Periodic Reporting for period 3 - REPRODAMH (Extra-gonadal roles of Anti-Müllerian Hormone in the aetiology of polycystic ovary syndrome: the domino effect to reproductive neuroendocrine dysfunctions)
Periodo di rendicontazione: 2020-05-01 al 2021-10-31
Polycystic ovary syndrome (PCOS) is a lifelong reproductive and metabolic disorder that affects up to 17% of women of reproductive age. Many women with PCOS have excessive ovarian and adrenal androgen secretion, oligo-anovulation and severe health consequences, including reduced fertility and type 2 diabetes, that are further aggravated by obesity.
Despite intensive research, the cause of PCOS is unknown. Moreover, there is no cure so far for PCOS and its management remains suboptimal because it relies on the ad hoc empirical management of symptoms only. In order to understand PCOS pathophysiology, its developmental origins, and how to predict and prevent PCOS onset, there is urgent need for safe and effective markers and treatments.
Historically, PCOS has been considered mainly as a gonadal pathology, although several pre-clinical and clinical investigations suggest that heightened release of gonadotropin hormone releasing hormone (GnRH) is an important pathophysiological characteristic in many cases of PCOS. Nevertheless, the origin of GnRH hypersecretion in PCOS women is unknown, and whether this increased pulse frequency is sufficient to trigger the cascade of events that leads to PCOS is unclear.
In a previous work, we have shown that the ovarian Anti-Müllerian Hormone (AMH) induces LH secretion by stimulating the activation of and neuropeptide secretion in GnRH neurons, which express AMH receptors in both rodents and humans (Figures 1, 2; Cimino et al., 2016; Barbotin et al., 2019). Interestingly, previous studies have shown that plasma AMH levels in PCOS patients are 2-3-fold higher than levels in women with normal ovaries and that the severity of the PCOS phenotype is correlated with AMH production, which is higher in anovulatory than in ovulatory PCOS patients.
The overall objectives of this translational ERC project are: 1) to identify the origin of PCOS; 2) to determine whether AMH, the circulating levels of which are abnormally elevated in PCOS patients with severe oligo-anovulation, might contribute significantly to the hormonal and gonadal alterations that are observed in PCOS; 3) to address whether AMH can negatively impact on fertility by centrally affecting GnRH neuronal excitability prenatally and/or postnatally; 4) to study the hypothalamic changes that may occur in the brain of PCOS women; 5) to design and test new preclinical therapeutic strategies to treat PCOS.
Despite intensive research, the cause of PCOS is unknown. Moreover, there is no cure so far for PCOS and its management remains suboptimal because it relies on the ad hoc empirical management of symptoms only. In order to understand PCOS pathophysiology, its developmental origins, and how to predict and prevent PCOS onset, there is urgent need for safe and effective markers and treatments.
Historically, PCOS has been considered mainly as a gonadal pathology, although several pre-clinical and clinical investigations suggest that heightened release of gonadotropin hormone releasing hormone (GnRH) is an important pathophysiological characteristic in many cases of PCOS. Nevertheless, the origin of GnRH hypersecretion in PCOS women is unknown, and whether this increased pulse frequency is sufficient to trigger the cascade of events that leads to PCOS is unclear.
In a previous work, we have shown that the ovarian Anti-Müllerian Hormone (AMH) induces LH secretion by stimulating the activation of and neuropeptide secretion in GnRH neurons, which express AMH receptors in both rodents and humans (Figures 1, 2; Cimino et al., 2016; Barbotin et al., 2019). Interestingly, previous studies have shown that plasma AMH levels in PCOS patients are 2-3-fold higher than levels in women with normal ovaries and that the severity of the PCOS phenotype is correlated with AMH production, which is higher in anovulatory than in ovulatory PCOS patients.
