Correlation between serum AMH, AFC, AND histologically quantified primordial follicles: a prospective study in premenopausal women.

In this prospectively designed study, a total of 89 premenopausal women aged 35–48 years who underwent elective oophorectomy for benign gynecological indications were included in the final analysis. The mean age of the cohort was 44.9 ± 4.1 years. The descriptive statistics for key demographic, hormonal, and ultrasonographic variables are presented in Table  1 . The median serum AMH level was 0.30 ng/mL, while the histologically determined median number of primordial follicles was 7. The AFC showed a median value of 4.0, with a range of 1 to 8 follicles. Table 1 Demographic, hormonal, and follicular characteristics of the study population Variable ( n  = 89) Minimum Maximum Mean ± SD Median Age (years) 35.00 48.00 44.93 ± 4.05 47.00 AMH (ng/mL) 0.03 1.31 0.43 ± 0.46 0.30 Primordial Follicle Count 0.00 62.00 14.38 ± 18.15 7.00 FSH (mIU/mL) 6.00 22.00 14.66 ± 4.65 16.00 Estradiol (E2, pg/mL) 15.00 223.00 93.27 ± 49.02 92.00 Antral Follicle Count (AFC) 1.00 8.00 3.97 ± 1.96 4.00 Data are presented as mean ± standard deviation (SD) for normally distributed variables and as median (minimum–maximum) for non-normally distributed variables. Normality was assessed using the Shapiro–Wilk test Demographic, hormonal, and follicular characteristics of the study population Data are presented as mean ± standard deviation (SD) for normally distributed variables and as median (minimum–maximum) for non-normally distributed variables. Normality was assessed using the Shapiro–Wilk test A Spearman correlation analysis was conducted to examine the associations between serum AMH levels, AFC, and histologically assessed primordial follicle counts. A strong and statistically significant positive correlation was identified between serum AMH concentrations and the number of primordial follicles (ρ = 0.75, p  < 0.001), suggesting that higher AMH levels are closely reflective of a greater reserve of histologically confirmed primordial follicles (Fig.  1 ). This finding underscores the potential of AMH as a reliable endocrine marker of ovarian reserve. Fig. 1 Correlation between serum AMH levels and histologically assessed primordial follicle count Correlation between serum AMH levels and histologically assessed primordial follicle count In addition, AMH levels demonstrated a very strong positive correlation with AFC (ρ = 0.93), indicating a high degree of concordance between hormonal and ultrasonographic markers of follicular reserve (Fig.  2 ). This alignment reinforces the clinical utility of AMH and AFC as surrogate tools for evaluating ovarian function in women approaching the perimenopausal period. Fig. 2 Correlation between serum AMH levels and antral follicle count (AFC) Correlation between serum AMH levels and antral follicle count (AFC) Serum AMH also exhibited strong inverse correlations with FSH levels (ρ = − 0.89, p  < 0.001) and moderate inverse correlations with E2 levels (ρ = − 0.62) (Figs.  3 and 4 ). These inverse relationships are consistent with the established hormonal feedback dynamics of the hypothalamic–pituitary–gonadal axis, where declining ovarian reserve leads to elevated FSH and altered estradiol production. Fig. 3 Correlation between serum AMH levels and serum FSH levels Correlation between serum AMH levels and serum FSH levels Fig. 4 Correlation between serum AMH levels and E2 levels Correlation between serum AMH levels and E2 levels Further analyses revealed that AFC was moderately negatively correlated with FSH levels (ρ = − 0.67) and weakly negatively correlated with E2 levels (ρ = − 0.49). These results suggest that AFC, while closely associated with AMH, may also reflect broader hormonal shifts associated with diminishing ovarian function. An age-related decline in ovarian reserve was also evident. Age was negatively correlated with both AMH (ρ = − 0.19) and primordial follicle count (ρ = − 0.39), indicating the expected trajectory of reproductive aging in the study population. Additionally, a moderate negative correlation was observed between FSH and E2 levels (ρ = − 0.65), further supporting the interplay between gonadotropins and ovarian steroidogenesis. All findings from the correlation analyses are summarized in Table  2 , and corresponding scatter plots with regression trendlines are provided in Figs.  1 , 2 , 3 and 4 . Taken together, these results provide robust evidence that serum AMH levels and AFC are strongly predictive of histologically assessed primordial follicle counts, reinforcing their role as clinically meaningful indicators of ovarian reserve. Table 2 Relationships between age, hormonal biomarkers, follicular imaging parameters, and histological follicle counts Variables ( n  = 89) Age AMH Primordial Follicle Count FSH E2 Age 1.00 –0.19 –0.39 0.49 –0.00 AMH –0.19 1.00 0.75 –0.90 –0.62 Primordial Follicle Count –0.39 0.75 1.00 –0.74 –0.44 FSH 0.49 –0.90 –0.74 1.00 0.37 E2 –0.00 –0.62 –0.44 0.37 1.00 Associations were evaluated using Spearman’s rank correlation coefficients. Bold values indicate statistically significant correlations ( p  < 0.05) Relationships between age, hormonal biomarkers, follicular imaging parameters, and histological follicle counts Associations were evaluated using Spearman’s rank correlation coefficients. Bold values indicate statistically significant correlations ( p  < 0.05) Exploratory multivariable linear regression analysis adjusting for age, BMI, and smoking history confirmed that both AMH (β = 0.46, p  < 0.001) and AFC (β = 0.51, p  < 0.001) were independent predictors of primordial follicle count. These findings are summarized in Table  3 . Table 3 Multivariable linear regression analysis of