Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of women in the reproductive years. It combines hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology in varying proportions, and its consequences reach well beyond the ovary into metabolic, cardiovascular and psychological health (1,2). In Pakistan, the condition is seen frequently in gynaecology and infertility clinics, yet adherence to prescribed treatment is often poor, with limited awareness, cost and side effects acting as practical barriers (3).

Insulin resistance and excess androgen sit at the centre of the reproductive problem in many of these women. Hyperinsulinaemia amplifies ovarian androgen production, arrests follicular development and keeps cycles anovulatory (4). The 2023 International Evidence-based Guideline therefore places lifestyle measures first and recommends letrozole as the first-line pharmacological agent for ovulation induction in women with PCOS and anovulatory infertility (5).

This recommendation rests on a solid evidence base. A meta-analysis of 29 randomised trials involving nearly 4,000 women showed that letrozole outperformed clomiphene citrate for ovulation, clinical pregnancy and live birth (6), and an earlier pooled analysis of 26 trials reached a similar conclusion for ovulation rate (7). Narrative reviews of fertility management in PCOS have also moved letrozole to the front of the treatment ladder, ahead of gonadotropins and laparoscopic ovarian drilling (8,9).

Even so, a substantial share of women do not ovulate on letrozole, and the reasons are not fully understood. PCOS is a heterogeneous disorder in which genetic, metabolic and environmental factors interact differently from one patient to another (10). Retrospective work from China found that late menarche, higher anti-Müllerian hormone (AMH), a raised LH/FSH ratio and a higher free androgen index predicted failure to respond to 2.5 mg letrozole (11). A prospective cohort from North India likewise reported that AMH and androgen markers helped to distinguish responders from non-responders (12).

Most of these studies were small, came from single centres and measured only part of the clinical picture. Few examined anthropometric, hormonal, metabolic, endometrial and treatment-related variables together, and data from Pakistani women are scarce. The present study was therefore conducted to identify the factors associated with successful ovulation after letrozole in women with PCOS, and to describe dominant follicle development and clinical pregnancy as secondary outcomes.

This cross-sectional study was conducted in the Department of Obstetrics and Gynaecology at the Tertiary care hospitals of Khyber Pakhtunkhwa. Informed consent was obtained from all participants, and patient identity was kept confidential throughout. A total of 200 women with PCOS who received letrozole for ovulation induction were included by non-probability consecutive sampling. PCOS was diagnosed according to the Rotterdam criteria, and women were classified into phenotypes A to D. Women with other causes of infertility, such as tubal or male factor, hyper-prolactinemia, uncorrected thyroid disease or ovarian cysts at baseline, were excluded. Data collected for each woman included age, body mass index (BMI), PCOS phenotype, serum AMH, LH/FSH ratio, fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR), total testosterone, thyroid-stimulating hormone (TSH), 25-hydroxyvitamin D, endometrial thickness, the number of previous failed ovulation-induction cycles, letrozole dose (2.5, 5.0 or 7.5 mg daily) and concurrent metformin use. Follicular growth was monitored by transvaginal ultrasonography. The primary outcome was successful ovulation, recorded as yes or no. Secondary outcomes were the number of follicles reaching ≥18 mm and clinical pregnancy, defined as an intrauterine gestational sac on ultrasound. Because follicle growth and pregnancy follow treatment, they were not treated as predictors of ovulation.

The dataset was complete, with no missing values. Data were analysed using SciPy (Python). Continuous variables were expressed as mean ± standard deviation, median and range, and categorical variables as frequencies and percentages. Proportions were reported with Wilson 95% confidence intervals (CI). Continuous variables were compared between ovulators and non-ovulators using the independent-samples t-test with Welch's correction for unequal variances, and the mean difference with its 95% CI and Cohen's d were calculated as measures of effect size. Categorical variables were compared using Pearson's chi-square test, with Yates' continuity correction for 2×2 tables and Fisher's exact test where expected cell counts were small. The strength of association was expressed as Cramér's V, and odds ratios (OR) with 95% CI were calculated where no cell contained zero. Multivariable binary logistic regression was attempted, but complete and quasi-complete separation produced non-convergent estimates, so adjusted odds ratios were not reported. A two-sided p-value <0.05 was considered statistically significant.

