{"paper_id":"c30ee2eb-2471-43dc-a5f1-a5ab398745f3","body_text":"Clin. Exp. Obstet. Gynecol. 2026; 53(2): 47356\nhttps://doi.org/10.31083/CEOG47356\nCopyright: © 2026 The Author(s). Published by IMR Press.\nThis is an open access article under the CC BY 4.0 license .\nPublisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nOriginal Research\nDiagnostic Evaluation of Primary Ovarian Insufficiency in a Cohort of\n290 Pakistani Women: Clinical, Hormonal, and Genetic Perspectives\nShamim Saleha1,*\n , Haleema Tahir1\n , Zainab Zaffar 2, Umme Nafeesa Tariq 3,\nMusarrat Jabeen4\n , Shajaat Ali Khan 5, Muhammad Y asin1\n1Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology (KUST), 26000 Kohat, Khyber Pakhtunkhwa,\nPakistan\n2Department of Accident and Emergency, HITEC Institute of Medical Sciences, 47080 Taxila, Punjab, Pakistan\n3Department of Obstetrics and Gynecology, HITEC Institute of Medical Sciences, 47080 Taxila, Punjab, Pakistan\n4Department of Obstetrics and Gynecology, Khyber Medical University Institute of Medical Sciences (KMU-IMS), 26000 Kohat, Khyber\nPakhtunkhwa, Pakistan\n5Department of Psychiatry, Khyber Medical University Institute of Medical Sciences (KMU-IMS), 26000 Kohat, Khyber Pakhtunkhwa, Pakistan\n*Correspondence: shamimsaleha@yahoo.com (Shamim Saleha)\nAcademic Editor: Camil Castelo-Branco\nSubmitted: 15 October 2025 Revised: 20 November 2025 Accepted: 8 December 2025 Published: 11 February 2026\nAbstract\nBackground: Primary ovarian insufficiency (POI) is a heterogeneous disorder with multifactorial etiologies. Accurate diagnosis requires\nan integrated clinical, hormonal, and genetic evaluation, yet data from Pakistan are limited, and the burden of idiopathic and genetically\npredisposed cases remains largely unknown. Methods: A total of 345 women under 40 years presenting with amenorrhea or menstrual\nirregularities were screened. After excluding pregnancy, cases not meeting the European Society of Human Reproduction and Embry-\nology (ESHRE) diagnostic criteria, and incomplete records, 290 women were included. Comprehensive clinical, hormonal, and genetic\ninvestigations were performed according to ESHRE guidelines to determine underlying etiologies. Results: The mean age at presentation\nwas 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\nindex (BMI) was 24.5 ± 3.4 kg/m2. Most women presented with amenorrhea (80%) or oligomenorrhea (20%). Secondary infertility was\nreported in 72.8% and primary infertility in 2.4%. A history of miscarriage was documented in 5.9% of participants. Common clinical\nfeatures included hot flushes (75.9%), depression (72.4%), high stress (65.5%), mood changes (62.1%), vaginal dryness or dyspareunia\n(55.2%), and night sweats (54.5%). Coexisting comorbidities were observed in 12.4%, most frequently migraines (4.1%). Hormonal\nevaluation confirmed elevated follicle-stimulating hormone (FSH) levels ( >25 IU/L) and low estradiol ( <50 pg/mL) in all participants.\nEtiological classification identified iatrogenic causes in 7.2%, genetic causes in 3.8% (confirmed in women with suggestive genetic\nfeatures or isolated POI), autoimmune causes in 6.6%, and idiopathic POI in 82.4%. Statistically significant differences in confirmed\ndiagnoses were observed among most etiological groups ( p < 0.0001), except for women with features suggestive of a genetic cause ( p\n≈ 0.8500). Conclusions: POI presents with diverse clinical features. Evaluation based on ESHRE guidelines enables identification of\niatrogenic, autoimmune, and genetic contributors, and highlights the high prevalence of idiopathic cases, which may have an underlying\ngenetic predisposition.