FOXL2 gene belongs to the winged-helix/forkhead transcription factor family, members of which are highly conservative. Pathogenic variants lead to either disordered subcellular distribution or dysregulation of downstream genes [10]. Apart from the eyelid, FOXL2 is most abundantly expressed in the ovary [11, 12]. It is an earliest granulosa cell marker, and plays a crucial role in sex determination, tumorigenesis, ovarian somatic cell proliferation and differentiation [13].
Both of the two patients presented to our reproductive medicine clinic for infertility with underlying ovarian function defect. Since their eyelid abnormality occurred early at birth and were surgically corrected before good understanding of the relationship between FOXL2 mutation and BPES [4], in addition to the absence of family history, genetic diagnosis was delayed until adulthood.
During their fertility treatment, they were clinically resistant to gonadotropins, but not completely unresponsive. Furthermore, both exhibited paradoxically normal AFC despite low AMH, and thus did not fall into any of the four groups under Poseidon criteria [14]. Beyond that, both experienced poor ORR in repeated IVF cycles despite multiple flushing [15], conform to poor ovarian responders according to Bologna criteria [16]. In spite of all these difficulties, they were lucky to have succeeded with their one and only embryo.
This is inspiring news considering the very limited successes reported in biological parenthood achieved by assisted reproductive technology (ART) for BPES patients (Table 3). In a large cohort of 164 probands, a 31-year-old BPES patient (c.672_701dup) without hypergonadotropic hypogonadism was mentioned to have achieved pregnancy after IVF treatment, but indication for IVF was not specified [17]. A case report in 2014 described a 30-year-old nulligravid woman with clinically diagnosed type I BPES, who delivered twins after artificial insemination with donor sperm (AID) [18]. Similar ovarian resistance as our patients was observed during her gonadotropin stimulation, and follicles were unresponsive to rFSH at 125IU/day. Monofollicular ovulation was achieved by 14-day course of rFSH at 300IU/day. Although genetic evidence was not provided in that case report, typical eyelid changes in one twin seemed to support the presence of genetic inheritance, probably FOXL2 mutation [3].
In patient 1 (c.843_859dup), insertion of 17 nucleotides (Fig. 2) resulted in frameshift in open reading frame (ORF) and a protein truncated after polyalanine tract (PAT), which belonged to group D according to classification suggested by De Baere E et al. [19]. It was a mutational hotspot responsible for approximately 13%-18% of all intragenic pathogenic variants [20, 17], but was reported for the first time here in East Asian populations. Both type II BPES phenotypes with naturally completed family and type I BPES phenotypes had been observed in pedigrees with this mutational variant, revealing phenotypic variability and even variable intrafamilial expressivity of POI [17].
The rarer c.178_192 duplication found in patient 2 leads to an elongated protein. Noteworthily, the insertion of five hydrophobic amino acids occurs in the regulatory forkhead domain (FHD) (Fig. 2). Studies have shown that missense mutations in the FHD impaired not only subcellular localization but also transactivation activity of FOXL2, with either loss-of-function or dominant- negative effect [21]. FHD-deleted FOXL2 mutants lost the direct binding ability to SF-1 in vitro, which mediated its repression on steroidogenic genes CYP17 [22]. Elongation from inside the FHD was not included in the classification suggested by De Baere E et a l. [19], but considering that it occurred immediately after the start of this functional domain, deranged protein function in regulatory activity was suspected. Clinically patient 2 manifested more severe phenotype with oligomenorrhea. Ovarian resistance was also stronger, even after adjustion for body weight. First described pedigree with this duplication was a mother and son with BPES eyelid features. The mother reported oligomenorrhea and menorrhagia. Ovarian function and age of reproduction was not mentioned. Since there was absence of POI and infertility history, the mother was grouped into type II BPES. Second case was a British boy aged 8 in 2019 [2], but family history was not stated. To our knowledge, this mutation was never reported in East Asian populations, and never reported to have caused classic type I BPES phenotype.
Further studies are needed to elucidate the pathophysiology of the distinctive ovarian characteristics in these FOXL2 mutants:
1) Actual follicular reserve behind the discordance between AFC and AMH: Considering the relative abundance of gonadotropin-responsive (even though resistant) antral follicles in both of our patients at their age, the mechanism of low AMH in type I BPES patients may differ from patients with non-genetic causes of ovarian insufficiency, such as advanced age, or acquired pathology (e.g endometriosis, surgery, or chemotherapy) [23]. AMH is produced in primordial follicles, preantral follicles and small antral follicles [24]. It works as an inhibitor of upstream primordial follicle recruitment [25]. FOXL2 works within a broader span of time along the follicular development, from multipotential stromal cells to cumulus cells in preovulatory follicles [13]. It was speculated that the mutual regulation of the two genes formed a positive feedback loop to maintain ovarian reserve [26]. Small follicles expressing mutant FOXL2 may have lowered expression of AMH, leading to the paradoxy.
