., 2012). AMH production is stimulated by the oocyte: evidence indicates that BMPs, such as BMP4, BMP6, and BMP15 originating from oocyte and theca cells, stimulate AMH mRNA expression by granulosa cells. On the contrary, FSH appears to have an inhibitory effect on AMH secretion, albeit still poorly understood. FSH decreased AMH mRNA expression in rat and cattle, but not in sheep (reviewed by Monniaux et al., 2012); however, FSH enhanced AMH gene expression and protein secretion in bovine granulosa cells in vitro at low concentrations, but inhibited them at higher concentrations (Scheetz et al., 2012). In summary, the regulation of AMH secretion by FSH is complex and the mechanism that regulates AMH secretion is unclear.
Alterations in Circulating AMH Concentrations From Birth to Puberty In healthy women, peripheral AMH concentrations change throughout life. A marked postnatal rise in AMH concentrations was reported in girls, with higher AMH at 3 months of age compared to those at birth, followed by a decrease at 12 months. This peak in early infancy was interpreted as an ovarian response to prevent FSH-induced follicle growth at a time when further differentiation of follicles would be inappropriate (Hagen et al., 2010).
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Fig. 1 Circulating AMH concentrations (ng/mL) in dairy (n ¼ 9) and cross bred beef (n ¼ 13) female calves. Results of ANOVA indicated that mean AMH concentrations were similar between dairy and beef calves (P ¼ 0.11), but varied with time (P < 0.01) (Mossa et al., 2017).
Several studies report that serum AMH levels rise steadily during childhood with greatest levels at around 15–18 years, but are stable during early adulthood (18–25 years). This pattern has been assessed in Caucasian and Chinese women, and may suggest that ethnicity does not have an impact on age-related AMH variations in women (Hagen et al., 2010). Nevertheless, the mechanisms responsible for the increase in AMH concentrations during childhood in girls remain to be elucidated. We conducted a study to illustrate the variations of AMH from birth to puberty in Holstein female calves (Mossa et al., 2017). Results depicted in Fig. 1 show that AMH concentrations increase during the first 2 months of age, decrease at 5 and are stable at 8– 9 months of age, around the time of first ovulation. We also described a similar pattern in beef calves (Mossa et al., 2017). In Maine-Anjou beef heifers, plasma AMH concentrations increased rapidly between 1 and 3 months of age, remained high at 6 months of age then declined slowly until 12 months of age, which corresponds to the age of ovulation for this breed (Monniaux et al., 2012). These findings are supported by results from others indicating that 3- to 4-month old calves have greater AMH levels compared to young adult heifers in both Holstein (14–16 months) and Bos indicus Nelore (18–24 months) cattle (reviewed in Mossa et al., 2017). In summary, AMH concentrations increase in the first months of life and decrease before puberty in cattle, like women, but the timing of such fluctuations may vary among breeds and genetic groups. Prepubertal heifers experience waves of antral follicular growth like adult cattle and number of follicles increases from 2 to 14 weeks of age (Evans et al., 1994). Thus, it is plausible that the variations in AMH concentrations observed before puberty are reflective of changes in growth patterns of small antral follicles. Another possible explanation is that prepubertal variations in circulating AMH concentrations are due to changes in the ability of granulosa cells to secrete AMH.