\nKeywords: primary ovarian insufficiency; idiopathic POI; genetic predisposition; autoimmune POI; iatrogenic causes; ESHRE guide-\nlines\n1. Introduction\nPrimary ovarian insufficiency (POI) is a heteroge-\nneous condition characterized by impaired ovarian func-\ntion occurring before the age of 40 years, affecting approxi-\nmately 1.1% of women of reproductive age [1]. POI is char-\nacterized by menstrual irregularities, amenorrhea, infertil-\nity, and hypoestrogenic symptoms such as hot flushes, night\nsweats, and vaginal dryness. It is further associated with a\nwide range of additional clinical features, including neu-\nropsychiatric, psychological, musculoskeletal, cardiovas-\ncular, and lifestyle-related factors [ 2–4]. POI often coex-\nists with thyroiditis, Addison’s disease or other autoimmune\ndisorders, sometimes accompanied by enlarged multifollic-\nular ovaries [ 2]. In addition, syndromic features of Turner\nsyndrome or blepharophimosis-ptosis-epicanthus inversus\nsyndrome (BPES) or fragile X syndrome and a family his-\ntory of early menopause or infertility may also be observed\nwith POI [ 5–7]. Beyond infertility, POI carries substan-\ntial health consequences, including increased risks of osteo-\nporosis, cardiovascular disease, metabolic disorders, and\ndiminished quality of life [8,9]. Associated symptoms, clin-\nical findings and comorbidities can be variable due to in-\ntermittent ovarian hormone secretion [ 2]. At presentation,\ndocumenting medical and family histories, along with key\nclinical features, is essential to help identify POI and its pos-\nsible underlying causes.\n\nAccording to the 2016 European Society of Human\nReproduction and Embryology (ESHRE) guidelines, POI\nshould be considered in women younger than 40 years\nwho present with oligo/amenorrhea or estrogen-deficiency\nsymptoms, with diagnosis confirmed by oligo/amenorrhea\nlasting at least four months, two elevated follicle-\nstimulating hormone (FSH) measurements ( >25 U/L), and\nlow estradiol levels (<50 pg/mL) obtained 4–6 weeks apart\nin the early follicular phase [ 10]. In contrast, the updated\n2024 ESHRE guidelines now indicate that a single ele-\nvated FSH measurement may suffice to establish the di-\nagnosis. The guidelines also recommend excluding preg-\nnancy and accounting for the effects of hormonal therapy,\nwhich can mask amenorrhea or alter FSH levels, before\nconfirming POI [ 11]. These recommendations outline fur-\nther testing to determine the cause of POI. Women with\na history of chemotherapy, radiotherapy, pelvic or ovar-\nian surgery, or bilateral salpingo-oophorectomy before age\n40 are considered to have iatrogenic POI and typically do\nnot require further evaluation. For all non-iatrogenic cases,\nfollowing genetic counseling and informed consent, chro-\nmosomal analysis and FMR1 premutation (PM) testing are\nrecommended, while broader genetic testing may be con-\nsidered where feasible. If no genetic cause is identified,\nscreening for 21-hydroxylase autoantibodies (21OH-Abs)\nand thyroid-stimulating hormone (TSH) levels is recom-\nmended to detect potential autoimmune etiologies. Cases\nwith no identifiable cause after these evaluations are classi-\nfied as idiopathic POI [ 10,11].\nDespite growing knowledge and international guide-\nlines, their implementation is limited in low- and middle-\nincome regions, and data on POI remain scarce. In Pakistan,\nonly a few studies have explored POI, focusing mainly on\npoor ovarian response, hormonal disturbances, and associ-\nated symptoms [12,13], without performing integrated clin-\nical, biochemical, or genetic assessments. Limited aware-\nness and resources have hindered guideline-based studies,\nleaving most cases unexplained. We hypothesized that\ncomprehensive clinical, hormonal, and genetic evaluation\naccording to ESHRE guidelines would enhance identifi-\ncation of underlying etiologies in POI, including potential\ngenetic contributions among idiopathic cases. Therefore,\nthis study aimed to comprehensively characterize the clini-\ncal, hormonal, and genetic features of POI among affected\nPakistani women and to classify its etiologies according to\nESHRE guidelines.\n2. Materials and Methods\n2.1 Study Design and Participants\nThis multicenter cross-sectional study was conducted\nbetween August 2023 and February 2025, and participat-\ning women were recruited from the obstetrics and gynecol-\nogy departments of tertiary care hospitals. Women under 40\nyears of age presenting with amenorrhea or oligomenorrhea\npersisting for at least four months and meeting the ESHRE\ndiagnostic criteria of FSH >25 IU/L and estradiol <50\npg/mL on two separate measurements taken more than four\nweeks apart, were included. Women who were pregnant,\ncurrently using hormonal therapy, diagnosed with polycys-\ntic ovary syndrome (PCOS), had malignant tumors, or had\nundergone ovarian removal were excluded (according to\n2016 and 2024 guidelines), as these conditions can con-\nfound menstrual irregularities or hormonal measurements\nrequired for POI evaluation [ 10,11]. A history of prior hor-\nmonal therapy was recorded, and participants were included\nonly if therapy had been discontinued for at least three\nmonths before recruitment. Written informed consent was\nobtained, and the study was approved by the Ethical Com-\nmittee of KUST (REF:/KUST/Ethical Committee/837).