The overall objectives of this translational ERC project are: 1) to identify the origin of PCOS; 2) to determine whether AMH, the circulating levels of which are abnormally elevated in PCOS patients with severe oligo-anovulation, might contribute significantly to the hormonal and gonadal alterations that are observed in PCOS; 3) to address whether AMH can negatively impact on fertility by centrally affecting GnRH neuronal excitability prenatally and/or postnatally; 4) to study the hypothalamic changes that may occur in the brain of PCOS women; 5) to design and test new preclinical therapeutic strategies to treat PCOS.
Summary of the work and major achievements:
AMH and neuroendocrine origins of PCOS
Familial clustering and twin studies have shown that PCOS has a strong heritable component. However, the polymorphisms identified in the AMH signalling pathway and in other genes do not explain the frequency of the disease, suggesting that environmental and epigenetic mechanisms may play far greater roles in the onset of PCOS.
In women with PCOS, endocrine alterations, such as hormonal imbalances, during gestation may contribute to an increased risk that their offspring will develop PCOS. Prenatal exposure to androgens, testosterone (T) or dihydrotestosterone, have been reported to generate the closest PCOS-like phenotype in a variety of animal models. This points to prenatal hyperandrogenism as one of the main triggers of the developmental programming of PCOS. Notably, our studies emerging during the first 30 months of this Project showed that pregnant women with PCOS have significantly higher circulating AMH levels, during the second trimester of gestation (Tata et al., 2018) as well as at term (Piltonen et al., 2019), as compared with pregnant women with normal fertility. This two-fold greater AMH levels detected in women with PCOS during pregnancy adds AMH to the list of potential candidates in the prenatal programming of PCOS. In support to this hypothesis, we have also shown in mice that excess prenatal AMH exposure leads to a PCOS-like phenotype in the female progeny, by inducing permanent hypothalamic and GnRH neuronal circuit changes in the brain, that are associated with impaired fertility in adulthood (Tata et al., 2018; Figure 3).
Another important finding emerging from this landmark study relates to the fact that the AMH-dependent prenatal hyperandrogenism leads to a persistent GnRH neuronal hyperactivity in the adult offspring. Importantly, we were able to reverse this effect in mice using a drug that “normalizes” the GnRH signalling pathway. After treatment with this drug, the mice stopped showing symptoms of polycystic ovary syndrome. Even though, these data need to be validated in humans, they offer a new therapeutic hope to treat PCOS and therefore they have been the object of a patent from our group that has been recently filed.
Hypothalamic structural and functional alterations in Polycystic Ovary Syndrome
We have previously shown (Cimino et al., 2016) that AMHR2 is widely expressed by neuronal hypothalamic populations and by non-neuronal specialized ependymo-glial cells, lining the wall of the third ventricle. These cells exhibited structural rearrangements during different phases of the ovulatory cycle which are critically important to regulate GnRH secretion (Prevot et al., Endocrine Review 2018). In female rats, under conditions involving low gonadotropin output, such as in diestrus, GnRH-secreting nerve terminals become completely surrounded by tanycytes. This prevents direct access to portal vessels. During the preovulatory surge on the day of proestrus tanycytes retract resulting in release of the engulfed axons and the establishment of a direct neurohaemal relationship between GnRH neuroendocrine neurons and pituitary portal blood. Thus, in adult women, before the preovulatory GnRH peak, retraction of tanycytes could allow the juxtaposition of the nerve endings of GnRH cells with the adjacent pericapillary space of the pituitary gland. These morphological changes occur in a few hours and are reversible. It has also been shown using metabolic resonance magnetic imagery (MRI) that women exhibit transient microstructural and metabolic changes in the hypothalamus across their ovarian cycle (Baroncini et al., 2010).
Based on these findings we hypothesised that AMH could also affect GnRH secretion acting on tanycytes, inducing tanycytic end-feet retraction and thus facilitating GnRH secretion, as well as positively acting on other GnRH excitatory afferents.