Alterations in Circulating AMH Concentrations during Estrous and Menstrual Cycles In women, AMH peripheral concentrations remain relatively constant during the menstrual cycle (La Marca et al., 2009). For example, in a study conducted on young (aged 19–35 years) normo-ovulatory women, the fluctuations of serum AMH levels were not significant either when aligned to day of menstrual cycle or to day of ovulation. Also, another study reported that AMH concentrations were not different within individual young women (aged 24–40) across a number of up to four consecutive menstrual cycles, ranging between 5.7 and 6.0 ng/mL (van Disseldorp et al., 2010). The limited variation in AMH peripheral concentration during the menstrual cycle indicates that AMH serum levels are not affected by dominant follicle growth during the late follicular phase of the normal menstrual cycle despite the finding that AMH expression declines in large follicles. These findings taken together imply that a single measurement of AMH is predictive of circulating AMH concentration during the menstrual cycle. The static nature of AMH concentrations enhances its clinical usefulness for example to assess response to ovarian stimulation protocols during ART compared with other currently available clinical markers of reproductive function, such as inhibin B, estradiol (E2), and FSH, which are all menstrual cycle dependent. Evidence is also accumulating that AMH concentration varies minimally during estrous cycles in cattle. For example, we reported that a single AMH measurement in young adult beef heifers was highly correlated (r ¼ 0.97) with the average for multiple AMH measurements during different days of the same or multiple estrous cycles (Ireland et al., 2011). In Holstein cows, AMH varied
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minimally during the same estrous cycle and on different days of two estrous cycles (reviewed in Mossa et al., 2017). Taken together these findings illustrate the static nature of AMH during the estrous cycle and its repeatability across multiple estrous cycles in cattle. Conversely, in mice serum AMH levels were increased at estrus and gradually declined to basal levels during the following days of the cycle (Visser et al., 2006). Such differences between mice compared with women and cattle may be explained by species differences in the dynamics of follicular growth and associated AMH secretion during reproductive cycles.
Correlation of Circulating AMH Concentrations With Antral Follicle Count, Size of the Ovarian Reserve (All Healthy Follicles), and Aging In women, antral follicle count (AFC) is defined as the number of follicles measuring 2–10 mm in diameter and is assessed by transvaginal ultrasound. AFC is proportionally related to the size of the ovarian reserve (La Marca et al., 2009). Similar to AFC, circulating AMH is a marker of the size of the ovarian follicular reserve in humans and in mice. Several studies report that AFC and serum AMH are strongly (r ¼ 0.74) to moderately (r ¼ 0.57–0.47) positively associated. For example, when AFC and AMH were repeatedly measured during a menstrual cycle, the moderate fluctuations in serum AMH concentration paralleled the variation in number of follicles 2–8 mm in diameter (Depmann et al., 2016b). This positive correlation between AFC and AMH can be explained by the AMH production by the small antral follicles that are also detected by ultrasonography. Both AFC and AMH are often used independently or jointly to estimate the size of the ovarian reserve and to predict the ovarian response to hormonal stimulation during ART (La Marca et al., 2009). A high positive correlation was observed between the variation in AMH, AFC and histological determination of total number of morphologically healthy follicles and oocytes in ovaries of young adult cattle (Ireland et al., 2008). In beef heifers, circulating AMH concentrations were approximately six- and twofold greater in animals with high ( 25 follicles, 3 mm in diameter) or intermediate (16–24 follicles) compared with a low ( 15 follicles) AFC during follicular waves (Fig. 2, left panel). Also, the overall average AMH concentration during ovulatory follicular waves per animal was highly correlated (r ¼ 0.88; Fig. 2, right panel) with average peak AFC during the two or three waves of an estrous cycle (Ireland et al., 2008). In another study, AMH plasma concentrations were highly positively correlated with the numbers of 3–7 mm antral follicles detected by ovarian ultrasonography in primiparous dairy cattle (Rico et al., 2009). Also, a positive association was detected between the antral follicle population and circulating AMH concentrations in Murrah (Bubalus bubalus), Holstein (Bos taurus), and Gyr (Bos indicus) heifers (Mossa et al., 2017). It can be 0.50
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Fig. 2 Alterations in circulating AMH concentrations during ovulatory follicular waves in cattle. Serial ovarian ultrasonography was used to identify animals with a consistently low (15 follicles 3 mm in diameter per wave, n ¼ 4 animals), intermediate (16–24 follicles, n ¼ 8), or high (25 follicles, n ¼ 4 animals) peak AFC during ovarian follicular waves. Prostaglandin F2a then was used to synchronize estrus, and ovarian ultrasonography was used to reconfirm that animals were correctly identified and to determine day of ovulation. Blood samples were taken daily at 1100 h beginning on Day 6 of the estrous cycle and ending 1 day after ovulation. Serum samples obtained 6–8 days preceding ovulation, which corresponded with ovulatory follicular waves, and on the day of ovulation were analyzed for AMH concentrations. In the left graph, data for each animal were aligned relative to day of ovulation, and data are plotted based on results of linear regression analysis. Each point represents the mean SEM for four or eight animals. In the right graph, average AMH concentration per animal was plotted relative to the average peak number of antral follicles per wave during the estrous cycle for each animal; r ¼ correlation coefficient and n ¼ total number of animals (Ireland et al., 2008).