\n2.2 Clinical Evaluation\nDetailed medical and reproductive histories were\nobtained, including age at menarche, age at symp-\ntom onset, type of menstrual disturbance (amenor-\nrhea/oligomenorrhea), reproductive history, miscarriage\nhistory, and infertility. Body mass index (BMI) was mea-\nsured for all participants. Hypoestrogenic symptoms such\nas hot flushes, night sweats, vaginal dryness, and sys-\ntemic manifestations were recorded. Features suggestive\nof Turner syndrome, BPES, autoimmune disorders, or iatro-\ngenic causes (chemotherapy, radiotherapy, ovarian surgery)\nwere carefully recorded. Coexisting comorbidities were\ndocumented based on patient history, prior diagnoses, man-\nagement records, laboratory investigations, imaging, and\nreview of medical records as appropriate. Family history of\ninfertility or early menopause, as well as lifestyle and psy-\nchosocial factors, were also recorded. Depression and emo-\ntional instability were evaluated using the Patient Health\nQuestionnaire-9 (PHQ-9) [14].\n2.3 Diagnostic Evaluation\nFollowing ESHRE recommendations, menstrual dis-\nturbance was required to persist for at least four months\nto support a diagnosis of POI. For classification purposes,\namenorrhea was defined as the complete absence of men-\nstruation for ≥ six months, while oligomenorrhea was de-\nfined as menstrual cycles longer than 35 days or fewer than\nnine cycles per year. Hormonal testing (FSH and estra-\ndiol) was performed in all participants, in the early follicu-\nlar phase (days 2–5) for women with ongoing menstrual cy-\ncles, and on a random day for those presenting with amen-\norrhea. Thyroid function was assessed in all participants\nby measuring serum TSH levels, with a reference range of\n0.4–4.0 mU/L. After genetic counseling and obtaining in-\nformed consent, peripheral blood samples were collected\nin EDTA tubes, appropriately labeled, and transported un-\nder cold-chain conditions. Samples were stored at 4 °C\nand processed within 24–48 hours. Genomic DNA was ex-\ntracted using the standard phenol–chloroform protocol or\nthe QIAamp DNA Blood Mini Kit (Catalog No. 51306;\n2\n\n\nTable 1. Symptoms, clinical features, psychological factors, and lifestyle characteristics in women with POI (N = 290).\nCategory Specific symptom(s)/Findings Frequency (n, %)\nEstrogen deficiency\nHot flushes 220 (75.9%)\nNight sweats 158 (54.5%)\nV aginal dryness/dyspareunia 160 (55.2%)\nNeuropsychiatric Mood changes (mood swings, mental fog, melancholia) 180 (62.1%)\nSleep disturbances Insomnia/irregular sleep–wake cycle 115 (39.7%)\nSexual dysfunction Reduced libido, dyspareunia 108 (37.2%)\nFatigue Generalized fatigue 103 (35.5%)\nDermatological Hair loss, skin dryness 92 (31.7%)\nOphthalmological Dry eyes 88 (30.3%)\nEndocrine/Metabolic Cold intolerance 83 (28.6%)\nMusculoskeletal Joint clicking, muscle/joint pain 75 (25.9%)\nNeurological Headaches, vertigo, tingling limbs 72 (24.8%)\nCardiovascular Palpitations 65 (22.4%)\nPsychological\nDepression 210 (72.4%)\nHigh stress levels 190 (65.5%)\nAnxiety 120 (41.4%)\nSocial withdrawal 98 (33.8%)\nLifestyle Poor diet/nutrition 85 (29.3%)\nLow physical activity 65 (22.4%)\nNote: V alues represent frequency (n) and percentage (%) of participants reporting each manifestation.\nPOI, primary ovarian insufficiency.\nTable 2. Comorbidities observed in women with POI.