Diagnostic Evaluation of Primary Ovarian Insufficiency in a Cohort of 290 Pakistani Women: Clinical, Hormonal, and Genetic Perspectives

Background: Primary ovarian insufficiency (POI) is a heterogeneous disorder with multifactorial etiologies. Accurate diagnosis requires an integrated clinical, hormonal, and genetic evaluation, yet data from Pakistan are limited, and the burden of idiopathic and genetically predisposed cases remains largely unknown. Methods: A total of 345 women under 40 years presenting with amenorrhea or menstrual irregularities were screened. After excluding pregnancy, cases not meeting the European Society of Human Reproduction and Embry- ology (ESHRE) diagnostic criteria, and incomplete records, 290 women were included. Comprehensive clinical, hormonal, and genetic investigations were performed according to ESHRE guidelines to determine underlying etiologies. Results: The mean age at presentation was 33 ± 4.5 years, with a median symptom duration of 6 months. The mean age at menarche was 13 ± 1 years, and the mean body mass index (BMI) was 24.5 ± 3.4 kg/m2. Most women presented with amenorrhea (80%) or oligomenorrhea (20%). Secondary infertility was reported in 72.8% and primary infertility in 2.4%. A history of miscarriage was documented in 5.9% of participants. Common clinical features included hot flushes (75.9%), depression (72.4%), high stress (65.5%), mood changes (62.1%), vaginal dryness or dyspareunia (55.2%), and night sweats (54.5%). Coexisting comorbidities were observed in 12.4%, most frequently migraines (4.1%). Hormonal evaluation confirmed elevated follicle-stimulating hormone (FSH) levels ( >25 IU/L) and low estradiol ( <50 pg/mL) in all participants. Etiological classification identified iatrogenic causes in 7.2%, genetic causes in 3.8% (confirmed in women with suggestive genetic features or isolated POI), autoimmune causes in 6.6%, and idiopathic POI in 82.4%. Statistically significant differences in confirmed diagnoses were observed among most etiological groups ( p < 0.0001), except for women with features suggestive of a genetic cause ( p ≈ 0.8500). Conclusions: POI presents with diverse clinical features. Evaluation based on ESHRE guidelines enables identification of iatrogenic, autoimmune, and genetic contributors, and highlights the high prevalence of idiopathic cases, which may have an underlying genetic predisposition.

primary ovarian insufficiency; idiopathic POI; genetic predisposition; autoimmune POI; iatrogenic causes; ESHRE guide- lines 1. Introduction Primary ovarian insufficiency (POI) is a heteroge- neous condition characterized by impaired ovarian func- tion occurring before the age of 40 years, affecting approxi- mately 1.1% of women of reproductive age [1]. POI is char- acterized by menstrual irregularities, amenorrhea, infertil- ity, 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 neu- ropsychiatric, psychological, musculoskeletal, cardiovas- cular, and lifestyle-related factors [ 2–4]. POI often coex- ists with thyroiditis, Addison’s disease or other autoimmune disorders, sometimes accompanied by enlarged multifollic- ular ovaries [ 2]. In addition, syndromic features of Turner syndrome or blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) or fragile X syndrome and a family his- tory of early menopause or infertility may also be observed with POI [ 5–7]. Beyond infertility, POI carries substan- tial health consequences, including increased risks of osteo- porosis, cardiovascular disease, metabolic disorders, and diminished quality of life [8,9]. Associated symptoms, clin- ical findings and comorbidities can be variable due to in- termittent ovarian hormone secretion [ 2]. At presentation, documenting medical and family histories, along with key clinical features, is essential to help identify POI and its pos- sible 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 ele- vated FSH measurement may suffice to establish the di- agnosis. The guidelines also recommend excluding preg- nancy and accounting for the effects of hormonal therapy, which can mask amenorrhea or alter FSH levels, before confirming POI [ 11]. These recommendations outline fur- ther testing to determine the cause of POI. Women with a history of chemotherapy, radiotherapy, pelvic or ovar- ian 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, chro- mosomal analysis and FMR1 premutation (PM) testing are recommended, while broader genetic testing