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concluded, therefore, that AMH and AFC are positively correlated in cattle and both AFC and AMH can be used interchangeably and reliably to estimate total number of morphologically healthy follicles and oocytes in the ovaries of an individual.
Diagnostic Use of AMH to Predict Fertility, Age at Menopause and Response to ART Predict Fertility In cattle, in our recent work, AMH concentrations were determined in young adult Holstein heifers at 11–12 months of age and a variety of fertility measurements made before and after calving in the same individuals. Results showed that conception rates to first artificial insemination (AI), services per conception and days open after calving until pregnant were similar among individuals in the different AMH quartiles before calving and during the first, second and third lactations. However, the quartile (Q) of cows with the lowest AMH concentrations (Q1) as heifers tended (P < 0.10) to be lower at each lactation and had the lowest overall average for total percentage pregnant compared with cows in Q2 or Q3 but not Q4 AMH quartiles (Jimenez-Krassel et al., 2015). These findings contrast somewhat with our other study that examined fertility in dairy cows (up to eight parities) with low, intermediate or high AFC and presumably corresponding differences in AMH concentrations. In this study, dairy cows with a low AFC had a lower conception rate to first AI, greater number of AI to conceive and higher calving interval compared with cows with an intermediate or high AFC (Mossa et al., 2012). Like our finding for dairy cows with low AMH as a heifer (Jimenez-Krassel et al., 2015) overall pregnancy rate was lower for the dairy cows with a low compared with a high AFC (Mossa et al., 2012). Another study shows that dairy cows with high AMH had greater pregnancy rates and lower incidence of pregnancy loss between days 30 and 65 of gestation. Lower fertility for cattle with a relatively low AFC is further supported by evidence of lower pregnancy rates in beef heifers with a low vs. a higher AFC and by a study showing that lactating cows with a low AFC had a longer interval from calving to conception and lower pregnancy rates than cows with a high AFC (reviewed in Mossa et al., 2017). The reasons for the differences between some fertility measurements in the studies that classified cattle based on AMH or AFC are unknown but likely caused by relatively small numbers of cattle in AMH quartiles and some AFC groups coupled with mixed ages of cows in AFC groups. In addition, cross-sectional studies using older cows may be biased by previous removal (culling) of individuals from the herd with a relatively low AFC or AMH. Nevertheless, combined results (Mossa et al., 2017) imply that fertility is suboptimal in cows with low AMH concentrations as heifers and in cows with low AMH or a low AFC compared with herd mates with higher AMH concentrations or a higher AFC.
Age at Menopause In women, the size of the ovarian reserve declines with age until there are too few follicles to sustain menstrual activity and menopause (final menstrual period) is reached. Menopause occurs at a mean age of 51 years when the ovarian reserve drops to approximately 1000 follicles, but the age at which natural menopause occurs varies considerably between 40 and 60 years (te Velde & Pearson, 2002). Similarly, the rate of decline of the ovarian reserve varies significantly among individuals and the high variability of age at menopause is believed to be caused by the high variation in the number of ovarian follicles and oocytes at birth. Since AMH is a marker of the ovarian reserve, it is considered a promising marker to predict age at natural menopause. So far, six studies showed that AMH concentration is predictive of age at natural menopause (hazard ratio, 5.6–9.2) (Depmann et al., 2016), but long-term studies on large numbers of women are lacking.