\nComorbidity Frequency (n, %) Documentation/Diagnosis\nMigraine 12 (4.1%) Patient history and documented past diagnosis and management records\nHypertension 8 (2.8%) Systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg on two occasions, or on anti-\nhypertensive therapy\nDiabetes mellitus 5 (1.7%) Fasting plasma glucose ≥126 mg/dL, HbA1c ≥6.5%, or prior physician diagnosis\nObesity 4 (1.4%) BMI ≥30 kg/m2\nOsteoporosis 3 (1.0%) T-score ≤−2.5 measured by dual-energy X-ray absorptiometry (DEXA) scan\nBreast cancer 2 (0.7%) Histopathology confirmation and clinical oncology records\nIschemic heart 1 (0.3%) History of myocardial infarction or angina, confirmed by ECG or imaging\nGastric ulcers 1 (0.3%) Endoscopic confirmation with biopsy\nTotal 36 (12.4%)\nBMI, body mass index; BP , blood pressure; HbA1c, glycated hemoglobin; ECG, electrocardiogram.\nQiagen, Hilden, Germany), following recommended pro-\ncedures. DNA concentration and purity were measured us-\ning a NanoDrop Lite Plus spectrophotometer (Catalog No.\nNDLPLUSGL; Thermo Fisher Scientific, Waltham, MA,\nUSA) by assessing A260/A280 and A260/A230 ratios, and\nthe DNA was stored at –20 °C for subsequent use. Testing\nincluded karyotyping and FMR1 PM analysis [ 15], as well\nas direct sequencing of established POI-associated genes\nsuch as BMP15, NOBOX, and GDF9 [16] for all partic-\nipants. FOXL2 sequencing was performed selectively in\nwomen presenting with features suggestive of both POI and\nBPES, in whom no variants were identified in the other es-\ntablished POI-associated genes . Segregation analysis was\nperformed in available family members to assess inheri-\ntance patterns of identified variants. In silico prediction\ntools were used to assess the potential impact of identi-\nfied variants, and each variant was classified according to\nthe American College of Medical Genetics and Genomics\n(ACMG) guidelines [17]. Autoimmune evaluation (21OH-\nAbs) was carried out selectively in women without an iden-\ntified genetic or iatrogenic cause, and in those presenting\nwith symptoms suggestive of autoimmune involvement.\n2.4 Classification of Cases\nCases were categorized according to the underlying\ncause, in line with ESHRE 2024 guidelines: iatrogenic\nPOI (history of chemotherapy, radiotherapy, or ovarian\nsurgery), genetic POI (chromosomal abnormalities, FMR1\nPM alleles, or pathogenic/likely pathogenic variants), au-\ntoimmune POI (positive 21OH-Abs or abnormal TSH lev-\nels), and idiopathic POI (no identifiable cause after a com-\nprehensive diagnostic workup).\n3\n\n2.5 Data Analysis\nData was analyzed using IBM SPSS Statistics, V er-\nsion 30.0 (IBM Corp., Armonk, NY , USA). Continuous\nvariables are presented as mean ± SD (standard devia-\ntion) or median, and categorical variables as frequencies\nand percentages. Associations between clinical presenta-\ntion groups and diagnostic outcomes (confirmed vs. non-\nconfirmed) were assessed using two-tailed Chi-square or\nFisher’s exact tests, applied as appropriate. The null hy-\npothesis assumed no association between clinical presenta-\ntion and diagnosis. A p-value < 0.05 was considered statis-\ntically significant, indicating an association between clini-\ncal presentation and diagnostic outcome.\n3. Results\n3.1 Study Cohort\nA total of 345 women under 40 years presenting with\namenorrhea or menstrual irregularities and/or estrogen-\ndeficiency symptoms were initially recruited. Women who\nwere pregnant, currently using hormonal therapy, or had not\nprovided consent for the diagnostic workup were excluded.\nAfter these exclusions, 290 women were included in the fi-\nnal analysis. The mean age at presentation was 33 ± 4.5\nyears, with a median symptom duration of 6 months.\n3.2 Clinical Features\nMost women presented with amenorrhea (80%, n =\n232) and oligomenorrhea (20%, n = 58), with a mean du-\nration of amenorrhea of 6 ± 2 months. The mean age at\nsymptom onset was 31.6 ± 3.8 years, and the mean age at\nmenarche was 13 ± 1 years. The mean BMI of the partici-\npants was 24.5 ± 3.4 kg/m2. Reproductive history revealed\nsecondary infertility in 72.8% (n = 211) and primary infer-\ntility in 2.4% (n = 7). Miscarriage history was documented\nin 5.9% women (n = 17). A family history of infertility or\nearly menopause was documented in 9.3% (n = 27).\nWomen exhibited a wide spectrum of clinical features,\nincluding manifestations of estrogen deficiency, neuropsy-\nchiatric symptoms, and musculoskeletal, neurological, and\ncardiovascular complaints. The most prevalent features\nwere hot flushes (75.9%), depression (72.4%), high stress\nlevels (65.5%), mood changes (62.1%), vaginal dryness or\ndyspareunia (55.2%), and night sweats (54.5%) (Table 1).