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\DOI{10.5802/crbiol.147}
\datereceived{2023-06-22}
\daterevised{2023-11-06}
\dateaccepted{2023-11-17}
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\dateposted{2024-04-19}
\begin{document}

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\CDRsetmeta{articletype}{review}

\title{Polycystic ovary syndrome (PCOS): progress towards~a~better
understanding and treatment of~the~syndrome}

\alttitle{Le syndrome des ovaires polykystiques (SOPK) : avanc\'{e}es
vers une meilleure compr\'{e}hension et traitement du syndrome}
                                                    
\author{\firstname{Nour El Houda} \lastname{Mimouni}\CDRorcid{0000-0001-8471-8551}\IsCorresp}
\address{Univ. Lille, Inserm, CHU Lille, Laboratory of Development and
Plasticity of the Postnatal Brain, Lille Neuroscience \& Cognition,
UMR-S1172, FHU 1000 days for health, 59000 Lille, France}
\address{Mortimer B. Zuckerman Mind Brain and Behavior Institute,
Columbia University, New York, NY, 10027, USA}
\email[N. E. H. Mimouni]{dr.nourmimouni@gmail.com}

\author{\firstname{Paolo} \lastname{Giacobini}\CDRorcid{0000-0002-3075-1441}}
\addressSameAs{1}{Univ. Lille, Inserm, CHU Lille, Laboratory of Development and
Plasticity of the Postnatal Brain, Lille Neuroscience \& Cognition,
UMR-S1172, FHU 1000 days for health, 59000 Lille, France}
\email[P. Giacobini]{paolo.giacobini@inserm.fr} 

\def\thanksname{Note}
\thanks{Nour El Houda Mimouni received the Grandes Avanc\'{e}es en
Biologie 2022 award.}

\thanks{\textbf{Funding.} European Research Council (ERC) under the
European Union's Horizon 2020 research and innovation program
(ERC-2016-CoG, grant agreement no. 725149/REPRODAMH); Institut National
de la Sant\'e et de la Recherche M\'edicale (INSERM), France (grant number
U1172); Centre Hospitalier R\'egional Universitaire, CHU de Lille,
France; Fondation pour la Recherche M\'edicale, France (FRM); Centre
National de la Recherche Scientifique (CNRS), France; and Universit\'e
de Strasbourg, France; Bourse France \textit{L'Or\'eal-UNESCO Pour les
Femmes et la Science}.}

\begin{abstract} 
Polycystic ovary syndrome (PCOS) is the most common endocrine and
metabolic disorder in women of reproductive age. It has a strong
hereditary component estimated at 60 to 70\% in daughters. It has been
suggested that environmental factors during the fetal period may be
involved in the development of the syndrome in adulthood. However, the
underlying mechanisms of its transmission remain unknown, thus limiting
the development of effective therapeutic strategies.

This article highlights how an altered fetal environment (prenatal
exposure to high levels of anti-M\"{u}llerian hormone) can contribute
to the onset of PCOS in adulthood and lead to the transgenerational
transmission of neuroendocrine and metabolic traits through alterations
in the DNA methylation process. 

The originality of the translational findings summarized here involves
the identification of potential biomarkers for early diagnosis of the
syndrome, in addition to the validation of a promising therapeutic
avenue in a preclinical model of PCOS, which can improve the management
of patients suffering from the syndrome.
\end{abstract}


\begin{altabstract} 
Le syndrome des ovaires polykystiques (SOPK) est le trouble endocrinien
et m\'{e}tabolique le plus r\'{e}pandu chez les femmes en \^{a}ge de
procr\'{e}er, avec une forte composante h\'{e}r\'{e}ditaire estim\'{e}e
entre 60 et 70\%. Les facteurs environnementaux pendant la p\'{e}riode
f\oe{}tale pourraient \^{e}tre impliqu\'{e}s dans l'apparition du
syndrome \`{a} l'\^{a}ge adulte. N\'{e}anmoins, les m\'{e}canismes
sous-jacents \`{a} sa transmission demeurent inconnus, limitant ainsi
le d\'{e}veloppement de th\'{e}rapies efficaces.

Cet article met en lumi\`{e}re comment un environnement f\oe{}tal
alt\'{e}r\'{e} (exposition pr\'{e}natale \`{a} des taux \'{e}lev\'{e}s
d'hormone anti-m\"{u}llerienne) pourrait contribuer \`{a} la survenue
du SOPK chez la descendance ainsi qu'\`{a} la transmission
transg\'{e}n\'{e}rationnelle des caract\'{e}ristiques
neuroendocriniennes et m\'{e}taboliques du SOPK, par le biais d'une
alt\'{e}ration du processus de la m\'{e}thylation de l'ADN.

