Endocrinology of Primary Ovarian Insufficiency: Diagnostic and Therapeutic Clues

Abstract

Background: This paper briefly reviews the most important endocrine features of primary ovarian insufficiency (POI) and shows their relevance for the diagnosis and treatment of this condition. Introduction: Endocrine disturbances in POI cause problems for both the fertility and general health status of the affected women. Both subfertility and infertility result from the depletion of growing ovarian follicles which, in its turn, is the causative factor of hypoestrogenism; this is responsible for most of the general health problems affecting women. Method: Search of literature. Results and conclusion: A combination of high-serum follicle-stimulating hormone (FSH) and low 17β-estradiol (E2) concentrations is a key feature characterizing POI and is the decisive element for POI diagnosis. However, an in-depth search for possible genetic and non-genetic causes is important for adequate counseling regarding prevention and early intervention. The treatment of general health problems, based on correcting hypoestrogenism through hormone replacement therapy (HRT), is relatively easy. On the other hand, resolving infertility is a much more difficult task, and oocyte donation is the only really efficient instrument. Fertility preservation is a suitable alternative in patients with early POI diagnosis, in whom some viable follicles are still present in the ovaries. In patients who refuse oocyte donation, intraovarian injection of autologous platelet-rich plasma and in vitro activation of dormant follicles may be considered. Other innovative treatments, such as stem cell therapies or nuclear transfer, are currently under investigation.

1. Introduction

Primary ovarian insufficiency (POI), sometimes also referred to as “primary ovarian failure”, is a condition characterized by the appearance of insufficient ovarian function before the age of 40 years [1,2]. Though not very frequent, POI is important to look for because of its multiple negative consequences, not only for the sake of fertility status but also for the patient’s health in general. The estimated global prevalence of POI is 3.7% [3], but this has been steadily increasing in recent years [4]. The negative consequences of POI for the general health of affected women have been extensively reviewed recently [1,2,5,6], and include an increase in the long-term risk of developing cardio-metabolic disease, type 2 diabetes, hyperlipidemia, coronary heart disease, stroke, centripetal obesity, chronic inflammation, hypertension, vasoconstriction, endothelial dysfunction, autonomic nervous system dysfunction, skeletal fragility, neurocognitive disorders, and total and cancer-related mortality. Improving the diagnostic and therapeutic methods to be employed in cases of POI is thus an important challenge for future research. The analysis of the endocrine profile of the affected patients, the search for possible genetic backgrounds and the development of new treatment strategies for POI-associated fertility problems are fundamental to achieving this goal.

2. Causes of POI

Causes of POI have also been reviewed recently [2,5,6,7,8,9]. They may be related to both genetic and non-genetic factors.

Although POI caused by genetic factors is mostly detected early in the third decade of life, it can also be found in adolescents and even in children [10]. The detection of POI of genetic origin at ages before menarche has been made possible by using next-generation sequencing (NGS) techniques including a POI-associated gene panel [11].

The genetic factors causing POI show marked heterogeneity and include chromosomal abnormalities, mainly those concerning the X chromosome, genetic polymorphism, and single-gene mutations (both in genes of the X chromosome and in non-X genes) [2,5,6,7,8,9].

Mutations of the newborn ovary homeobox (NOBOX), factor in germline alpha (FIGLA), genes located in forkhead box L2 (FOXL2), nuclear receptor subfamily 5 group A member 1 (NR5A1), bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF-9), transforming growth factor-β superfamily, the FSH and LH receptor, steroidogenic factor 1 (SF-1), cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1), and inhibin alpha subunit (INHA) genes were all demonstrated to cause infertility and loss of follicles [2,5,6,9,12]. Alterations of non-coding RNA (ncRNA) expression, leading to epigenetic dysregulation, were also found in women with POI [13,14], making them potential biomarkers of the disease and targets for therapy.