may be con- sidered where feasible. If no genetic cause is identified, screening for 21-hydroxylase autoantibodies (21OH-Abs) and thyroid-stimulating hormone (TSH) levels is recom- mended to detect potential autoimmune etiologies. Cases with no identifiable cause after these evaluations are classi- fied as idiopathic POI [ 10,11]. Despite growing knowledge and international guide- lines, 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 associ- ated symptoms [12,13], without performing integrated clin- ical, biochemical, or genetic assessments. Limited aware- ness 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 identifi- cation of underlying etiologies in POI, including potential genetic contributions among idiopathic cases. Therefore, this study aimed to comprehensively characterize the clini- cal, hormonal, and genetic features of POI among affected Pakistani women and to classify its etiologies according to ESHRE guidelines. 2. Materials and Methods 2.1 Study Design and Participants This multicenter cross-sectional study was conducted between August 2023 and February 2025, and participat- ing women were recruited from the obstetrics and gynecol- ogy departments of tertiary care hospitals. Women under 40 years of age presenting with amenorrhea or oligomenorrhea persisting for at least four months and meeting the ESHRE diagnostic criteria of FSH >25 IU/L and estradiol <50 pg/mL on two separate measurements taken more than four weeks apart, were included. Women who were pregnant, currently using hormonal therapy, diagnosed with polycys- tic ovary syndrome (PCOS), had malignant tumors, or had undergone ovarian removal were excluded (according to 2016 and 2024 guidelines), as these conditions can con- found menstrual irregularities or hormonal measurements required for POI evaluation [ 10,11]. A history of prior hor- monal therapy was recorded, and participants were included only if therapy had been discontinued for at least three months before recruitment. Written informed consent was obtained, and the study was approved by the Ethical Com- mittee of KUST (REF:/KUST/Ethical Committee/837). 2.2 Clinical Evaluation Detailed medical and reproductive histories were obtained, including age at menarche, age at symp- tom onset, type of menstrual disturbance (amenor- rhea/oligomenorrhea), reproductive history, miscarriage history, and infertility. Body mass index (BMI) was mea- sured for all participants. Hypoestrogenic symptoms such as hot flushes, night sweats, vaginal dryness, and sys- temic manifestations were recorded. Features suggestive of Turner syndrome, BPES, autoimmune disorders, or iatro- genic causes (chemotherapy, radiotherapy, ovarian surgery) were carefully recorded. Coexisting comorbidities were documented based on patient history, prior diagnoses, man- agement records, laboratory investigations, imaging, and review of medical records as appropriate. Family history of infertility or early menopause, as well as lifestyle and psy- chosocial factors, were also recorded. Depression and emo- tional instability were evaluated using the Patient Health Questionnaire-9 (PHQ-9) [14]. 2.3 Diagnostic Evaluation Following ESHRE recommendations, menstrual dis- turbance was required to persist for at least four months to support a diagnosis of POI. For classification purposes, amenorrhea was defined as the complete absence of men- struation for ≥ six months, while oligomenorrhea was de- fined as menstrual cycles longer than 35 days or fewer than nine cycles per year. Hormonal testing (FSH and estra- diol) was performed in all participants, in the early follicu- lar phase (days 2–5) for women with ongoing menstrual cy- cles, and on a random day for those presenting with amen- orrhea. Thyroid function was assessed in all participants by measuring serum TSH levels, with a reference range of 0.4–4.0 mU/L. After genetic counseling and obtaining in- formed consent, peripheral blood samples were collected in EDTA tubes, appropriately labeled, and transported un- der cold-chain conditions. Samples were stored at 4 °C and processed within 24–48 hours. Genomic DNA was ex- tracted using the standard phenol–chloroform protocol or the QIAamp DNA Blood Mini Kit (Catalog No. 51306; 2 Table 1. Symptoms, clinical features, psychological factors, and lifestyle characteristics in women with POI (N = 290). Category Specific symptom(s)/Findings Frequency (n, %) Estrogen deficiency Hot flushes 220 (75.9%) Night