L'originalit\'{e} des travaux translationnels pr\'{e}sent\'{e}s ici
repose d'une part sur l'identification de potentiels biomarqueurs de
diagnostic pr\'{e}coce du syndrome. Et d'autre part, sur la validation
d'une piste th\'{e}rapeutique prometteuse dans un mod\`{e}le
pr\'{e}clinique de SOPK, offrant ainsi des perspectives
d'am\'{e}lioration de la prise en charge des patientes atteintes de ce
syndrome.
\end{altabstract} 

\keywords{\kwd{PCOS} \kwd{AMH} \kwd{Fetal programming} \kwd{Heritability} 
\kwd{Biomarkers} \kwd{Epigenetics} \kwd{Neuroendocrinology}}

\altkeywords{\kwd{SOPK} \kwd{AMH} \kwd{Programmation f\oe{}tale} 
\kwd{H\'{e}ritabilit\'{e}} \kwd{Biomarqueurs} 
\kwd{\'{E}pig\'{e}n\'{e}tique} \kwd{Neuroendocrinologie}}

\maketitle

\twocolumngrid

\end{noXML}


\section{Introduction}

Polycystic ovary syndrome (PCOS) is a complex, heritable,
reproductive-metabolic disorder affecting 10--20\% of reproductive age
women and is a leading cause of female
infertility~\cite{1,2,3}. Previously published International Guidelines
for the Assessment and Management of PCOS~\cite{3} recommend a clinical
diagnosis requiring at least two out of the following three Rotterdam
criteria: (i) the evidence of either biological or clinical
hyperandrogenism; (ii) irregular ovulatory function (oligomenorrhea or
amenorrhea); and (iii) polycystic ovarian morphology on ultrasound.

In addition to reproductive issues and neuroendocrine alteration of the
hypothalamic-pituitary-ovarian axis~\cite{4}, women with PCOS commonly
experience long-term metabolic health consequences such as diabetes
mellitus (both type 2 and gestational), hyperinsulinemia, glucose
intolerance, systemic inflammation and obesity~\cite{3,5,6,7,8}. Despite
its high prevalence and detrimental impact on women's health, the exact
etiology and underlying mechanisms associating the neuroendocrine and
metabolic traits of PCOS remain unclear, hampering the development of
effective therapeutics.


Recently, the diagnostic criteria for PCOS have incorporated the
measurement of Anti-M\"{u}llerian hormone (AMH) levels in adult women
as an alternative to ultrasound~\cite{9}. Elevated AMH levels,
typically two to three times higher in women with PCOS~\cite{10,11,12},
are associated with the severity of the condition and various clinical
features. AMH is a glycoprotein secreted by the granulosa cells of the
ovaries~\cite{13}. Its primary role is to negatively regulate the
transition from primordial follicles to primary follicles~\cite{14},
protecting growing follicles from premature maturation by counteracting
the effects of FSH~\cite{15}. This may contribute to other PCOS
symptoms such as hyperandrogenism and insulin resistance.

Interestingly, AMH receptor is expressed in hypothalamic
gonadotropin-releasing hormone (GnRH) neurons~\cite{16,17},
pituitary~\cite{18,19} and the placenta~\cite{20} suggesting that AMH
could exert extragonadal actions~\cite{21} on the placenta and the
brain (especially on the hypothalamus~\cite{16,17,18,19,20} and
pituitary~\cite{18}), which could be implicated in the central
dysregulations observed in patients with the syndrome. In addition, AMH
receptor is expressed by endothelial cells, tanycytes and the majority
of arcuate nucleus neurons. This suggests that peripheral AMH could act
either directly or indirectly to activate GnRH neurons in the
brain~\cite{17,22}.

Familial clustering and twin studies have shown that PCOS has a strong
heritable component~\cite{23,24,25}. However, the mutations that have been
identified~\cite{26} so far do not account for its high prevalence in
the population, implying that fetal environmental factors such as
androgens~\cite{27,28} and AMH~\cite{20,29,30,31} excess might play
important roles in the onset of PCOS.