There is also evidence showing that POI (both isolated and syndromic) may result from specific variants of pleiotropic genes (genes that exhibit multiple phenotypic expression), underscoring the importance of individualized genetic counseling with regard to prediction, diagnosis, and intervention for women with POI or at high risk of developing this pathology [15,16]. Perrault syndrome, a rare autosomal recessive disorder characterized by ovarian dysgenesis in females and sensorineural hearing loss in both genders, sometimes also including ataxia, neuropathies, and intellectual disability, is an example of such a pleiotropic action [17]. To date, biallelic pathogenic variants in one of six different genes (CLPP, ERAL1, HARS2, HSD17B4, LARS2, or TWNK), most of which are involved in mitochondrial metabolism and renewal, have been linked to this disease [17]. In the absence of complete Perrault syndrome, mutations in nuclear genes causing the instability of mitochondrial DNA (mtDNA) [18] can also be associated with isolated POI [19,20]. Interestingly, blood cell mtDNA depletion is a frequent finding among women with POI, making the determination of mtDNA content in blood a useful tool for POI risk prediction [21].

Mutations in the genes involved in meiosis and DNA repair can also cause POI by interfering with different stages of meiotic recombination, including double-strand break (DSB) formation, DSB end processing, single-strand invasion, intermediate processing, recombination, synaptonemal complex and cohesion complex formation, inadequate chromosome segregation, and Fanconi anemia pathway. They can be evaluated to facilitate risk prediction, ovarian protection, and early intervention for POI women [8,22]. In fact, inadequate chromosome segregation during oocyte meiosis, resulting from the functional impairment of DNA repair, was reported in POI patients [23]. Cell cycle checkpoint controls (pachytene checkpoint and metaphase checkpoint) activate apoptosis in affected oocytes [24], which eventually leads to oocyte depletion and POI [25].

The non-genetic causes of POI can be related to autoimmune, iatrogenic, or metabolic factors. Autoimmune thyroiditis, adrenal insufficiency, polyglandular syndrome, and Addison’s disease are the most common immune factors associated with POI [2,5,6]. Chemotherapy and radiotherapy are the main iatrogenic causes of POI, while the most well-known metabolic causes of this disease are galactosemia, myotonic dystrophy, and hydroxylase deficiency [2,5,6]. Knowledge of possible non-genetic factors causing POI is essential, since, once POI is diagnosed, their involvement can be investigated, and the underlying pathology can be treated adequately.

3. Hormonal Regulation of the Ovarian Function

Most biologically relevant ovarian activity is exerted by different cells in the ovarian follicles. Ovarian follicles are dynamic structures that undergo important changes over time, related to their growth, cell differentiation, and hormonal dependence. At birth, only small primordial follicles are present. Later in life, primordial follicles grow and sequentially transform into growing pre-antral, early antral and preovulatory follicles [26]. Individual primordial follicles have different fates: some enter the growth phase, while others fall prey to atresia.

Two phases of follicular growth can be distinguished: a gonadotropin-independent one, and a gonadotropin-dependent one. The gonadotropin-independent phase is characterized by multiplication of granulosa cells, begins before puberty, and is driven by PI3K/AKT/mTOR activation and PTEN inhibition, while the activation of specific transcription factors, such as NOBOX, in the oocyte is essential for its growth [2]. After puberty, the growing blood levels of pituitary gonadotropins, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH), take over the main control of follicle development, namely the formation of follicular antrum, through increased follicular fluid production by granulosa cells, and initiation of meiotic maturation of the oocyte [26]. Pituitary synthesis of FSH and LH is stimulated by secretion of GnRH and inhibited by E2 and inhibin released from growing follicles [27]. Ovulation is triggered by preovulatory LH peak which, in turn, is provoked by a sudden switch from an inhibitory to a stimulatory action of E2 when persistently increasing E2 concentrations reach a critical point [27,28]. The LH peak is also responsible for resumption of meiosis and its progression to metaphase II in preovulatory oocytes [29].