predictors of primordial follicle count Variable ( n  = 89) β coefficient 95% CI p -value AMH (ng/mL) 0.46 0.32–0.60 < 0.001 AFC 0.51 0.37–0.64 < 0.001 Age (years) –0.08 –0.21–0.05 0.21 BMI (kg/m²) –0.05 –0.18–0.07 0.40 Smoking (Yes vs. No) –0.07 –0.19–0.09 0.31 β: standardized regression coefficient; CI: confidence interval; BMI: body mass index Multivariable linear regression analysis of predictors of primordial follicle count β: standardized regression coefficient; CI: confidence interval; BMI: body mass index AMH and AFC were both positively associated with primordial follicle count, while negative correlations were observed with FSH and E2 levels. Age demonstrated a weak negative correlation with AMH and primordial follicle numbers. All findings are consistent with established markers of ovarian reserve dynamics. Age-stratified analysis demonstrated progressive declines in AMH, AFC, and primordial follicle counts across increasing age groups (35–40, 41–45, 46–48 years). These findings are shown in Supplementary Fig. 1, with the number of observations indicated above each group.

This was a prospective, cross-sectional, observational study conducted at Kayseri City Hospital, a tertiary referral center located in central Turkey. The study protocol received ethical approval from the Non-Interventional Clinical Research of the University of Health Sciences Kayseri City Hospital (Approval No: 354, dated 11 March 2025). All participants provided written informed consent prior to inclusion, and the study was conducted in accordance with the principles outlined in the Declaration of Helsinki and Good Clinical Practice guidelines. The sample size was calculated using G*Power 3.1 software. Based on prior data reporting a moderate correlation ( r  = 0.30–0.50) between serum AMH levels and histologically determined primordial follicle counts, a minimum sample of 84 participants was required to detect a correlation coefficient of r  = 0.35 with 80% power at a two-tailed significance level of α = 0.05. To accommodate potential exclusions and missing data, a total of 89 participants were recruited. Eligible participants were premenopausal women aged between 35 and 48 years who were scheduled for elective unilateral or bilateral oophorectomy as part of benign gynecological surgical interventions, including hysterectomy for fibroids, pelvic organ prolapse, or benign adnexal masses. Inclusion criteria included regular menstrual cycles, absence of systemic illness, and no history of malignancy. Patients were excluded if they had undergone prior chemotherapy or pelvic radiation therapy, had known ovarian pathology (e.g., endometriosis, PCOS), or used hormonal medications within the preceding 3 months. AFC was evaluated using transvaginal ultrasonography (TVUS) during the early follicular phase (day 2–5 of the menstrual cycle) using a 5–9 MHz endovaginal transducer (GE Voluson E10, GE Healthcare, Austria). All scans were performed by the same board-certified gynecologist to reduce interobserver variability. AFC was defined as the total number of follicles measuring 2–10 mm in both ovaries. On the day of surgery, fasting venous blood samples were obtained from each participant between 08:00 and 10:00 AM. Samples were centrifuged at 3000 rpm for 10 min and stored at − 80 °C until batch analysis. Serum AMH, FSH, and estradiol (E2) levels were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits: AMH (Beckman Coulter Access 2, USA), FSH and E2 (Roche Elecsys 2010, Germany). The detection limit of the AMH assay was 0.02 ng/mL. Values below this threshold were assigned as 0.02 ng/mL for analysis. All assays were conducted in duplicate, and the intra-assay and inter-assay coefficients of variation were < 8% and < 10%, respectively. During surgery, ovarian tissues were excised and immediately fixed in 10% neutral buffered formalin for at least 24 h. The specimens were processed using standard histopathological protocols and embedded in paraffin blocks. Serial sections of 5 μm thickness were obtained and stained with hematoxylin and eosin (H&E). Every fifth section was selected for analysis to prevent duplicate counting of follicles. Primordial follicles were defined morphologically as oocytes surrounded by a single layer of flattened pre-granulosa cells. Follicle identification and enumeration were performed under a light microscope (Olympus BX53) at ×100 and ×400 magnifications by a single senior pathologist who was blinded to all clinical and laboratory data. For patients undergoing bilateral oophorectomy, follicle counts were averaged. To assess reproducibility, 10 randomly selected ovaries were recounted five times, yielding an intra-observer coefficient of variation of 7.5%. Primordial follicle counts were expressed as the number of follicles per ovary. For patients who underwent bilateral oophorectomy, counts were determined separately for each ovary, and the mean value was used in the analysis. Data were analyzed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA). Normality of the variables was assessed using the Shapiro–Wilk test. Continuous variables were expressed as mean ± standard deviation (SD) for normally distributed data and as median with interquartile range (IQR) for non-normally distributed data. The associations between serum AMH levels, AFC, and histologically confirmed primordial follicle counts were evaluated using Spearman’s rank correlation coefficient (ρ). A two-tailed p -value of < 0.05 was considered statistically significant. Subgroup analyses were also performed to assess the robustness of correlations within different age groups (35–40, 41–45, and 46–48 years). Multicollinearity was assessed using variance inflation factor (VIF) values in exploratory regression analyses.