Using different PCOS animal models (rodents and sheep), we have first demonstrated (Barbotin et al., submitted; Chasles et al., in preparation) that AMH enhances GnRH/LH secretion, not only acting directly on GnRH neurons and increasing their neuronal activity, but also inducing the retraction of the tanycytic end-feet and activating some neuronal populations that send excitatory afferents to GnRH cells. Then using proton MR spectroscopy (MRS) as a non-invasive approach to compare neuronal hypothalamic and thalamus activity between PCOS women and control women, we have shown that women with PCOS present an increased hypothalamic activity as compared to normo-ovulatory women. Finally, we have been able to measure for the first time in humans the amount of GnRH in the general circulation using an ultra-sensitive method (High Pressure Liquid Chromatography–High Resolution Mass Spectrometry or Nano-HPLC-HRMS) and we have shown that women with PCOS have higher GnRH levels that control women. These novel findings show for the first time the existence of a GnRH deregulation and overt secretion in PCOS women.
Sheep model of PCOS
Animal models of PCOS have provided researchers with valuable insight into the pathogenesis of this syndrome, especially in large animals, such as sheep or rhesus monkeys. Prenatal androgenized (PNA) sheep have enabled multiple longitudinal studies aimed at careful characterization of PCOS-like traits at multiple developmental time points, identifying pre-PCOS-like traits and biomarkers of high translational interest to pediatricians. Because they are predominantly mono-ovulatory species, these species most strongly resemble humans, and they are amenable to repeated sampling to determine GnRH and LH levels. Since the beginning of the Project we have established a fairly large colony of PNA and control sheep in Nouzilly (INRA, Tours, France). These animals have been thoroughly characterized (hormonal measurements, behaviour, gonadal anatomy by Ultra-Sound) since their birth up to now.
PNA sheep will be used to complete Aim3 and Aim4 of the project. In particular, several preclinical therapeutic strategies will be tested in this animal model to determine which is the most efficient to rescue the neuroendocrine and reproductive phenotypes of these animals.
AMH and neuroendocrine origins of PCOS
Familial clustering and twin studies have shown that PCOS has a strong heritable component. However, the polymorphisms identified in the AMH signalling pathway and in other genes do not explain the frequency of the disease, suggesting that environmental and epigenetic mechanisms may play far greater roles in the onset of PCOS.
In women with PCOS, endocrine alterations, such as hormonal imbalances, during gestation may contribute to an increased risk that their offspring will develop PCOS. Prenatal exposure to androgens, testosterone (T) or dihydrotestosterone, have been reported to generate the closest PCOS-like phenotype in a variety of animal models. This points to prenatal hyperandrogenism as one of the main triggers of the developmental programming of PCOS. Notably, our studies emerging during the first 30 months of this Project showed that pregnant women with PCOS have significantly higher circulating AMH levels, during the second trimester of gestation (Tata et al., 2018) as well as at term (Piltonen et al., 2019), as compared with pregnant women with normal fertility. This two-fold greater AMH levels detected in women with PCOS during pregnancy adds AMH to the list of potential candidates in the prenatal programming of PCOS. In support to this hypothesis, we have also shown in mice that excess prenatal AMH exposure leads to a PCOS-like phenotype in the female progeny, by inducing permanent hypothalamic and GnRH neuronal circuit changes in the brain, that are associated with impaired fertility in adulthood (Tata et al., 2018; Figure 3).
Another important finding emerging from this landmark study relates to the fact that the AMH-dependent prenatal hyperandrogenism leads to a persistent GnRH neuronal hyperactivity in the adult offspring. Importantly, we were able to reverse this effect in mice using a drug that “normalizes” the GnRH signalling pathway. After treatment with this drug, the mice stopped showing symptoms of polycystic ovary syndrome. Even though, these data need to be validated in humans, they offer a new therapeutic hope to treat PCOS and therefore they have been the object of a patent from our group that has been recently filed.