\nCoexisting comorbidities were observed in 12.4% of cases,\nwith migraine being the most common (4.1%) (Table 2).\n3.3 Etiological Clues\nClinical evaluation revealed iatrogenic factors (pre-\nvious ovarian surgery, chemotherapy, or radiotherapy) in\n7.2% (n = 21), features suggestive of a genetic etiology in\n16.6% (n = 48), and coexisting autoimmune disorders in\n14.5% (n = 42) (Table 3).\n3.4 Hormonal and Genetic Findings\nAll included women had elevated FSH ( >25 IU/L)\nconfirmed on two occasions, with a mean FSH of 30 ±\n12 IU/L and mean estradiol of 25 ± 10 pg/mL. Among\nthe 290 women with POI, 21 had iatrogenic causes due\nto ovarian surgery, chemotherapy, or radiotherapy, all of\nwhom had confirmed diagnoses (100%). Among women\nwithout iatrogenic causes, pathogenic genetic variants were\ndetected in two groups. In 48 women with Turner syn-\ndrome stigmata, BPES, a family history of early menopause\nor infertility, or early age at onset, 8 had confirmed diag-\nnoses, including karyotypic abnormalities consistent with\nTurner syndrome (2 cases), FMR1 PM alleles with 50–65\nCGG repeats (5 cases), and a heterozygous FOXL2 vari-\nant [c.223C >T, p.(Leu75Phe)] identified in one woman\nwith both POI and BPES. The same FOXL2 variant was\npresent in her father and brother, who exhibited BPES fea-\ntures. Based on ACMG criteria (PM1, PM2, PP1, PP3),\nthe variant is classified as a variant of uncertain signifi-\ncance (VUS). Among 179 women with POI features only\n3 rare genetic variants were detected, including FMR1 PM\nalleles with 50, 57 CGG repeats in 2 cases and a homozy-\ngous GDF9 variant [c.604C>T, p.(Gln202*)] identified in\none woman with POI. This GDF9 variant segregated in an\nautosomal recessive manner, with both parents being het-\nerozygous carriers. Based on ACMG criteria (PVS1, PM3,\nPP3), the variant is classified as likely pathogenic. Au-\ntoimmune evaluation confirmed the diagnosis in 19 women\nwith autoimmune disease, while no confirmed cases were\nobserved in women with features suggestive of a genetic\netiology or POI features only. Statistically significant dif-\nferences in the proportion of confirmed diagnoses were ob-\nserved among most clinical presentation groups, except for\nwomen with features suggestive of a genetic etiology ( p ≈\n0.8500). Overall, the proportion of confirmed diagnoses\ndiffered significantly across the underlying etiological cat-\negories, with highly significant associations observed for\niatrogenic (p < 0.0001), autoimmune (p < 0.0001), and id-\niopathic/other genetic cases ( p < 0.0001) (Table 3).\n3.5 Etiological Classification\nConfirmed causes of POI were identified in a subset of\nwomen: iatrogenic 7.2%, genetic 3.8%, and autoimmune\n6.6%. The remaining women without a definitive cause,\nor with suggestive genetic or autoimmune features, were\nclassified as idiopathic POI, accounting for 82.4% of the\ncohort (Table 4).\n4. Discussion\nPOI is a heterogeneous reproductive disorder, char-\nacterized by diverse etiologies and a wide range of clini-\ncal manifestations [ 18], yet in many cases the underlying\ncause remains unidentified despite advances in understand-\ning. Studies from high-income countries have clarified the\ngenetic, autoimmune, and iatrogenic factors associated with\n4\n\n\nTable 3. Underlying causes of POI in 290 women, determined by clinical features and diagnostic investigations.\nClinical presentation (n, %) Investigations confirmed diagnosis Confirmed diagnosis\n(n, %) (N = 51)\nNon-confirmed diagnosis\n(n, %) (N = 239)\nStatistical test (p-value) Underlying etiology of POI\nHistory of ovarian surgery,\nchemotherapy, or radiotherapy\n(21, 7.2%)\nConfirmation from medi-\ncal/surgical history and records\n21 (100.0%) 0 (0.0%) <0.0001* Iatrogenic\nTurner syndrome stigmata, family his-\ntory of infertility/early menopause,\nblepharophimosis, early age at onset\n(48, 16.6%)\nGenetic testing: FMR1 PM analy-\nsis, FOXL2, BMP15, NOBOX, and\nGDF9 genes sequencing\n8 (16.7%) 40 (83.3%) ≈0.8500 Genetic (Turner syndrome,\nFMR1 