Without the guarding by properly-expressed AMH and FOXL2 at different stages, is there imminently exhausted primordial follicle pool behind the ultrasonographic normal AFC? We do not have ovarian tissue histology to uncover the real situation in our two patients, but previous studies suggested risk of follicular depletion at an alarming rate, which eventually led to the well-known final stage of POI in type I BPES. Two siblings with a strong family history of autosomal dominant inheritance of BPES were reported in 1988, before establishment of genetic causation. The elder sister aged 28 and the younger sister aged 20 presented with secondary amenorrhea and hypergonadotropic hypogonadism. Ovarian pathological sections obtained from laparoscopy respectively showed true premature menopause with total absence of primordial follicles in the elder sister, and normal number of primordial follicles in the younger sister [27]. In Foxl2lacZ homozygous murine ovaries, the presence of defected squamous-to-cuboidal transition and premature expression of Gdf9 indicated accelerated depletion of primordial follicles and oocytes atresia, providing plausible explanation for the elder sister’s histological finding. In another paper, two patients with FOXL2 deletions after PAT and resultant putative elongated proteins were reported. The 19-year-old patient suffered from primary amenorrhea with underlying low AMH and abnormal ovarian histology (polyovular follicles, defective basal lamina, deposition of cholesterol crystals, advanced expression of proliferative marker Ki67, and intracytoplasmic FOXL2 aggregation). The 25-year-old patient presented with infertility, but had normal AMH and normal ovarian reserve clinically, ultrasonographically, and histologically. But there was also evidence of uncommon advanced expression of Ki67 in transitional follicles implying accelerated recruitment [28].
2) Gonadotropin resistance: Our patients were resistant to gonadotropins, yet responsive at high doses with extended courses. Target serum FSH seemed to be higher than 25 to 30 mIU/mL. Doubled to quadrupled total gonadotropin doses in comparison to general population was required in various COH protocols in order to awaken follicular response, and clomiphene citrate was added to patient 2 for enhancement. The eventual FORTs were still low. Apart from the AID case mentioned above, few case reports, before and after FOXL2 gene localization, also described the phenomenon of ovarian resistance to gonadotropins in BPES patients [29, 27, 30–32], but failed either in ovulation induction or in conception. Is the resistance universal to type I BPES patients? Is it the major cause of infertility? Answers are not clear yet. We speculate that the genes involved in FSH responsiveness and/or antral follicle growth, such as FSH receptor (FSHR), might be dysregulated in mutants through direct or indirect mechanisms.
3) Abnormal ovulation and low ORR: Less studies focused on ovulation in BPES patients. As described, patient 2 was oligomenorrheic secondary to anovulation. Patient 1 maintained overall natural normal cycle with sonographically confirmed ovulation. It was at least once followed by successful natural fertilization and implantation but unluckily ended up with first-trimester miscarriage. However, she also reported to have experienced cycles judged as luteinized unruptured follicle syndrome (LUFS). Furthermore, the history of endometrial hyperplasia implied possible preceding anovulation. There might be some mechanism behind the disordered ovulation of graafian (preovulatory) follicles as well as the difficulty in harvesting oocytes from them in IVF. Hypotheses include hampered oocyte maturation, abnormal expression of Luteinizing hormone receptor (LHR) [33], defected gap junction, and deranged activity of local factors such as prostaglandin and proteases [34]. Individualized ovulation triggering-to-harvesting interval and two-chamber needle flushing may have a role in improving the ORR.
Currently mainstream recommendation at genetic counseling for type I BPES patients includes donation of eggs or embryos, adoption or fostering, sacrificing biological parenthood. This is probably based on the following considerations: 1) missed reproductive window at presentation; 2) limited translation of in vitro maturation (IVM) into clinical use; 3) ethical concerns about precautionary ovarian tissue cryopreservation or oocyte cryopreservation for young girls with unclear ovarian phenotype; 4) risk of inheritance of pathogenic mutations.
Noteworthily on the other hand, patients manifesting either type of BPES usually have normal menarche and secondary sex characteristics. Association with major defects including intellectual disability were rare with inconclusive causation. Even type I BPES phenotypes with primary or secondary amenorrhea possess potential capacity of natural conception and deserve a childbearing opportunity. Early intervention may rescue their threatened fertility.
Clinicians and scientists were making efforts to specify the phenotype-genotype correlation in ovarian function [19]. Studies suggested high POI risk from mutations with proteins truncated upstream of PAT and from mutations involving inward-pointing of relevant amino acids towards the hydrophobic core. Meanwhile PAT expansion predominantly leads to type II BPES [10]. However, considering the inter-individual variability with even varied intrafamilial penetrance of ovarian phenotypes from identical genotypes, POI risk should be emphasized to all female patients as early as possible while an efficient prediction model is still on the way. As the putative acceleration of follicular depletion takes place without clear red flag for onset and exhaustion, it is unrational to declare innocence and passively wait till the age of 40 to diagnose POI, especially considering the much higher difficulty they might encounter like our patients, compared to patients with other causes of low ovarian reserve.
In summary, we described detailed IVF treatment to two Chinese type I BPES patients, whose FOXL2 mutations were new to East Asian population database. Their ovarian profiles seem to differentiate type I BEPS from either pure resistant ovarian syndrome (ROS) or pure POI, and offers materials for further basic research on FOXL2 gene and type I BPES. Of course, due to the rarity of the disease, we look forward to more cases to reproduce the success and the observed phenomena. Nevertheless, our report confirmed that IVF in type I BPES is difficult, but still feasible with individualized treatment. It also serves as a stepping-stone to future attempts on IVM and PGT in this population.