sweats 158 (54.5%) V aginal dryness/dyspareunia 160 (55.2%) Neuropsychiatric Mood changes (mood swings, mental fog, melancholia) 180 (62.1%) Sleep disturbances Insomnia/irregular sleep–wake cycle 115 (39.7%) Sexual dysfunction Reduced libido, dyspareunia 108 (37.2%) Fatigue Generalized fatigue 103 (35.5%) Dermatological Hair loss, skin dryness 92 (31.7%) Ophthalmological Dry eyes 88 (30.3%) Endocrine/Metabolic Cold intolerance 83 (28.6%) Musculoskeletal Joint clicking, muscle/joint pain 75 (25.9%) Neurological Headaches, vertigo, tingling limbs 72 (24.8%) Cardiovascular Palpitations 65 (22.4%) Psychological Depression 210 (72.4%) High stress levels 190 (65.5%) Anxiety 120 (41.4%) Social withdrawal 98 (33.8%) Lifestyle Poor diet/nutrition 85 (29.3%) Low physical activity 65 (22.4%) Note: V alues represent frequency (n) and percentage (%) of participants reporting each manifestation. POI, primary ovarian insufficiency. Table 2. Comorbidities observed in women with POI. Comorbidity Frequency (n, %) Documentation/Diagnosis Migraine 12 (4.1%) Patient history and documented past diagnosis and management records Hypertension 8 (2.8%) Systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg on two occasions, or on anti- hypertensive therapy Diabetes mellitus 5 (1.7%) Fasting plasma glucose ≥126 mg/dL, HbA1c ≥6.5%, or prior physician diagnosis Obesity 4 (1.4%) BMI ≥30 kg/m2 Osteoporosis 3 (1.0%) T-score ≤−2.5 measured by dual-energy X-ray absorptiometry (DEXA) scan Breast cancer 2 (0.7%) Histopathology confirmation and clinical oncology records Ischemic heart 1 (0.3%) History of myocardial infarction or angina, confirmed by ECG or imaging Gastric ulcers 1 (0.3%) Endoscopic confirmation with biopsy Total 36 (12.4%) BMI, body mass index; BP , blood pressure; HbA1c, glycated hemoglobin; ECG, electrocardiogram. Qiagen, Hilden, Germany), following recommended pro- cedures. DNA concentration and purity were measured us- ing a NanoDrop Lite Plus spectrophotometer (Catalog No. NDLPLUSGL; Thermo Fisher Scientific, Waltham, MA, USA) by assessing A260/A280 and A260/A230 ratios, and the DNA was stored at –20 °C for subsequent use. Testing included karyotyping and FMR1 PM analysis [ 15], as well as direct sequencing of established POI-associated genes such as BMP15, NOBOX, and GDF9 [16] for all partic- ipants. FOXL2 sequencing was performed selectively in women presenting with features suggestive of both POI and BPES, in whom no variants were identified in the other es- tablished POI-associated genes . Segregation analysis was performed in available family members to assess inheri- tance patterns of identified variants. In silico prediction tools were used to assess the potential impact of identi- fied variants, and each variant was classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines [17]. Autoimmune evaluation (21OH- Abs) was carried out selectively in women without an iden- tified genetic or iatrogenic cause, and in those presenting with symptoms suggestive of autoimmune involvement. 2.4 Classification of Cases Cases were categorized according to the underlying cause, in line with ESHRE 2024 guidelines: iatrogenic POI (history of chemotherapy, radiotherapy, or ovarian surgery), genetic POI (chromosomal abnormalities, FMR1 PM alleles, or pathogenic/likely pathogenic variants), au- toimmune POI (positive 21OH-Abs or abnormal TSH lev- els), and idiopathic POI (no identifiable cause after a com- prehensive diagnostic workup). 3 2.5 Data Analysis Data was analyzed using IBM SPSS Statistics, V er- sion 30.0 (IBM Corp., Armonk, NY , USA). Continuous variables are presented as mean ± SD (standard devia- tion) or median, and categorical variables as frequencies and percentages. Associations between clinical presenta- tion groups and diagnostic outcomes (confirmed vs. non- confirmed) were assessed using two-tailed Chi-square or Fisher’s exact tests, applied as appropriate. The null hy- pothesis assumed no association between clinical presenta- tion and diagnosis. A p-value < 0.05 was considered statis- tically significant, indicating an association between clini- cal presentation and diagnostic outcome. 3. Results 3.1 Study Cohort A total of 345 women under 40 years presenting with amenorrhea or menstrual irregularities and/or estrogen- deficiency symptoms were initially recruited. Women who were pregnant, currently using hormonal therapy, or had not provided consent for the diagnostic workup were excluded. After these exclusions, 290 women were included in the fi- nal analysis. The mean age at presentation was 33 ± 4.5 years, with a median symptom duration of 6 months. 