PM alleles, one FOXL2\nvariant)\nCoexisting autoimmune disorders:\nthyroid dysfunction, systemic lupus\nerythematosus, rheumatoid arthritis,\nautoimmune haemolytic anaemia, vi-\ntiligo, Addison’s disease (42, 14.5%)\nAutoimmune serological testing for\n21OH-Abs and serum TSH levels\n19 (45.2%) 23 (54.8%) <0.0001* Autoimmune (Positive 21OH-\nAbs and/or abnormal TSH levels)\nPOI features without additional con-\nditions (179, 61.7%)\nGenetic and autoimmune serologi-\ncal testing\n3 (1.7%) 176 (98.3%) <0.0001* Idiopathic (no definitive cause\nidentified)/Genetic ( FMR1 PM\nalleles in 2 cases, a GDF9 variant\nin 1 case)\nNote: 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\np-value (p < 0.05). Bold = significant values and underlying POI etiology. PM, premutation; 21OH-Abs, 21-hydroxylase autoantibodies; TSH, thyroid-stimulating hormone.\n5\n\nTable 4. Distribution of underlying causes of POI among women (N = 290).\nUnderlying etiology Number of cases (n) Percentage (%)\nIatrogenic (surgery, chemotherapy, radiotherapy) 21 7.2\nGenetic (Turner syndrome, FMR1 PM alleles, one FOXL2 variant, one GDF9 variant) 11 3.8\nAutoimmune (thyroid dysfunction, lupus, rheumatoid arthritis, Addison’s disease, etc.) 19 6.6\nIdiopathic (no definitive cause identified) 239 82.4\nTotal 290 100.0\nNote: V alues represent frequency (n) and percentage (%) of participants in each etiological category.\nPOI [ 7,19,20], but large gaps remain in low- and middle-\nincome regions. In Pakistan, only a single study by Izhar\net al . [ 12] has explored POI, focusing on poor ovarian\nresponse criteria to detect occult POI in women with in-\nfertility and oligomenorrhea. More recently, Kazi et al .\n[13] assessed the diagnostic and management challenges\nof POI in women attending a tertiary hospital in Lahore,\nhighlighting the association of infertility, hot flashes, mood\nswings, vaginal dryness, and insomnia with disturbed hor-\nmonal and ovarian reserve markers. However, these studies\nrelied mainly on clinical symptoms and surrogate biochemi-\ncal markers, without integrating broader clinical, hormonal,\nand genetic investigations. To our knowledge, the present\nstudy is the first in Pakistan to comprehensively evaluate\nPOI by integrating detailed clinical assessment, hormonal\nprofiling, and genetic testing according to ESHRE diagnos-\ntic criteria.\nConsistent with ESHRE recommendations, all women\nof reproductive age (under 40 years) in this study fulfilled\nthe diagnostic criteria for POI, presenting with at least four\nmonths of oligo/amenorrhea, repeated serum FSH levels\n>25 IU/L, and low estradiol levels of approximately 50\npg/mL. Although POI can occur across a broad age range,\nfrom early adolescence to 40 years [ 21], the age of onset\nand age at diagnosis remain key factors for timely recogni-\ntion and management. In the current cohort, the mean age\nat symptom onset was 31 years, and the mean age at clinical\npresentation was 33 years, indicating an approximate delay\nof 24 months. This delay is shorter than the 48-month me-\ndian time to diagnosis reported by Minis et al . [ 22], but\ncomparable to findings from Sun et al. [ 23], who reported\nan average interval of approximately 28 months between\nsymptom onset and POI diagnosis, and to Bakhsh [24], who\nobserved diagnostic delays exceeding 18 months in a sub-\nstantial proportion of patients. Such delays are often related\nto variability and inconsistency in presenting features, as\nhighlighted by Kapoor [ 25]. In Pakistan, additional factors\nsuch as limited awareness of POI, sociocultural barriers af-\nfecting care-seeking, and variability in access to specialized\ndiagnostic services may further contribute to delayed diag-\nnosis.\nThe clinical spectrum observed was broad, with va-\nsomotor symptoms such as hot flushes, vaginal dryness or\ndyspareunia, and night sweats, alongside mood changes and\npsychological disturbances including depression and stress,\nbeing highly prevalent. Comparable clinical presentations\nhave been observed in studies of other populations, where\nvasomotor and neuropsychiatric symptoms dominate the\nclinical profile [ 2–4]. The high prevalence of depression\n(72.4%) and stress (65.5%) in our cohort is consistent with\nprevious reports of psychological disturbances in women\nwith POI [26], highlighting the substantial psychosocial im-\npact of POI, which may be further aggravated by cultural\nstigma surrounding infertility in South Asian societies.