POI is a primary ovarian defect characterized by primary or secondary amenorrhea before the age of 40 with hypergonadotropism and hypoestrogenism.
All the women of our study presented secondary amenorrhea before 40 years old. 2 patients presented primary amenorrhea. The mean age of menopause is 26.6 years. FSH value was over 25 mUI/ml in the whole group.
Characteristically, almost half (44%) of patients who underwent transvaginal pelvic ultrasonography had poor follicle assets with few millimetric follicles or small ovaries’ size.
Three patients (17%) had a family history of POI.
Screening for autoimmunity was performed, measuring anti-ovarian antibodies, anti-thyroid antibodies (anti-thyroperoxydase (TPO) and anti-thyroglobulin (TG) antibodies), anti-adrenal antibodies, anti-gastric parietal cells antibodies and antitissue transglutaminase (tTG) antibodies. 1 patient presented anti-ovarian autoantibodies; 7 patients had anti-TG antibodies and 3 of them presented anti-TPO autoantibodies too; 1 woman had anti-gastric parietal cells autoantibodies.
Autoimmunity etiology characterized about 5% of total POI cases. Prevalence of association between POI and many autoimmune diseases was estimated in 20–55% [20, 21]. POI is frequently associated with autoimmune thyroiditis (20%) and until 24% of women with POI present anti-TPO autoantibodies [8]. In our group, nine patients (50%) presented with a personal history of one or more autoimmune diseases: two patients presented Graves’ disease, seven patients had autoimmune thyroiditis. One patient had celiac disease and one had an history of diffused giant urticaria. Finally in our study, we found an association between POI and autoimmune thyroiditis of 38.8% and between POI and autoimmune diseases of 50%. In total 16.7% of patients presented anti-TPO autoantibodies.
In order to evaluate the effect of hypoestrogenism on bone, 15/18 women underwent femoral and lumbar spine osteodensitometry and 2 of them (13%) showed reduced bone density. In this study we realized genetic analysis of all the 18 patients in order to identify the karyotype, possible X chromosomal abnormalities, mutations/polymorphism of BMP15 and GDF-9 genes, and FMR1 premutation.
The analysis of the kariotype of the whole cohort of women did not show any abnormality (46XX).
There weren’t any abnormalities of the X chromosome such as X monosomy (Turner’s syndrome), X deletions, chromosome X translocations.
No genetic abnormalities of GDF-9, and FMR1 premutation were found in our group.
In one patient with secondary amenorrhea and diagnosis of POI at age of 18 years we found a pathogenetic mutation of BMP-15 gene, not previously described in literature (Fig. 1). The new variant involves second exon of BMP15 gene and causes heterozygous substitution c.406G > C, determining the V136L modification (leucine in the place of valine). The mutation is predicted to be possibly damaging by PolyPhen-2 software. The in vitro functional study showed a significant decrease of luciferase activity when COV434 cells were cotransfected with BMP15-V136L variant in the homozygous and in the heterozygous status, confirming the pathogenetic role of the mutation in development of POI.
As described previously, BMP15 is an oocyte-specific growth/differetiation factor and his role is critical to ovarian reserve determination. It promotes follicle development, granulosa cell mitosis, ovulation rate physiology and modulating granulosa cell sensitivity to FSH [22]. Prevalence of BMP15 gene mutations in POI is variable between 1.5% and 12% among studies [19, 22, 23]. Di Pasquale reported the first mutation of BMP15 gene in two sisters with hypergonadotropic ovarian failure characterized by primary amenorrhea and ovarian dysgenesis [24]. Subsequently, they have extended the genetic screening and identified new BMP15 nonsynonymous variants affecting 4.2% of 46XX idiopathic POI women [23]. In a previous study we reported a heterozygous missense substitution c.538G > A determining the A180T modification in the pro-region of the protein in one patient who developed secondary amenorrhea at the age of 31 years [9]. Mayer et al. described a family with two sisters with POI, harboring a compound heterozygous deletion of each allele of the BMP15 gene, transmitted in a recessive mode, as a “knockout-like” effect with complete lack of mature BMP15 and consequentely precocious follicle degeneration [25]. A number of other mutations and rare deletions in this gene have been described in women with POI [19, 22, 26], confirming his role as a major determinant of ovulation quota and dominant follicle selection in mammals. Several other genes have been reported to contribute to the genetic etiology of POI, including FMR1, FSHR, NOBOX, PGRMC1, GDF9, FOXO3, FIGLA, NR5A1, FOXL2, STAG3, SYCE1, HFM1, NUP107, MCM8, MCM9, MSH5, MSH4, KHDRBS1, EIF4ENIF1, PSMC3IP, CLPP and abnormal Y chromosome [3]. These genes are involved in various processes, including primordial germ cell development, DNA repair and meiosis, oocyte transcription and translational control during folliculogenesis, granulosa cell development and mitochondrial function [27]. The availability of the next-generation sequencing technology has resulted in a growing list of genes causing POI in the last years, revealing that it is a genetically complex disease [28, 29].