Clinical and animal model studies implicate hyperandrogenic gestational
origins~\cite{32,33} ith an altered maternal endocrine-metabolic
environment that could promote epigenomic developmental programming of
PCOS inheritance, consolidating the ``developmental origins of health
and disease'' (DOHaD) theory~\cite{34}. Furthermore, increasing
evidence indicates that fetal environment influences gene expression
through transgenerational epigenetic modifications, leading to altered
gene expression which could ultimately increase disease
susceptibility~\cite{35}. Our team has developed a preclinical mouse
model called ``PAMH'' (prenatal exposure to elevated anti-M\"{u}llerian
hormone)~\cite{20,36}. This model successfully reproduces key features
of PCOS, including hyperandrogenism, ovarian dysfunction,
neuroendocrine abnormalities, and metabolic disturbances. This article
summarizes and puts into perspective the previously published 
study~\cite{refcellmetab}, which challenges the developmental origins of
PCOS and investigates the implication of AMH in the onset and
transgenerational epigenetic transmission of the condition across
subsequent generations.

\section{Results}

\subsection{Preclinical transgenerational model of PCOS}
How
does prenatal AMH exposure drive the transmission of reproductive and
metabolic PCOS-like alterations across multiple generations in
adulthood?

Throughout the history of modern medicine, from the rabies vaccine to
gene therapy, preclinical models have played a crucial role in helping
researchers to understand diseases and to develop effective preventive
and therapeutic strategies. In the case of PCOS, significant efforts
have been invested in generating animal models that accurately mirror
the human condition and facilitate the optimization of potential
treatments~\cite{33}.


We have previously shown that women with PCOS exhibit higher levels of
AMH during pregnancy~\cite{20,29,31}. To replicate these 
findings in a preclinical setting, we have established
a promising preclinical PCOS model in rodents called ``prenatal AMH
treated'', also named ``PAMH''~\cite{36} which effectively mimics the
human PCOS condition. This model involves exposing pregnant mice to
high AMH levels towards the end of the gestational period (Embryonic
day E16.5--E18.5) (Figure~\ref{fig1}A). Such treatment drives maternal
hyperandrogenism with subsequent changes in the hypothalamic pituitary
gonadal (HPG) axis and in hormone levels of both the dams and the
progeny~\cite{20}. The temporal window of AMH treatment was selected
because it falls beyond the developmental stages during which gonadal
and genital tract differentiation occurs in mice (E12.5--E14.5), thus
excluding any potential morphogenetic effects of exogenous
AMH~\cite{37}.

\begin{figure*}
{\vspace*{-3pt}}
\includegraphics{fig01}
{\vspace*{-3pt}}
\caption{\label{fig1}Polycystic ovary syndrome (PCOS): progress towards
a better understanding and treatment of the syndrome. (A) Schematic
representation of preclinical and clinical transgenerational
investigations of Polycystic Ovary Syndrome (PCOS). (B) Proposed
physiopathological mechanism of transgenerational PCOS inheritance and
epigenetic-based therapeutic approach. Created with 
\mbox{\href{https://www.BioRender.com}{BioRender.com}}.}
{\vspace*{-3pt}}
\end{figure*}


Considering the strong heritability of PCOS and the documented
inheritance of the key neuroendocrine and metabolic characteristics
observed in close relatives of women with PCOS~\cite{28,38,39}, we
sought to examine whether female offspring with PCOS-like traits (F1)
from pregnant dams exposed to elevated levels of AMH during pregnancy
(F0) are prone to transmitting PCOS-like traits to subsequent
generations, specifically F2 (intergenerational) and F3
(transgenerational) offspring (Figure~\ref{fig1}A).

Using a large panel of reproductive, endocrine, and metabolic
phenotyping techniques, our study demonstrated that prenatal exposure
to high levels of AMH during gestation in mice results in the
intergenerational and transgenerational transmission of PCOS-like
traits in female offspring of the F2 and F3 generations. Remarkably,
these offspring exhibited all the major diagnostic manifestations of
PCOS, including hyperandrogenism, oligo-anovulation, central
alterations, and fertility impairments. Additionally, the study found
significant metabolic alterations in the PAMH lineage, spanning from
the F1 to F3 generations, compared to the control groups. These
metabolic changes included an alteration in body composition (increase
in body weight and fat mass), as revealed by nuclear magnetic resonance
(NMR) analyses in the PAMH offspring. Notably, higher fasting glucose
levels support a hyperglycemic phenotype, and its association with high
insulin levels is indicative of insulin resistance. higher fasting
glucose and insulin levels indicative of a hyperglycemic phenotype.
Moreover, the study employed tissue-clearing techniques and whole-organ
3D imaging to analyze the distribution of insulin-producing ${\upbeta}$
cells and glucagon-producing ${\upalpha}$ cells in the pancreas,
revealing hypertrophic pancreatic islets in PAMH F1 female mice
compared to the controls.