\nThe association between POI and long-term comor-\nbidities, such as osteoporosis, metabolic syndrome, and car-\ndiovascular disease, is well established [ 8,9]. Comorbidi-\nties of POI were documented in 12.4% of cases, with mi-\ngraine being the most common (4.1%). Although the preva-\nlence of metabolic and cardiovascular comorbidities in our\ncohort was lower than reported in other studies [ 27,28],\nthis is not solely explained by age, as participants in these\nstudies were also predominantly below 40 years. In con-\ntrast, women aged ≥45 years with a prior diagnosis of POI\nhave been reported to exhibit a higher prevalence of car-\ndiometabolic comorbidities [29,30], highlighting the cumu-\nlative risk associated with longer duration of estrogen defi-\nciency. These findings emphasize that age and duration of\nPOI are important factors influencing the manifestation of\nlong-term systemic comorbidities.\nThe etiological distribution of POI in our cohort il-\nlustrates its complexity. Iatrogenic causes accounted for\n7.2% of cases, consistent with a study reporting 6–47%\n[31], and aligning with another study reporting a ~8% cu-\nmulative risk of POI by age 40 in female cancer survivors\n[32]. The highest risk was observed after alkylating agents\nand ovarian radiotherapy [ 31], influenced by age at ex-\nposure, treatment dose, and baseline ovarian reserve [ 2].\nIn our cohort, the difference in confirmed iatrogenic diag-\nnoses was highly significant (p < 0.0001), highlighting that\nthis subgroup represents a clearly distinguishable and reli-\nably diagnosed etiology. Genetic causes were confirmed\nin 3.8%, collectively from women with features sugges-\ntive of a genetic etiology (16.6%) and those presenting with\nPOI features only (61.7%), which is markedly lower than\nthe 20–25% reported in a previous study [ 6]. FMR1 PM\nalleles were detected in seven cases, Turner syndrome in\ntwo, one FOXL2 variant [c.223C >T, p.(Leu75Phe)] pre-\nviously reported in POI with BPES [ 5] in one case, and\na GDF9 variant [c.604C >T, p.(Gln202*)] previously re-\n6\n\n\nported in POI [ 7] in sisters. The frequency of confirmed\ncases was not statistically significant among women with\nsuggestive genetic features ( p ≈ 0.8500), whereas the dif-\nference was highly significant among those with POI fea-\ntures only ( p < 0.0001), indicating a substantial burden of\nundiagnosed genetic predisposition in the idiopathic group\nand emphasizing the limitations of conventional diagnos-\ntic approaches. This underscores the importance of ad-\nvanced genetic testing, particularly whole-exome sequenc-\ning (WES), in women with idiopathic POI or subtle clini-\ncal indicators such as Turner stigmata, BPES, family his-\ntory of infertility/early menopause, or early disease onset.\nScreening for 21OH-Abs and TSH confirmed an associa-\ntion of POI with autoimmune disease in 19 women (6.6%),\nwith no cases detected in women with features suggestive\nof a genetic etiology or POI features only. This difference\nwas highly significant (p < 0.0001), indicating that autoim-\nmune testing effectively identified women with POI asso-\nciated with autoimmune disease and highlighting its utility\nin detecting cases with an underlying autoimmune etiology.\nHowever, a causal relationship could not be established, as\nautoimmune disorders are common and autoantibody pos-\nitivity may not always reflect a direct cause of ovarian in-\nsufficiency [2].