Primary ovarian insufficiency (POI) is a heterogeneous condition characterized by impaired ovarian function occurring before the age of 40 years, affecting approximately 1.1% of women of reproductive age [1]. POI is characterized by menstrual irregularities, amenorrhea, infertility, and hypoestrogenic symptoms such as hot flushes, night sweats, and vaginal dryness. It is further associated with a wide range of additional clinical features, including neuropsychiatric, psychological, musculoskeletal, cardiovascular, and lifestyle-related factors [2, 3, 4]. POI often coexists with thyroiditis, Addison’s disease or other autoimmune disorders, sometimes accompanied by enlarged multifollicular ovaries [2]. In addition, syndromic features of Turner syndrome or blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) or fragile X syndrome and a family history of early menopause or infertility may also be observed with POI [5, 6, 7]. Beyond infertility, POI carries substantial health consequences, including increased risks of osteoporosis, cardiovascular disease, metabolic disorders, and diminished quality of life [8, 9]. Associated symptoms, clinical findings and comorbidities can be variable due to intermittent ovarian hormone secretion [2]. At presentation, documenting medical and family histories, along with key clinical features, is essential to help identify POI and its possible underlying causes.

According to the 2016 European Society of Human Reproduction and Embryology (ESHRE) guidelines, POI should be considered in women younger than 40 years who present with oligo/amenorrhea or estrogen-deficiency symptoms, with diagnosis confirmed by oligo/amenorrhea lasting at least four months, two elevated follicle-stimulating hormone (FSH) measurements ($>$25 U/L), and low estradiol levels (50 pg/mL) obtained 4–6 weeks apart in the early follicular phase [10]. In contrast, the updated 2024 ESHRE guidelines now indicate that a single elevated FSH measurement may suffice to establish the diagnosis. The guidelines also recommend excluding pregnancy and accounting for the effects of hormonal therapy, which can mask amenorrhea or alter FSH levels, before confirming POI [11]. These recommendations outline further testing to determine the cause of POI. Women with a history of chemotherapy, radiotherapy, pelvic or ovarian surgery, or bilateral salpingo-oophorectomy before age 40 are considered to have iatrogenic POI and typically do not require further evaluation. For all non-iatrogenic cases, following genetic counseling and informed consent, chromosomal analysis and FMR1 premutation (PM) testing are recommended, while broader genetic testing may be considered where feasible. If no genetic cause is identified, screening for 21-hydroxylase autoantibodies (21OH-Abs) and thyroid-stimulating hormone (TSH) levels is recommended to detect potential autoimmune etiologies. Cases with no identifiable cause after these evaluations are classified as idiopathic POI [10, 11].

Despite growing knowledge and international guidelines, their implementation is limited in low- and middle-income regions, and data on POI remain scarce. In Pakistan, only a few studies have explored POI, focusing mainly on poor ovarian response, hormonal disturbances, and associated symptoms [12, 13], without performing integrated clinical, biochemical, or genetic assessments. Limited awareness and resources have hindered guideline-based studies, leaving most cases unexplained. We hypothesized that comprehensive clinical, hormonal, and genetic evaluation according to ESHRE guidelines would enhance identification of underlying etiologies in POI, including potential genetic contributions among idiopathic cases. Therefore, this study aimed to comprehensively characterize the clinical, hormonal, and genetic features of POI among affected Pakistani women and to classify its etiologies according to ESHRE guidelines.

POI is a heterogeneous reproductive disorder, characterized by diverse etiologies and a wide range of clinical manifestations [18], yet in many cases the underlying cause remains unidentified despite advances in understanding. Studies from high-income countries have clarified the genetic, autoimmune, and iatrogenic factors associated with POI [7, 19, 20], but large gaps remain in low- and middle-income regions. In Pakistan, only a single study by Izhar et al. [12] has explored POI, focusing on poor ovarian response criteria to detect occult POI in women with infertility and oligomenorrhea. More recently, Kazi et al. [13] assessed the diagnostic and management challenges of POI in women attending a tertiary hospital in Lahore, highlighting the association of infertility, hot flashes, mood swings, vaginal dryness, and insomnia with disturbed hormonal and ovarian reserve markers. However, these studies relied mainly on clinical symptoms and surrogate biochemical markers, without integrating broader clinical, hormonal, and genetic investigations. To our knowledge, the present study is the first in Pakistan to comprehensively evaluate POI by integrating detailed clinical assessment, hormonal profiling, and genetic testing according to ESHRE diagnostic criteria.

Consistent with ESHRE recommendations, all women of reproductive age (under 40 years) in this study fulfilled the diagnostic criteria for POI, presenting with at least four months of oligo/amenorrhea, repeated serum FSH levels $>$25 IU/L, and low estradiol levels of approximately 50 pg/mL. Although POI can occur across a broad age range, from early adolescence to 40 years [21], the age of onset and age at diagnosis remain key factors for timely recognition and management. In the current cohort, the mean age at symptom onset was 31 years, and the mean age at clinical presentation was 33 years, indicating an approximate delay of 24 months. This delay is shorter than the 48-month median time to diagnosis reported by Minis et al. [22], but comparable to findings from Sun et al. [23], who reported an average interval of approximately 28 months between symptom onset and POI diagnosis, and to Bakhsh [24], who observed diagnostic delays exceeding 18 months in a substantial proportion of patients. Such delays are often related to variability and inconsistency in presenting features, as highlighted by Kapoor [25]. In Pakistan, additional factors such as limited awareness of POI, sociocultural barriers affecting care-seeking, and variability in access to specialized diagnostic services may further contribute to delayed diagnosis.