3.2 Clinical Features Most women presented with amenorrhea (80%, n = 232) and oligomenorrhea (20%, n = 58), with a mean du- ration of amenorrhea of 6 ± 2 months. The mean age at symptom onset was 31.6 ± 3.8 years, and the mean age at menarche was 13 ± 1 years. The mean BMI of the partici- pants was 24.5 ± 3.4 kg/m2. Reproductive history revealed secondary infertility in 72.8% (n = 211) and primary infer- tility in 2.4% (n = 7). Miscarriage history was documented in 5.9% women (n = 17). A family history of infertility or early menopause was documented in 9.3% (n = 27). Women exhibited a wide spectrum of clinical features, including manifestations of estrogen deficiency, neuropsy- chiatric symptoms, and musculoskeletal, neurological, and cardiovascular complaints. The most prevalent features were hot flushes (75.9%), depression (72.4%), high stress levels (65.5%), mood changes (62.1%), vaginal dryness or dyspareunia (55.2%), and night sweats (54.5%) (Table 1). Coexisting comorbidities were observed in 12.4% of cases, with migraine being the most common (4.1%) (Table 2). 3.3 Etiological Clues Clinical evaluation revealed iatrogenic factors (pre- vious ovarian surgery, chemotherapy, or radiotherapy) in 7.2% (n = 21), features suggestive of a genetic etiology in 16.6% (n = 48), and coexisting autoimmune disorders in 14.5% (n = 42) (Table 3). 3.4 Hormonal and Genetic Findings All included women had elevated FSH ( >25 IU/L) confirmed on two occasions, with a mean FSH of 30 ± 12 IU/L and mean estradiol of 25 ± 10 pg/mL. Among the 290 women with POI, 21 had iatrogenic causes due to ovarian surgery, chemotherapy, or radiotherapy, all of whom had confirmed diagnoses (100%). Among women without iatrogenic causes, pathogenic genetic variants were detected in two groups. In 48 women with Turner syn- drome stigmata, BPES, a family history of early menopause or infertility, or early age at onset, 8 had confirmed diag- noses, including karyotypic abnormalities consistent with Turner syndrome (2 cases), FMR1 PM alleles with 50–65 CGG repeats (5 cases), and a heterozygous FOXL2 vari- ant [c.223C >T, p.(Leu75Phe)] identified in one woman with both POI and BPES. The same FOXL2 variant was present in her father and brother, who exhibited BPES fea- tures. Based on ACMG criteria (PM1, PM2, PP1, PP3), the variant is classified as a variant of uncertain signifi- cance (VUS). Among 179 women with POI features only 3 rare genetic variants were detected, including FMR1 PM alleles with 50, 57 CGG repeats in 2 cases and a homozy- gous GDF9 variant [c.604C>T, p.(Gln202*)] identified in one woman with POI. This GDF9 variant segregated in an autosomal recessive manner, with both parents being het- erozygous carriers. Based on ACMG criteria (PVS1, PM3, PP3), the variant is classified as likely pathogenic. Au- toimmune evaluation confirmed the diagnosis in 19 women with autoimmune disease, while no confirmed cases were observed in women with features suggestive of a genetic etiology or POI features only. Statistically significant dif- ferences in the proportion of confirmed diagnoses were ob- served among most clinical presentation groups, except for women with features suggestive of a genetic etiology ( p ≈ 0.8500). Overall, the proportion of confirmed diagnoses differed significantly across the underlying etiological cat- egories, with highly significant associations observed for iatrogenic (p < 0.0001), autoimmune (p < 0.0001), and id- iopathic/other genetic cases ( p < 0.0001) (Table 3). 3.5 Etiological Classification Confirmed causes of POI were identified in a subset of women: iatrogenic 7.2%, genetic 3.8%, and autoimmune 6.6%. The remaining women without a definitive cause, or with suggestive genetic or autoimmune features, were classified as idiopathic POI, accounting for 82.4% of the cohort (Table 4). 