\nLimitations\nDespite providing a comprehensive evaluation of POI\nin women across multiple centers in Pakistan, this study has\nseveral limitations. First, the cohort included only Pakistani\nwomen, which may limit generalizability to other popula-\ntions. Second, although our study aimed to identify under-\nlying etiologies and potential undetected genetic predispo-\nsitions in POI using ESHRE-guided evaluation, no control\ngroup was included, limiting comparative analyses. Third,\nin Pakistan, genetic testing is generally not performed for\nPOI cases, and due to high costs, our study focused on a\nlimited gene panel, which may underestimate rare or novel\nvariants and highlights the need for broader genetic evalu-\nation. Fourth, autoimmune assessment relied on specific\nserological markers, which may not capture all relevant\nmechanisms. Finally, the cross-sectional design precludes\nassessment of long-term outcomes, including the progres-\nsion of comorbidities and reproductive or metabolic conse-\nquences. These limitations underscore the need for larger,\nmulti-center, longitudinal studies with broader genetic and\nimmunological testing to fully characterize POI.\n5. Conclusions\nIn conclusion, this study provides the first comprehen-\nsive evaluation of POI in Pakistan, integrating clinical, hor-\nmonal, and genetic assessments in line with ESHRE guide-\nlines. The findings highlight delayed diagnosis, a high psy-\nchological burden, and underrecognized comorbidities in\naffected women. Iatrogenic causes were clearly identifi-\nable and statistically significant, while genetic causes were\nconfirmed in a small proportion, with many women show-\ning subtle features or idiopathic presentation, indicating a\nsubstantial burden of undiagnosed genetic predisposition.\nAutoimmune contributors were confirmed in a subset of\nwomen, with testing proving effective in identifying true\ncases. Overall, the high proportion of idiopathic cases un-\nderscores the need for broader genetic evaluation, including\nWES, and earlier diagnostic approaches to improve recog-\nnition and management of POI, particularly in resource-\nlimited settings.\nAvailability of Data and Materials\nAll data reported in this study are included within the\nmanuscript.\nAuthor Contributions\nSS contributed to the conceptualization and study de-\nsign, genetic investigations, data validation and interpre-\ntation, manuscript drafting, and final approval. HT per-\nformed data collection, analysis, and laboratory work. UNT\nand MJ were responsible for participant recruitment and\nclinical evaluation, including investigations and interpreta-\ntion. ZZ contributed to the clinical evaluation and interpre-\ntation of participants presenting with acute or overlapping\nsymptoms such as palpitations, dizziness, and headaches.\nSAK performed psychiatric and psychological evaluations\nand their interpretation. MY contributed to data collection,\nanalysis, laboratory work, and oversight of commercial hor-\nmonal and genetic testing. All authors contributed to criti-\ncal revision of the manuscript for important intellectual con-\ntent. All authors read and approved the final manuscript.\nAll authors have participated sufficiently in the work and\nagreed to be accountable for all aspects of the work.\nEthics Approval and Consent to Participate\nThe study was conducted in accordance with the Dec-\nlaration of Helsinki and was approved by the Ethical Com-\nmittee of Kohat University of Science and Technology\n(KUST), Kohat, Pakistan (REF:/KUST/Ethical Commit-\ntee/837). Written informed consent was obtained from all\nparticipants prior to inclusion in the study.\nAcknowledgment\nWe sincerely acknowledge the contributions of the\nparticipating women and their families. We also thank the\nstaff at the gynecology and infertility clinics for their sup-\nport in recruitment and sample collection.\nFunding\nThis research received no external funding.\nConflict of Interest\nThe authors declare no conflict of interest.\n7\n\nDeclaration of AI and AI-Assisted\nTechnologies in the Writing Process\nDuring the preparation of this work, the authors used\nChatGPT-3.5 to check spelling and grammar. After using\nthis tool, the authors reviewed and edited all content as\nneeded and take full responsibility for the content of the\npublication.\nReferences\n[1] Luborsky JL, Meyer P , Sowers MF, Gold EB, Santoro N. Pre-\nmature menopause in a multi-ethnic population study of the\nmenopause transition. Human Reproduction. 2003; 18: 199–\n206. https://doi.org/10.1093/humrep/deg005.\n[2] Federici S, Rossetti R, Moleri S, Munari EV , Frixou M, Bonomi\nM, et al . Primary ovarian insufficiency: update on clinical\nand genetic findings. 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