Hypothalamic structural and functional alterations in Polycystic Ovary Syndrome
We have previously shown (Cimino et al., 2016) that AMHR2 is widely expressed by neuronal hypothalamic populations and by non-neuronal specialized ependymo-glial cells, lining the wall of the third ventricle. These cells exhibited structural rearrangements during different phases of the ovulatory cycle which are critically important to regulate GnRH secretion (Prevot et al., Endocrine Review 2018). In female rats, under conditions involving low gonadotropin output, such as in diestrus, GnRH-secreting nerve terminals become completely surrounded by tanycytes. This prevents direct access to portal vessels. During the preovulatory surge on the day of proestrus tanycytes retract resulting in release of the engulfed axons and the establishment of a direct neurohaemal relationship between GnRH neuroendocrine neurons and pituitary portal blood. Thus, in adult women, before the preovulatory GnRH peak, retraction of tanycytes could allow the juxtaposition of the nerve endings of GnRH cells with the adjacent pericapillary space of the pituitary gland. These morphological changes occur in a few hours and are reversible. It has also been shown using metabolic resonance magnetic imagery (MRI) that women exhibit transient microstructural and metabolic changes in the hypothalamus across their ovarian cycle (Baroncini et al., 2010).
Based on these findings we hypothesised that AMH could also affect GnRH secretion acting on tanycytes, inducing tanycytic end-feet retraction and thus facilitating GnRH secretion, as well as positively acting on other GnRH excitatory afferents.
Using different PCOS animal models (rodents and sheep), we have first demonstrated (Barbotin et al., submitted; Chasles et al., in preparation) that AMH enhances GnRH/LH secretion, not only acting directly on GnRH neurons and increasing their neuronal activity, but also inducing the retraction of the tanycytic end-feet and activating some neuronal populations that send excitatory afferents to GnRH cells. Then using proton MR spectroscopy (MRS) as a non-invasive approach to compare neuronal hypothalamic and thalamus activity between PCOS women and control women, we have shown that women with PCOS present an increased hypothalamic activity as compared to normo-ovulatory women. Finally, we have been able to measure for the first time in humans the amount of GnRH in the general circulation using an ultra-sensitive method (High Pressure Liquid Chromatography–High Resolution Mass Spectrometry or Nano-HPLC-HRMS) and we have shown that women with PCOS have higher GnRH levels that control women. These novel findings show for the first time the existence of a GnRH deregulation and overt secretion in PCOS women.
Sheep model of PCOS
Animal models of PCOS have provided researchers with valuable insight into the pathogenesis of this syndrome, especially in large animals, such as sheep or rhesus monkeys. Prenatal androgenized (PNA) sheep have enabled multiple longitudinal studies aimed at careful characterization of PCOS-like traits at multiple developmental time points, identifying pre-PCOS-like traits and biomarkers of high translational interest to pediatricians. Because they are predominantly mono-ovulatory species, these species most strongly resemble humans, and they are amenable to repeated sampling to determine GnRH and LH levels. Since the beginning of the Project we have established a fairly large colony of PNA and control sheep in Nouzilly (INRA, Tours, France). These animals have been thoroughly characterized (hormonal measurements, behaviour, gonadal anatomy by Ultra-Sound) since their birth up to now.
PNA sheep will be used to complete Aim3 and Aim4 of the project. In particular, several preclinical therapeutic strategies will be tested in this animal model to determine which is the most efficient to rescue the neuroendocrine and reproductive phenotypes of these animals.
1) Most women with PCOS are hyperandrogenic during pregnancy, yet the cause of this remains enigmatic. Our recent work (Tata et al., Nature Medicine 2018) highlighted that the origin of the gestational hyperandrogenism of women with PCOS could to be caused by elevated AMH levels during pregnancy and inhibition of aromatase expression/activity.