Of the 200 women analysed, 128 ovulated after letrozole, giving an ovulation rate of 64.0% (95% CI 57.1–70.3), while 72 (36.0%) did not. Table 1 summarises the baseline profile of the whole cohort. The mean age was 30.4 years and the mean BMI 28.8 kg/m², so the typical participant was overweight. Mean HOMA-IR was 3.47, mean AMH 8.13 ng/mL and mean vitamin D 22.5 ng/mL, the last value falling in the insufficient range.

Table 1: Baseline characteristics of the study population (n=200)

Table 2 compares ovulators with non-ovulators. Every continuous variable differed significantly between the groups (p<0.001). Women who ovulated were on average 6.5 years younger and 7.0 kg/m² lighter in BMI. Their AMH was lower by 5.7 ng/mL, their HOMA-IR was roughly half that of non-ovulators, and total testosterone was about 29 ng/dL lower.

 In the opposite direction, ovulators had higher vitamin D (by 12.6 ng/mL), a thicker endometrium (by 2.1 mm) and a slightly higher TSH. The effect sizes were very large throughout, with Cohen's d ranging from 2.80 to 3.73 in absolute value.

Table 2: Comparison of continuous variables between women with and without ovulation

Values are mean ± SD. Welch's independent-samples t-test. Mean difference = ovulation minus no ovulation.

The categorical analysis is shown in Table 3. PCOS phenotype had a strong association with ovulation (χ²=126.84, df=3, p<0.001; Cramér's V=0.80). All 95 women classified as phenotype A ovulated, compared with 60.6% of phenotype C, 31.0% of phenotype B and none of phenotype D. Between the two phenotypes with responses in both directions, phenotype B carried lower odds of ovulation than phenotype C (OR 0.29; 95% CI 0.11–0.76; Fisher's p=0.018). Previous ovulation failure showed the strongest association of any variable (χ²=159.08, df=4, p<0.001; V=0.89). Every woman with no prior failure ovulated, 85.7% ovulated after one failure, and none ovulated after two or more failures; the difference between zero and one prior failure was itself significant (Fisher's p=0.001). Ovulation fell across dose groups, from 84.2% with 2.5 mg to 68.1% with 5.0 mg and 0% with 7.5 mg (χ²=67.49, df=2, p<0.001; V=0.58). Women on 5.0 mg had lower odds of ovulation than those on 2.5 mg (OR 0.40; 95% CI 0.19–0.85). Metformin users also ovulated less often than non-users (56.6% vs 73.6%; χ²=5.40, p=0.020; OR 0.47; 95% CI 0.26–0.86).

Table 3: Association of PCOS phenotype and treatment-related factors with ovulation

Pearson's chi-square test (Yates-corrected for metformin). V = Cramér's V. *Phenotype B vs C, Fisher's exact p=0.018. OR not estimable for categories with a zero cell.

Secondary outcomes are presented in Table 4. A single follicle ≥18 mm developed in 34 women (17.0%) and two follicles in 94 (47.0%), while 72 (36.0%) had none. Every woman with at least one mature follicle ovulated. Clinical pregnancy was confirmed in 71 women, a rate of 35.5% (95% CI 29.2–42.3) for the whole cohort and 55.5% (95% CI 46.8–63.8) among those who ovulated.

Table 4: Secondary reproductive outcomes

95% CI calculated by the Wilson score method.

In this study, close to two-thirds of women with PCOS ovulated after letrozole. A prospective cohort from North India, using 5 mg letrozole for up to three cycles, reported almost the same figure, with 63.75% of women classed as responders (13). Response rates elsewhere vary with dose schedules; in a South Indian cohort receiving sequential dose escalation, ovulation rose from 33.3% at 2.5 mg to 78.9% at 7.5 mg (14). Our rate therefore sits comfortably within the published range.