The results of our study present convincing evidence supporting the
inheritance of neuroendocrine, reproductive, and metabolic dysfunctions
associated with PCOS across several generations in PAMH mice. To our
knowledge, PAMH is currently the sole rodent model that completely
satisfies the mouse equivalents of the Rotterdam criteria for
PCOS---encompassing metabolic abnormalities like
hyperglycemia, glucose intolerance, hyperinsulinemia, and type-2
diabetes---without the need for additional environmental
factors such as a high-fat diet. These findings solidify the
involvement of AMH in the pathogenesis of PCOS.

\subsection{Molecular pathways underlying PCOS transgenerational
transmission} 

Next, we investigated how these neuroendocrine, reproductive, and
metabolic dysfunctions of PCOS are passed down through generations
(Figure~\ref{fig1}A).

To unravel the molecular mechanisms and gene pathways involved in the
``fetal reprogramming'' of PCOS and its inheritance, we conducted RNA
sequencing (RNA-seq) analysis to identify differentially expressed
genes (DEGs) in ovaries obtained from control offspring (CNTR) and
third-generation females (PAMH F3). This analysis revealed 102 DEGs (54
downregulated and 48 upregulated) in PAMH F3 ovaries compared to
control ovaries.

Given that environmental factors have the potential to affect
epigenetic mechanisms, including DNA methylation, we sought to examine
the influence of ancestral prenatal AMH exposure during gestation on
the epigenome of PAMH F3 offspring, using a genome-wide methylated DNA
immunoprecipitation (MeDIP) approach. 

The profiling of transcriptomic and methylomic landscapes in ovaries of
both control and PCOS-like females from the third generation---which
represented the first generation of offspring not exposed to
AMH---revealed an overall decrease in methylation in PAMH F3 females.
Moreover, many affected genes in the ovarian tissue of PCOS-like
females are associated with inflammatory and metabolic \mbox{pathways} typical
of the human PCOS condition~\cite{32,39,40}.

Importantly, these alterations in gene expression were detected in
animals as early as 2 months of age, preceding the appearance of
metabolic manifestations observed at 6 months, which suggests that
molecular changes could serve as potential predictors of physiological
changes well before they occur. Our preclinical findings conclude that
the DNA methylation profile reveals several key molecules associated
with the PCOS phenotype that are epigenetically regulated through DNA
hypomethylation. This is consistent with clinical studies that have
reported DNA methylation changes associated with steroidogenesis,
inflammation, hormone-related processes, and glucose and lipid
metabolism in different tissues of women with PCOS~\cite{40,41}.

\subsection{Hope for early diagnosis for PCOS patients?} 
Identification of common epigenetic biomarkers in a cohort of women
with PCOS and their daughters, as well as in the preclinical model of
PCOS.

To investigate how our preclinical findings might relate to the human
PCOS condition, we searched by MeDIP-PCR for common epigenetic
signatures in blood samples collected from both mothers and daughters
diagnosed with PCOS in the Reproductive Medicine Department of Jeanne
de Flandre in Lille University Hospital, France.

The study reports that several of the differentially methylated genes
identified in ovarian tissues of PCOS-like mice of the third generation
were also altered in blood samples from women with PCOS and from
daughters of women with PCOS compared with healthy women.

These findings highlighted reveal common epigenetic signatures between
females in the preclinical model and patients with PCOS compared to
control groups (Figure~\ref{fig1}A). This signature is \mbox{characterized} by
hypomethylation of genes associated with: DNA demethylation
(\textit{TET1}), axon guidance (\textit{ROBO-1}), inhibition of cell
proliferation (\textit{CDKN1A}), inflammation (\textit{HDC}), and
insulin signaling (\textit{IGFBPL1}, \textit{IRS4}). Among these
potential biomarkers, three genes are also hypomethylated in daughters
diagnosed with PCOS (\textit{ROBO-1,} \textit{HDC}, and
\textit{IGFBPL1}).