The clinical spectrum observed was broad, with vasomotor symptoms such as hot flushes, vaginal dryness or dyspareunia, and night sweats, alongside mood changes and psychological disturbances including depression and stress, being highly prevalent. Comparable clinical presentations have been observed in studies of other populations, where vasomotor and neuropsychiatric symptoms dominate the clinical profile [2, 3, 4]. The high prevalence of depression (72.4%) and stress (65.5%) in our cohort is consistent with previous reports of psychological disturbances in women with POI [26], highlighting the substantial psychosocial impact of POI, which may be further aggravated by cultural stigma surrounding infertility in South Asian societies.

The association between POI and long-term comorbidities, such as osteoporosis, metabolic syndrome, and cardiovascular disease, is well established [8, 9]. Comorbidities of POI were documented in 12.4% of cases, with migraine being the most common (4.1%). Although the prevalence of metabolic and cardiovascular comorbidities in our cohort was lower than reported in other studies [27, 28], this is not solely explained by age, as participants in these studies were also predominantly below 40 years. In contrast, women aged $\ge$45 years with a prior diagnosis of POI have been reported to exhibit a higher prevalence of cardiometabolic comorbidities [29, 30], highlighting the cumulative risk associated with longer duration of estrogen deficiency. These findings emphasize that age and duration of POI are important factors influencing the manifestation of long-term systemic comorbidities.

The etiological distribution of POI in our cohort illustrates its complexity. Iatrogenic causes accounted for 7.2% of cases, consistent with a study reporting 6–47% [31], and aligning with another study reporting a ~8% cumulative risk of POI by age 40 in female cancer survivors [32]. The highest risk was observed after alkylating agents and ovarian radiotherapy [31], influenced by age at exposure, treatment dose, and baseline ovarian reserve [2]. In our cohort, the difference in confirmed iatrogenic diagnoses was highly significant (p 0.0001), highlighting that this subgroup represents a clearly distinguishable and reliably diagnosed etiology. Genetic causes were confirmed in 3.8%, collectively from women with features suggestive of a genetic etiology (16.6%) and those presenting with POI features only (61.7%), which is markedly lower than the 20–25% reported in a previous study [6]. FMR1 PM alleles were detected in seven cases, Turner syndrome in two, one FOXL2 variant [c.223C$>$T, p.(Leu75Phe)] previously reported in POI with BPES [5] in one case, and a GDF9 variant [c.604C$>$T, p.(Gln202*)] previously reported in POI [7] in sisters. The frequency of confirmed cases was not statistically significant among women with suggestive genetic features (p $\approx$ 0.8500), whereas the difference was highly significant among those with POI features only (p 0.0001), indicating a substantial burden of undiagnosed genetic predisposition in the idiopathic group and emphasizing the limitations of conventional diagnostic approaches. This underscores the importance of advanced genetic testing, particularly whole-exome sequencing (WES), in women with idiopathic POI or subtle clinical indicators such as Turner stigmata, BPES, family history of infertility/early menopause, or early disease onset. Screening for 21OH-Abs and TSH confirmed an association of POI with autoimmune disease in 19 women (6.6%), with no cases detected in women with features suggestive of a genetic etiology or POI features only. This difference was highly significant (p 0.0001), indicating that autoimmune testing effectively identified women with POI associated with autoimmune disease and highlighting its utility in detecting cases with an underlying autoimmune etiology. However, a causal relationship could not be established, as autoimmune disorders are common and autoantibody positivity may not always reflect a direct cause of ovarian insufficiency [2].

In conclusion, this study provides the first comprehensive evaluation of POI in Pakistan, integrating clinical, hormonal, and genetic assessments in line with ESHRE guidelines. The findings highlight delayed diagnosis, a high psychological burden, and underrecognized comorbidities in affected women. Iatrogenic causes were clearly identifiable and statistically significant, while genetic causes were confirmed in a small proportion, with many women showing subtle features or idiopathic presentation, indicating a substantial burden of undiagnosed genetic predisposition. Autoimmune contributors were confirmed in a subset of women, with testing proving effective in identifying true cases. Overall, the high proportion of idiopathic cases underscores the need for broader genetic evaluation, including WES, and earlier diagnostic approaches to improve recognition and management of POI, particularly in resource-limited settings.

All data reported in this study are included within the manuscript.

SS contributed to the conceptualization and study design, genetic investigations, data validation and interpretation, manuscript drafting, and final approval. HT performed data collection, analysis, and laboratory work. UNT and MJ were responsible for participant recruitment and clinical evaluation, including investigations and interpretation. ZZ contributed to the clinical evaluation and interpretation of participants presenting with acute or overlapping symptoms such as palpitations, dizziness, and headaches. SAK performed psychiatric and psychological evaluations and their interpretation. MY contributed to data collection, analysis, laboratory work, and oversight of commercial hormonal and genetic testing. All authors contributed to critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of Kohat University of Science and Technology (KUST), Kohat, Pakistan (REF:/KUST/Ethical Committee/837). Written informed consent was obtained from all participants prior to inclusion in the study.

We sincerely acknowledge the contributions of the participating women and their families. We also thank the staff at the gynecology and infertility clinics for their support in recruitment and sample collection.

This research received no external funding.

The authors declare no conflict of interest.

During the preparation of this work, the authors used ChatGPT-3.5 to check spelling and grammar. After using this tool, the authors reviewed and edited all content as needed and take full responsibility for the content of the publication.