Since we could show for the first time that high AMH levels persisted (Tata et al., Nature Medicine 2018) and increased throughout pregnancy in women with PCOS (Piltonen et al., Fertility and Sterility, 2019) compared with weight-matched, non-PCOS controls, this led to the hypothesis that AMH levels were implicated in the prenatal foetal ovarian ‘programming’ that results in PCOS later in life. Our hypothesis is that high circulating maternal AMH levels reduce the activity of aromatase P450 in the placenta. Reduced aromatase activity combined with high maternal circulating testosterone levels allows increased placental passage and hyperexposes the foetus to androgens. This then has an epigenetic effect on the developing ovaries, predisposing them to intrauterine growth restriction, and the metabolic dysfunction, insulin resistance, and oligo-ovulatory menstrual cycles of PCOS. Current investigations in my team are aiming at addressing whether these PCOS-traits could be transferred over multiple generations through epigenetic modifications, thus uncovering novel aspects underlying PCOS transmission from mother to daughter. The aim of this study is to dissect the epigenetic mechanisms of the disease and to provide biomarkers for the early detection of PCOS in daughters born to PCOS mothers to allow in the future preventive interventions.
Another important progress made since the beginning of this Project is the finding that intermittent delivery of a GnRH antagonist to adult PCOS-like animals, corrected their neuroendocrine and reproductive alterations. Given the fact that GnRH antagonists are frequently used in the clinic, pharmacological antagonism aimed at tempering GnRH–LH secretion is an attractive therapeutic strategy to restore ovulation and fertility in individuals with PCOS characterized by high LH levels. Based on these findings, we have patented the use of GnRH antagonist as a new drug to treat PCOS infertility (N° de publication: WO2018177746; Inventors: Paolo Giacobini, Vincent Prevot; Applicant: Institut National de la Santé et de la Recherche Medicale (INSERM); Title of the invention: TREATMENT OF PREGNANT WOMEN AFFECTED WITH POLYCYSTIC OVARY SYNDROME).
In 2020 we will test whether the intermittent use of low doses of GnRH antagonists could rescue the neuroendocrine and metabolic disturbances that characterize the PNA sheep as a proof of concept for a first clinical trial in women with PCOS.
2) The possibility that structural plasticity could be altered in GnRH neurons and the multiple neuronal and glial networks that are involved in the control of GnRH secretion in PCOS has never before been investigated. This issue therefore represents a novel and relevant scientific question. To the best of our knowledge no previous imaging study of the hypothalamus in PCOS-patients has been conducted. Our ongoing investigation (Barbotin et al., submitted) will provide a better understanding of the hypothalamic structural plasticity and activity in PCOS women. For the first time, we employed MRI techniques using proton MR spectroscopy (MRS) as a non-invasive approach to compare neuronal hypothalamic and thalamus activity between PCOS women and controls women. As previously described (Florent et al., 2019), voxel-based spectroscopic analyses of freely moving cerebral metabolites were performed both in the hypothalamus and in the thalamus, and data were normalized to creatine, in a relative quantitative approach. Our results point to a stronger neuronal hypothalamic hyperactivity in PCOS women as compared to controls, which is followed by increased GnRH/secretion in the former group.
Our findings provide novel insights into the AMH-dependent regulation of GnRH release and propose that it occurs through multiple coordinated hypothalamic pathways and mechanisms. These translational physiological studies may pave the way for the development of new diagnostic and treatment strategies in PCOS.
In the next years, we will also investigate whether prenatal reprogramming of the hypothalamus that is mediated by androgen exposure promotes similar changes in mono-ovulatory (PNA sheep) species and we will correlate the observed changes with levels of GnRH and LH secretion by ELISA.
The results emerging from these studies will provide important insights into how brain deregulations could be key events in the origin of PCOS. We expect that the pharmacological strategies that we are developing for PCOS could ameliorate these deregulations and lead to restoration of normal reproductive functions.