4. Endocrine Disturbances in POI and Their Clinical Consequences

The depletion of growing follicles in the ovaries of women with POI leads to extremely low levels of circulating E2 and inhibin. This, in turn, causes abnormally high levels of pituitary gonadotropins, mainly FSH. POI-related hypoestrogenism is also the cause of most clinical symptoms concerning the patients’ general health status, such as hot flashes, dyspareunia, genitourinary syndrome of menopause (vulvovaginal atrophy, urinary incontinence), decreased sexual desire, dry eyes, night sweats, insomnia, as well as the risks for cardiovascular health, metabolic abnormalities, diabetes, obesity, chronic inflammation, hypertension, and autonomic nervous system dysfunction [1,2,5,6,30]. Most POI patients are subfertile or infertile, depending on the duration of the POI condition, and some of them show symptoms supposedly related to the genetic background of the disease, such as the Turner syndrome-like phenotype [5]. In fact, Turner syndrome (45,X) and other abnormalities of the X chromosome, such as mosaicisms, partial loss of critical terminal regions of the long arm of the X chromosome, and X-autosomal translocations, are well-known chromosomal abnormalities causing POI [2].

5. Diagnosis of POI

The diagnosis of POI is mainly based on the evaluation of endocrine parameters. Patients usually present with irregularities of the menstrual cycle and symptoms related to hypoestrogenism (see Section 4). The diagnosis of POI is dependent on evidence of hypergonadotropic hypogonadism in women aged younger than 40. In particular, the assessment of serum concentrations of FSH is the most important parameter. When a POI diagnosis is suspected, serum levels of FSH should be measured twice, at least one month apart, with persistently elevated FSH levels greater than 25 IU/L confirming the diagnosis of POI [31,32]. Together with low-serum antimullerian hormone (AMH) concentration [33] and low numbers of antral ovarian follicles detected by pelvic ultrasound scan, these hormonal assays can reliably identify patients with POI or those at risk of POI. However, according to a recently updated evidence-based guideline [34], AMH should not be used as the primary diagnostic test for POI, and should instead be reserved to confirm this condition where FSH results are inconclusive.

Based on the same source, the diagnostic workup recommended for all women with non-iatrogenic POI should include the following tests: chromosomal analysis, FMR1 premutation (Fragile X syndrome gene), screening for 21-hydroxylase autoantibodies (21OH-Abs), thyroid-stimulating hormone (TSH) measurement, and, where available and after comprehensive genetic counselling, additional genetic testing (e.g., NGS) to identify other potential genes involved [34].

Early diagnosis is particularly important in women with a familial history of POI in whom the disease risk is significantly higher as compared to general population; this risk is 18-fold, 4-fold, and 2.7-fold higher for first-degree, second-degree, and third-degree relatives, respectively [35].

6. Treatment of POI

6.1. General Health Problems

Since virtually all of the general health problems associated with POI are caused by hypoestrogenism, hormonal replacement therapy (HRT) with estrogenic preparation is the first choice of treatment to restore physiological estrogen levels, in line with the patient’s age [36]. Among the available formulations, preparations based on E2 (no less than 2 mg oral or 100 μg transdermal E2 or equivalent) are preferable, as compared with those based on ethinyl-estradiol or conjugated equine estrogens [34]. Transdermal administration is more suitable than oral treatment because it bypasses the hepatic first-pass effect, reducing liver exposure to supraphysiologic doses of estrogen and thus avoiding the resulting increase in pro-coagulant factors, SHBG, triglycerides, and markers of inflammation [37]. For long-term management of HRT, the use of “new estrogen” estetrol (E4), first discovered and identified in 1965 [38], a natural estrogenic compound with a low impact on the liver, mammary gland, and hemostasis balance, and recently approved for use in human subjects [39], might be preferable to E2 because of its favorable benefit-to-risk ratio, especially with regard to breast cancer risk.

Adding progestin is recommended to prevent endometrial hyperplasia and minimize irregular bleeding [40]. Micronized natural progesterone, taken orally or vaginally, is safer than traditional synthetic progestins regarding breast cancer risk, metabolic impact, and thromboembolic events [40]. Vaginal estrogen therapy should be offered to relieve genitourinary and sexual symptoms [34]. In addition to these specific treatments, patients should be advised to observe a well-balanced diet with adequate physical activity, to avoid smoking and to minimize alcohol consumption [41], with supplementary calcium and vitamin D treatment in cases with inadequate dietary intake [42]. In fact, the consumption of oily fish, mitochondria nutrient therapy, dairy and antioxidant vitamins should be recommended for at-risk women [43].