4. Discussion POI is a heterogeneous reproductive disorder, char- acterized by diverse etiologies and a wide range of clini- cal manifestations [ 18], yet in many cases the underlying cause remains unidentified despite advances in understand- ing. Studies from high-income countries have clarified the genetic, autoimmune, and iatrogenic factors associated with 4 Table 3. Underlying causes of POI in 290 women, determined by clinical features and diagnostic investigations. Clinical presentation (n, %) Investigations confirmed diagnosis Confirmed diagnosis (n, %) (N = 51) Non-confirmed diagnosis (n, %) (N = 239) Statistical test (p-value) Underlying etiology of POI History of ovarian surgery, chemotherapy, or radiotherapy (21, 7.2%) Confirmation from medi- cal/surgical history and records 21 (100.0%) 0 (0.0%) <0.0001* Iatrogenic Turner syndrome stigmata, family his- tory of infertility/early menopause, blepharophimosis, early age at onset (48, 16.6%) Genetic testing: FMR1 PM analy- sis, FOXL2, BMP15, NOBOX, and GDF9 genes sequencing 8 (16.7%) 40 (83.3%) ≈0.8500 Genetic (Turner syndrome, FMR1 PM alleles, one FOXL2 variant) Coexisting autoimmune disorders: thyroid dysfunction, systemic lupus erythematosus, rheumatoid arthritis, autoimmune haemolytic anaemia, vi- tiligo, Addison’s disease (42, 14.5%) Autoimmune serological testing for 21OH-Abs and serum TSH levels 19 (45.2%) 23 (54.8%) <0.0001* Autoimmune (Positive 21OH- Abs and/or abnormal TSH levels) POI features without additional con- ditions (179, 61.7%) Genetic and autoimmune serologi- cal testing 3 (1.7%) 176 (98.3%) <0.0001* Idiopathic (no definitive cause identified)/Genetic ( FMR1 PM alleles in 2 cases, a GDF9 variant in 1 case) Note: V alues are presented as frequency (n) and percentage (%). Statistical significance was assessed using Chi-square or Fisher’s exact tests. An asterisk (*) indicates a statistically significant p-value (p < 0.05). Bold = significant values and underlying POI etiology. PM, premutation; 21OH-Abs, 21-hydroxylase autoantibodies; TSH, thyroid-stimulating hormone. 5 Table 4. Distribution of underlying causes of POI among women (N = 290). Underlying etiology Number of cases (n) Percentage (%) Iatrogenic (surgery, chemotherapy, radiotherapy) 21 7.2 Genetic (Turner syndrome, FMR1 PM alleles, one FOXL2 variant, one GDF9 variant) 11 3.8 Autoimmune (thyroid dysfunction, lupus, rheumatoid arthritis, Addison’s disease, etc.) 19 6.6 Idiopathic (no definitive cause identified) 239 82.4 Total 290 100.0 Note: V alues represent frequency (n) and percentage (%) of participants in each etiological category. 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 in- fertility 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 hor- monal and ovarian reserve markers. However, these studies relied mainly on clinical symptoms and surrogate biochemi- cal 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 diagnos- tic 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 recogni- tion 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 me- dian 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 sub- stantial 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 af- fecting care-seeking, and variability in access to specialized diagnostic services may further contribute to delayed diag- nosis. The clinical spectrum observed was broad, with va- somotor 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 [ 2–4]. 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 im- pact of POI, which may be further aggravated by cultural stigma surrounding infertility in South Asian societies. The association between POI and long-term comor- bidities, such as osteoporosis, metabolic syndrome, and car- diovascular disease, is well established [ 8,9]. Comorbidi- ties of POI were documented in 12.4% of cases, with mi- graine being the most common (4.1%). Although the preva- lence 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 con- trast, women aged ≥45 years with a prior diagnosis of POI have been reported to exhibit a higher prevalence of car- diometabolic comorbidities [29,30], highlighting the cumu- lative risk associated with longer duration of estrogen defi- ciency. 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 il- lustrates 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% cu- mulative 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 ex- posure, treatment dose, and baseline ovarian reserve [ 2]. In our cohort, the difference in confirmed iatrogenic diag- noses was highly significant (p < 0.0001), highlighting that this subgroup represents a clearly distinguishable and reli- ably diagnosed etiology. Genetic causes were confirmed in 3.8%, collectively from women with features sugges- tive 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)] pre- viously reported in POI with BPES [ 5] in one case, and a GDF9 variant [c.604C >T, p.