Background
Primary Ovarian Insufficiency (POI) is an important human disease model that provided substantial understanding into the factors involved in differentiation and ovarian development. This rare disease is characterized by amenorrhea with elevated gonadotropin levels, and affects 1% of women before the age of 40 years [
1]. POI encompasses a heterogeneous spectrum of conditions, through two major mechanisms, follicle dysfunction and follicle depletion [
2]. Although, the majority of cases remain idiopathic, POI can be triggered by autoimmune disease, viral, iatrogenic or genetic causes [
1]. The disorder may be associated in syndromic diseases, such as Turner’s syndrome, X-Fragile syndrome or BPES syndrome [
3]. Genetic component such as X-chromosome abnormalities, deletions,
FMR1 premutations,
BMP15 variants, were identified as the first genetic causes of the pathophysiology [
4]. To date, about 30 autosomal genes were identified as involved in the pathogenesis of POI, and more recently a digenic form has been observed in large cohort of POI women [
5]. For instance, recent publications reported mutations in genes involved in meiosis like
STAG3 [
6],
HFM1 [
7] or
SYCE1 [
8] and in transcription factors including
FIGLA [
9] or
SOHLH2 [
10]. However, except the high prevalence of
NOBOX [
11] and
BMP15 mutations [
4] no gene has been shown implicated in more than 6% and 3%, respectively, of POI cases.
Most POI are idiopathic, thus it seems to be important to research additional factors. Potential candidates could be members of R-spondin (RSPO) family that are ligands activating Wnt pathway [
12]. Among them, R-spondin1 acts on ovarian differentiation and
RSPO1 mutations have been found in patients with sex differentiation disorders [
13]. It has recently been suggested that Rspo2, another member of this family, could be important in regulating ovarian follicle development [
14]. Rspo2 is expressed in mouse oocytes from primary follicles to growing follicles. An ex vivo and in vivo treatment of mouse ovary with Rspo2 promoted development of follicles. Moreover,
Rspo2 heterozygous deficient female mice become infertile around 4 months of age, then mimicking POI [
15‐
17].
So, the aim of this study was to analyze RSPO2 regulation by NOBOX, a master-transcription factor of ovarian folliculogenesis and to determine whether RSPO2 genetic variation is associated with idiopathic POI women.
Discussion
More than 80% of POI causes are unknown until now, it is of interest that multiple approaches are required to discover new responsible candidate genes. Mouse forward genetics is a powerful tool for the identification of genes and pathways important in biological process and has been very valuable for the characterization of key players in ovarian folliculogenesis [
23]. Recently transgenic mouse studies supported the role of
Rspo2 in follicle development. Human
RSPO2 gene is located on chromosome 8 and encodes a member of the R-Spondin family of secreted proteins involved in β-catenin activation through the canonical Wnt pathway leading to T-cell factor-dependent gene activation [
24‐
26].
Footless mutant mice with a transgene insertion in
the Rspo2 gene showed many malformations [
15], although
Footless heterozygous animals were indistinguishable from wild-type animals through weaning, female animals were only fertile until 4 months of age. Similarly, heterozygous
Rspo2 mutant female mice were 25% sterile at 4 months and 85% sterile at 5 months [
25]. This infertile phenotype associated with the oocyte
Rspo2 expression and follicular growth function led us to consider this gene as a possible candidate gene in POI. It is likely that the initial waves of follicle growth were maintained in
Rspo2 heterozygous mutants, but inappropriate levels of Rspo2 in oocytes eventually led to the failure of follicle development during late reproductive life. It was recently shown that the administration of Rspo2 agonist promotes human early follicle development after xenografted ovarian cortical pieces in immunodeficient mice [
14]. Loss of function mutations in
RSPO2 has not previously been associated with any phenotype in women.
Of interest, we described a number of mutations of the oocyte-specific homeobox gene
NOBOX associated with POI patients [
11,
27]. Interestingly, the ovarian
Rspo2 expression was reported very low in
Nobox-null mice [
22]. Otherwise, by an in silico analysis of
RSPO2 promoter the presence of NOBOX Binding Element (NBE) is revealed. Here, the transcriptional assays showed that NOBOX was able to activate the
RSPO2 expression. This suggests that
RSPO2 is a novel target gene of NOBOX, a master regulator of the folliculogenesis. It was, thus, of interest to look at
RSPO2 variants and to evaluate the role of RSPO2 in POI patients.
Among 100 independent idiopathic POI patients, we identified 9 Single Nucleotide Polymorphisms (SNPs) in RSPO2, four of them had never been described and one caused an amino-acid change but none of them seems to be deleterious when analyzed in silico.
The variations of
RSPO2 gene found in this cohort seem to be polymorphisms with current technics, and
RSPO2 mutations are therefore rare. However, RSPO2 appears to have an important role in ovary and it should be interesting to search
RSPO2 mutations in a larger cohort of POI patients. Moreover, RSPO2 is also a local intraovarian factor expressed at specific stages of follicular development. Taking into account, it would be rare to find a pathogenic variant or polymorphism in the peripheral blood of patients with POI. The disruption mediated through RSPO2 is most likely a local disruption which is temporally associated with folliculogenesis. Since it is challenging to study RSPO2 variations in ovarian tissue, we cannot exclude some somatic variants. Identically, the search of mutations in genes largely involved in different folliculogenesis steps such as
NANOS3, FOXO3a and PRLR [
28‐
30] has also been performed without any success. Moreover, many factors and regulators involved in folliculogenesis are intraovarian and work in a paracrine manner. The expression in oocytes and granulosa cells is more important than in somatic cells. Therefore, the paper demonstrating that R-spondin2 acts in folliculogenesis [
14] and the present study support and reinforce the importance of RSPO2 in this process.
Future high-throughput sequencing of such genes in women with idiopathic ovarian insufficiency should provide a better idea of the contribution that oocyte genes make to nonsyndromic POI.