Response to ART Assisted reproductive technology (ART), which includes hormonal stimulation, AI, in vitro fertilization and surrogacy, is used in both women and cattle. Ovarian stimulation consists of the artificial increase of FSH concentration that leads to the simultaneous growth of several follicles. The oocytes can be recovered from the stimulated follicles and fertilized in vitro or they can be allowed to ovulate (superovulation) and fertilized in vivo. In women oocytes are usually collected before ovulation, whereas in cattle superovulation is commonly followed by AI and the nonsurgical recovery of the embryos. The ovarian response to hormonal stimulation varies greatly among individuals in both women and cattle; such responsiveness is negatively associated with aging and growing evidence indicates that it can be predicted by measuring serum AMH concentrations (La Marca et al., 2009). In women AMH correlates strongly with the number of oocytes retrieved following ovarian stimulation for IVF (La Marca et al., 2009), hence it now widely used to assess the size of the ovarian reserve before assisted conception. Women with high AMH are likely to respond excessively to exogenous gonadotrophins (Dewailly et al., 2014), leading to a serious condition named ovarian hyperstimulation syndrome (OHSS), thus their treatment strategy can be modified by using a less intense ovarian stimulation regime. Conversely women with a low AMH are likely to respond poorly to stimulation with consequently a low chance of pregnancy (Dewailly et al., 2014) and their expectations can be managed appropriately, reducing the financial and emotional burden on couples seeking ART treatment. Evidence is now growing to support the use of AMH as a predictor of the responsiveness to ovarian stimulatory treatment in agricultural species. In primiparous dairy cows, AMH concentrations before the superovulatory treatment were positively correlated with the number of follicles before treatment and with the numbers of large follicles and corpora lutea (CL) after treatment (Rico et al., 2009). In nonlactating Holstein cows, plasma AMH concentrations before gonadotropin treatments were highly correlated
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with the numbers of large follicles and oocytes recovered at ovum pick up (OPU), as well as with the number of large follicles at estrus and the number of embryos collected from multiple ovulation and embryo transfer protocols. Further, in Japanese Black beef cattle, AMH concentrations were positively correlated with the number of follicles, number of oocytes/embryos recovered, fertilized embryos and transferable embryos. In lactating Holstein cows, AMH concentrations for each individual cow were correlated with superovulation response (number of CL on the day of the flush), total oocytes collected and total transferable embryos. Also, when cows were classified into quartiles of circulating AMH, Q4 cows had a > 2-fold greater response to superovulation including embryo production compared with cows in other quartiles (Mossa et al., 2017). In addition, a positive correlation existed between AMH with OPU and in vitro embryo production in Holstein, beef (Korean Hanwoo) and Bos indicus (Zebu) cattle (Mossa et al., 2017).
Summary and Conclusion Evidence from studies conducted in women, cattle and rodents indicates that AMH circulating concentration (1) increases in the first months of life and then decreases until puberty; (2) is repeatable in the same or multiple estrous/menstrual cycles within individuals; (3) is positively correlated with AFC; (4) is a reliable indicator of the total number of healthy follicles and oocytes; (5) is positively associated with measures of fertility (women and cattle: response to ovarian stimulation; cattle: pregnancy rate, herd longevity); (6) is a promising candidate to predict age at menopause. Taken together, these findings support the potential utility of AMH measurement in human ART and in agricultural species as a predictor of fertility. However, uncertainty still exists concerning AMH mechanism of action and regulation of secretion. Cattle may be used as a valid animal model to further corroborate the association between AMH and fertility in mammals.
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