(Gln202*)] previously re- 6 ported in POI [ 7] in sisters. The frequency of confirmed cases was not statistically significant among women with suggestive genetic features ( p ≈ 0.8500), whereas the dif- ference was highly significant among those with POI fea- tures only ( p < 0.0001), indicating a substantial burden of undiagnosed genetic predisposition in the idiopathic group and emphasizing the limitations of conventional diagnos- tic approaches. This underscores the importance of ad- vanced genetic testing, particularly whole-exome sequenc- ing (WES), in women with idiopathic POI or subtle clini- cal indicators such as Turner stigmata, BPES, family his- tory of infertility/early menopause, or early disease onset. Screening for 21OH-Abs and TSH confirmed an associa- tion 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 autoim- mune testing effectively identified women with POI asso- ciated 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 pos- itivity may not always reflect a direct cause of ovarian in- sufficiency [2].

Despite providing a comprehensive evaluation of POI in women across multiple centers in Pakistan, this study has several limitations. First, the cohort included only Pakistani women, which may limit generalizability to other popula- tions. Second, although our study aimed to identify under- lying etiologies and potential undetected genetic predispo- sitions in POI using ESHRE-guided evaluation, no control group was included, limiting comparative analyses. Third, in Pakistan, genetic testing is generally not performed for POI cases, and due to high costs, our study focused on a limited gene panel, which may underestimate rare or novel variants and highlights the need for broader genetic evalu- ation. Fourth, autoimmune assessment relied on specific serological markers, which may not capture all relevant mechanisms. Finally, the cross-sectional design precludes assessment of long-term outcomes, including the progres- sion of comorbidities and reproductive or metabolic conse- quences. These limitations underscore the need for larger, multi-center, longitudinal studies with broader genetic and immunological testing to fully characterize POI. 5. Conclusions In conclusion, this study provides the first comprehen- sive evaluation of POI in Pakistan, integrating clinical, hor- monal, and genetic assessments in line with ESHRE guide- lines. The findings highlight delayed diagnosis, a high psy- chological burden, and underrecognized comorbidities in affected women. Iatrogenic causes were clearly identifi- able and statistically significant, while genetic causes were confirmed in a small proportion, with many women show- ing 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 un- derscores the need for broader genetic evaluation, including WES, and earlier diagnostic approaches to improve recog- nition and management of POI, particularly in resource- limited settings. Availability of Data and Materials All data reported in this study are included within the manuscript. Author Contributions SS contributed to the conceptualization and study de- sign, genetic investigations, data validation and interpre- tation, manuscript drafting, and final approval. HT per- formed data collection, analysis, and laboratory work. UNT and MJ were responsible for participant recruitment and clinical evaluation, including investigations and interpreta- tion. ZZ contributed to the clinical evaluation and interpre- tation 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 hor- monal and genetic testing. All authors contributed to criti- cal revision of the manuscript for important intellectual con- tent. 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. Ethics Approval and Consent to Participate The study was conducted in accordance with the Dec- laration of Helsinki and was approved by the Ethical Com- mittee of Kohat University of Science and Technology (KUST), Kohat, Pakistan (REF:/KUST/Ethical Commit- tee/837). Written informed consent was obtained from all participants prior to inclusion in the study. Acknowledgment 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 sup- port in recruitment and sample collection. Funding This research received no external funding. Conflict of Interest The authors declare no conflict of interest. 7 Declaration of AI and AI-Assisted Technologies in the Writing Process 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.

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