6.2. Infertility

The probability of a spontaneous pregnancy is very low in women with POI. It is estimated to be 5% [44]. The reason for which pregnancy still may occur is that POI is not always permanent, and some POI patients can ovulate from time to time [6]. However, even in those women who can ovulate (about 25%), only 5–10% can conceive [45]. This is likely due to impaired oocyte quality. Oocyte donation is the recommended treatment for infertility due to POI, as it has been proven to achieve a 70–80% successful pregnancy rate in patients suffering from this pathology [6]. Fertility preservation strategies, such as the cryopreservation of oocytes, embryos, and ovarian tissue, are suitable alternatives in cases in which some viable follicles are still present in the ovaries [46]. Hence, the possibility of preserving fertility strongly depends on early diagnosis.

It is important to note that women who undergo infertility treatments, both those who had not received HRT before these treatments and those who interrupted it in the context of these treatments, should resume HRT after the attempt to conceive [34]. If they do not, the general health problems associated with POI (see Section 4) will reappear.

6.3. New Therapies

Stem cell therapy is a new exciting advance in POI [47]. Stem cells (SCs) can be derived from embryos (ESCs) or adult tissues (ASCs). They are basically fully or partially undifferentiated but possess the unique ability to self-renew and differentiate [48]. Therapies with ESCs, obtained from supernumerary in vitro fertilization-derived embryos, adult mesenchymal SCs originating from the bone marrow, adipose tissue, menstrual blood, umbilical cord, amniotic fluid, amniotic membrane, placenta, and endometrium, and adult cells transformed into SCs with the use of transcription factors (induced SCs) [48], are currently being tested in animal models.

Intra-ovarian injection of platelet-rich plasma, prepared from autologous blood, has been shown to support the viability and growth of preantral follicles and to increase the number of retrieved oocytes, presumably through the release of different growth factors including platelet-derived growth factor, epidermal growth factor, insulin-like growth factor β-I, vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor [49]. Successful conception and live birth with the use of this technique and in vitro fertilization have been reported [50].

In vitro activation (IVA) of dormant ovarian follicles by incubating ovarian strips, prepared from ovaries removed by laparoscopic surgery, with PTEN inhibitors and PI3K activators for 2 days, followed by laparoscopic autologous transplantation beneath the serosa of Fallopian tubes [51,52], is another novel intervention for POI patients. After subsequent ovarian stimulation and intracytoplasmic sperm injection (ICSI), normal babies were born [52]. Moreover, ovarian disruption by itself (drug-free in vitro activation) was also shown to activate dormant follicles [53]. A more recent systematic review and meta-analysis, involving 164 patients with POI treated by chemical or drug-free IVA, reported resumption of follicle development in 43 patients (26.2%) for whom the pregnancy and live birth rates were 35.6% (11/43) and 20.9% (9/43), respectively [54].

Traditional Chinese medicine [55] and oral intake of melatonin [56] can be of help to slow ovarian aging, and represent simple and patient-friendly treatment options. In fact, melatonin was shown to reduce autophagy and enhance the viability of ovarian granulosa cells from POI patients via the activation of FOXO3A [57].

The administration of growth hormone during ovarian stimulation with gonadotropins is known to enhance both the quantity and the quality of oocytes recovered by ovarian puncture in poor-prognosis patients [58], but it has not yet been specifically evaluated in women with POI. Other innovative techniques, based on nuclear transfer from patients’ oocytes or somatic cells into enucleated donor oocytes [59], are under investigation but still not clinically available.

7. Conclusions

A specific pattern of endocrine disorders, characterized by high-serum FSH and low E2 concentrations, is a typical feature of POI and is decisive for POI diagnosis. Low-serum E2 is responsible for most of the negative impacts of POI on patients’ general health status, while the depletion of growing ovarian follicles is often the cause of infertility. Restoring normal E2 levels is the main therapeutic action to combat the general health problems of POI patients. Oocyte donation is an efficient method for the treatment of POI-associated infertility. Other treatments, aimed at achieving pregnancy with the patients’ own oocytes, can be considered, but are much less reliable. Innovative treatment alternatives are currently under investigation.