Since we could show for the first time that high AMH levels persisted (Tata et al., Nature Medicine 2018) and increased throughout pregnancy in women with PCOS (Piltonen et al., Fertility and Sterility, 2019) compared with weight-matched, non-PCOS controls, this led to the hypothesis that AMH levels were implicated in the prenatal foetal ovarian ‘programming’ that results in PCOS later in life. Our hypothesis is that high circulating maternal AMH levels reduce the activity of aromatase P450 in the placenta. Reduced aromatase activity combined with high maternal circulating testosterone levels allows increased placental passage and hyperexposes the foetus to androgens. This then has an epigenetic effect on the developing ovaries, predisposing them to intrauterine growth restriction, and the metabolic dysfunction, insulin resistance, and oligo-ovulatory menstrual cycles of PCOS. Current investigations in my team are aiming at addressing whether these PCOS-traits could be transferred over multiple generations through epigenetic modifications, thus uncovering novel aspects underlying PCOS transmission from mother to daughter. The aim of this study is to dissect the epigenetic mechanisms of the disease and to provide biomarkers for the early detection of PCOS in daughters born to PCOS mothers to allow in the future preventive interventions.
Another important progress made since the beginning of this Project is the finding that intermittent delivery of a GnRH antagonist to adult PCOS-like animals, corrected their neuroendocrine and reproductive alterations. Given the fact that GnRH antagonists are frequently used in the clinic, pharmacological antagonism aimed at tempering GnRH–LH secretion is an attractive therapeutic strategy to restore ovulation and fertility in individuals with PCOS characterized by high LH levels. Based on these findings, we have patented the use of GnRH antagonist as a new drug to treat PCOS infertility (N° de publication: WO2018177746; Inventors: Paolo Giacobini, Vincent Prevot; Applicant: Institut National de la Santé et de la Recherche Medicale (INSERM); Title of the invention: TREATMENT OF PREGNANT WOMEN AFFECTED WITH POLYCYSTIC OVARY SYNDROME).
In 2020 we will test whether the intermittent use of low doses of GnRH antagonists could rescue the neuroendocrine and metabolic disturbances that characterize the PNA sheep as a proof of concept for a first clinical trial in women with PCOS.
2) The possibility that structural plasticity could be altered in GnRH neurons and the multiple neuronal and glial networks that are involved in the control of GnRH secretion in PCOS has never before been investigated. This issue therefore represents a novel and relevant scientific question. To the best of our knowledge no previous imaging study of the hypothalamus in PCOS-patients has been conducted. Our ongoing investigation (Barbotin et al., submitted) will provide a better understanding of the hypothalamic structural plasticity and activity in PCOS women. For the first time, we employed MRI techniques using proton MR spectroscopy (MRS) as a non-invasive approach to compare neuronal hypothalamic and thalamus activity between PCOS women and controls women. As previously described (Florent et al., 2019), voxel-based spectroscopic analyses of freely moving cerebral metabolites were performed both in the hypothalamus and in the thalamus, and data were normalized to creatine, in a relative quantitative approach. Our results point to a stronger neuronal hypothalamic hyperactivity in PCOS women as compared to controls, which is followed by increased GnRH/secretion in the former group.
Our findings provide novel insights into the AMH-dependent regulation of GnRH release and propose that it occurs through multiple coordinated hypothalamic pathways and mechanisms. These translational physiological studies may pave the way for the development of new diagnostic and treatment strategies in PCOS.
In the next years, we will also investigate whether prenatal reprogramming of the hypothalamus that is mediated by androgen exposure promotes similar changes in mono-ovulatory (PNA sheep) species and we will correlate the observed changes with levels of GnRH and LH secretion by ELISA.
The results emerging from these studies will provide important insights into how brain deregulations could be key events in the origin of PCOS. We expect that the pharmacological strategies that we are developing for PCOS could ameliorate these deregulations and lead to restoration of normal reproductive functions.