Furthermore, epigenetic changes such as DNA methylation, known for
their stability and ability to precede phenotypic
manifestations~\cite{42}, hold promise as diagnostic indicators for
PCOS risk and prognostic indicators for disease progression. The
identification of common epigenetic alterations in both human and mouse
samples provide valuable insights into the shared biological pathways
involved in PCOS development and supports the translational potential
of the mouse models, which could pave the way for future explorations
of targeted therapeutic interventions for this complex disorder.

{\vspace*{-2pt}}

\subsection{Therapeutical avenue for PCOS management: preclinical
efficiency of an epigenetic-based therapy}

{\vspace*{-1pt}}

Since our methylation studies pointed to a preponderance of
hypomethylation in PCOS condition, we challenged the reversible nature
of epigenetic modifications and examined the therapeutic potential of
using the universal methyl group donor S-adenosylmethionine (SAM) in an
epigenetic preclinical investigation in our rodent model.

To further explore an epigenomic basis for this transgenerational
transmission of PCOS-like phenotypic expression and in an attempt to
re-methylate, PAMH F3 females were exposed to SAM treatment and their
endocrine and metabolic parameters were monitored. Our findings
demonstrated that SAM treatment restored normal ovulation and
testosterone concentrations, body weight, and body mass composition to
control conditions in PCOS-like animals. However, circulating AMH
levels after treatment were not measured. SAM treatment also normalized
pancreatic islet hypertrophy but did not significantly lower total
glucose levels of PAMH F3 animals. Our study further explored the
effect of SAM treatment on gene expression levels in mice with
PCOS-like symptoms and found that SAM treatment may have a positive
effect on reducing inflammation associated with PCOS-like symptoms in
mice.

{\vspace*{-1pt}}

\section{Conclusion} 

These findings strengthen the DOHaD concept that an adverse
foeto-maternal environment in PCOS has lasting effects on the health of
future generations. These findings, together with those of Risal
{{and colleagues}}~\cite{28} indicate communication
between ovaries, germ cells, and serum, and support the translational
relevance of findings in mice. Collectively, our study highlighted the
role of elevated levels of AMH during pregnancy and established a new
PCOS model through AMH treatment, demonstrating the inheritance of key
characteristics. Our findings revealed novel insights into the
transgenerational transmission of PCOS-related reproductive and
metabolic dysfunctions, providing compelling evidence of its
association with altered DNA methylation patterns (Figure~\ref{fig1}B). This
study identified potential promising biomarkers shared between mothers
and daughters that could enhance early diagnosis of the disease and its
progression. Finally, the experimental effectiveness of a novel
epigenetic therapy opens new perspectives for the treatment of PCOS.

{\vspace*{-1pt}}

\section*{Declaration of interests}

{\vspace*{-1pt}}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

{\vspace*{-1pt}}

\section*{Funding}

{\vspace*{-1pt}}

This work was supported by the European Research Council (ERC) under
the European Union's Horizon 2020 research and innovation program
({ERC-2016-CoG}  {to PG, grant agreement no. 725149/REPRODAMH);
Institut National de la Sant\'{e} et de la Recherche M\'{e}dicale
(INSERM), France (grant number} {U1172} {to PG and VP); Centre
Hospitalier R\'{e}gional Universitaire, CHU de Lille, France (Bonus H
to PG and PhD fellowship to NEHM); Fondation pour la Recherche
M\'{e}dicale, France (FRM, fellowship to IP); Centre National de la
Recherche Scientifique (CNRS), France; and Universit\'{e} de
Strasbourg, France (to A-LB).} Bourse France \textit{L'Or\'{e}al-UNESCO
Pour les Femmes et la Science} to NEHM.

{\vspace*{-1pt}}

\section*{Acknowledgements}

{\vspace*{-1pt}}

We would like to express our sincere gratitude to all the authors
involved in this work: Dr. Isabel Paiva, Dr. Anne-Laure Barbotin,
Fatima Ezzahra Timzoura, Damien Plassard, Stephanie Le Gras, Ga\"{e}tan
Ternier, Professor Pascal Pigny, Professor Catteau-Jonard, Dr. Virginie Simon,
Dr. Vincent Pr\'{e}vot, and Dr. Anne-Laurence Boutillier. Your
contribution and dedication have been essential to the completion of
this study. We also thank M.-H. Gevaert (histology core facility of
Lille), J. Devassine (animal core facility, BICeL core facility of
Lille, University of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur
de Lille, US 41-UMS 2014-PLBS, F-59000 Lille, France), as well as the
IGBMC core facility of Strasbourg for their technical assistance.

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