The most striking finding was the near-total separation of outcome by phenotype. All phenotype A women ovulated and no phenotype D woman did. This runs against the usual expectation, because phenotype A carries the full burden of hyperandrogenism, anovulation and polycystic morphology, whereas phenotype D is the mildest, non-hyperandrogenic form. In the South Indian series, ovulation improved with escalating doses across phenotypes rather than being confined to one group (14). Women from South Asia also carry a higher background risk of insulin resistance than other ethnic groups (15), which may modify phenotype behaviour. We therefore believe that the phenotype coding in our records should be rechecked against the Rotterdam definitions before this result is given clinical weight.

Age, adiposity and insulin resistance separated the groups clearly. Grade II obesity independently reduced the likelihood of response to letrozole in the North Indian cohort (13), and a 2026 analysis linked pre-pregnancy insulin resistance with poorer fertility outcomes in PCOS (16). Overweight and obese women undergoing ovarian stimulation have also been shown to need more gonadotropin and longer stimulation (17). Excess insulin drives theca-cell androgen output and suppresses sex hormone-binding globulin, so the higher testosterone among our non-ovulators fits the same mechanism.

AMH was almost twice as high in non-ovulators. This agrees with Guo et al., who found that rising AMH and

LH/FSH ratio predicted non-response to 2.5 mg letrozole (11). A large American series showed that the probability of follicular response with letrozole fell steadily as AMH increased (18), and an Austrian prospective cohort found AMH of 9.7 versus 5.0 ng/mL in non-responders and responders (19). High AMH probably reflects a large pool of arrested follicles that resist FSH stimulation. Elevated LH, however, did not harm outcomes in a retrospective study of 835 letrozole cycles (20), and genetic variation in the FSH receptor may also shape ovarian sensitivity (21), so these markers are best read together rather than alone.

Vitamin D was considerably lower among non-ovulators. The Austrian cohort, in contrast, found no difference in 25-hydroxyvitamin D between women with and without follicular response (19). Supplementation improved ovulation in a small group of vitamin D-deficient letrozole failures (22), and a multicentre randomised trial is now testing vitamin D added to letrozole (23). Until that trial reports, low vitamin D is better regarded as a marker of overall metabolic health than as a proven cause of non-response.TSH was modestly higher among ovulators, although values in both groups stayed within the normal range. This is the reverse of a recent Chinese study, in which responders to clomiphene had lower baseline TSH and TSH predicted response together with T3 and T4 (24). Secondary analysis of two large American trials found that live birth fell when TSH was ≥2.0 mU/L in women who were also positive for thyroid peroxidase antibodies (25). We did not measure antibodies, so this small difference should not be over-interpreted.

The 7.5 mg group had no ovulations, and the 5.0 mg group did worse than the 2.5 mg group. This should not be read as harm from higher doses. In routine practice, the dose is raised after failure at a lower dose (14), so women on higher doses are, by selection, the more resistant cases. The same confounding by indication probably explains the lower ovulation rate with metformin, which is usually added for obese or insulin-resistant women. Cochrane evidence suggests that metformin can improve ovulation compared with placebo but that its benefit depends on BMI (26), and a meta-analysis of non-obese women reported improved pregnancy outcomes with metformin (27).

Women with two or more previous failures did not ovulate at all. This group resembles the letrozole- or clomiphene-resistant population for whom second-line options are needed. Combined ovulation-induction regimens and laparoscopic ovarian drilling have both been studied in clomiphene-resistant PCOS (28), and a recent randomised trial compared drilling with step-up gonadotropins in women resistant to letrozole (29). Gonadotropins remain an effective second-line choice (30), and earlier referral of such women may save time and cost.

This study has limitations. It was conducted at a single centre, and follow-up was limited to clinical pregnancy without live birth. Several variables completely separated the outcome groups, so multivariable logistic regression did not converge and independent effects could not be estimated. Penalised methods, such as Firth regression, should be applied in a larger prospective sample with a prespecified set of predictors.

About two-thirds of women with PCOS ovulated with letrozole. Younger age, lower BMI, lower AMH, milder insulin resistance and hyperandrogenism, higher vitamin D, a thicker endometrium and no previous ovulation failure characterised responders. Women with two or more failed cycles did not respond and may benefit from early second-line treatment. These associations should be confirmed in a larger prospective study using multivariable modelling

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