This study provides histological validation that AMH and AFC accurately reflect the size of the primordial follicle pool in premenopausal women. Among commonly used ovarian reserve markers, AMH and AFC demonstrated the strongest concordance with true follicular reserve. These findings reinforce their value as reliable, non-invasive tools for individualized reproductive assessment and clinical decision-making.

Ovarian reserve reflects the remaining quantity of oocytes contained within primordial follicles and represents a key determinant of reproductive potential in women. The clinical relevance of assessing ovarian reserve is particularly pronounced in the context of assisted reproductive technologies (ART), where it facilitates the prediction of ovarian responsiveness to stimulation protocols and informs the risk stratification for suboptimal or exaggerated responses, including ovarian hyperstimulation syndrome (OHSS) [ 17 ]. However, existing biomarkers provide only an indirect estimation of the functional oocyte pool and have limited precision in reflecting the biologically relevant reserve that impacts reproductive lifespan [ 18 ]. Given the limitations of current surrogate tests, evaluating the concordance between these clinical markers—such as AMH and AFC—and the actual number of primordial follicles remains critical to establishing their diagnostic validity. While numerous studies have hypothesized associations between AMH or AFC and primordial follicle populations, these indicators inherently serve as indirect reflections of the true ovarian reserve, which is histologically defined by the number of quiescent primordial follicles present in the ovarian cortex [ 19 ]. Direct enumeration of primordial follicles in vivo is not feasible in clinical practice due to their microscopic size and non-secretory nature; hence, much of the available evidence has been extrapolated from animal models and cultured human ovarian tissues. AMH is hypothesized to exert an inhibitory effect on the initiation of folliculogenesis by preventing the transition from the primordial to the primary follicle stage. Additionally, AMH modulates follicular sensitivity to gonadotropins, particularly FSH, thereby acting as a critical regulator of ovarian dynamics [ 20 ]. The present study adds novel histological evidence to support the utility of both AMH and AFC as valid indicators of ovarian reserve in premenopausal women. By aligning preoperative hormonal and ultrasonographic markers with direct postoperative histological counts, the study confirms a strong association between these surrogate biomarkers and the true primordial follicle pool. The results demonstrated that serum AMH levels and AFC were strongly correlated with histologically quantified primordial follicle numbers. These findings are consistent with prior studies, including Hansen et al., who reported similar correlation strengths between AMH ( r  = 0.72), AFC ( r  = 0.78), and follicle counts in surgically obtained ovarian specimens. Moreover, these associations remained statistically significant even after adjusting for chronological age (AMH: r  = 0.48; AFC: r  = 0.53), indicating that both markers independently reflect follicular reserve irrespective of age [ 21 ]. In parallel, mathematical models of ovarian aging have identified AMH as the most dynamic endocrine correlate of follicular decline across the female reproductive lifespan. The present study provides biological validation for these models, particularly within the perimenopausal age range, where rapid depletion of the follicular pool occurs [ 22 ]. AFC, as a non-invasive and real-time imaging parameter, also exhibited a strong positive correlation with AMH and histological follicle counts, reinforcing its relevance for use in clinical settings where hormonal testing may be inconclusive or variable. In contrast, FSH displayed only a moderate inverse correlation with primordial follicle counts and AMH levels, in line with previous reports that question its reliability as an ovarian reserve marker, particularly in late reproductive age. The cyclic variability and delayed feedback kinetics of FSH contribute to its limited utility in precisely estimating the remaining follicular pool [ 23 ]. A notable strength of this study lies in its methodological design, which included a demographically homogeneous cohort of premenopausal women evaluated uniformly during the early follicular phase, combined with blinded histological quantification of follicle numbers. Unlike earlier studies that relied solely on mathematical estimations or indirect surrogates, the direct microscopic assessment of primordial follicles in this study enhances the validity and robustness of the conclusions. Nevertheless, several limitations must be acknowledged. The study population consisted exclusively of women undergoing gynecologic surgery for benign indications, which may limit the generalizability of the results to broader reproductive-age populations. Despite efforts to reduce variability—such as performing all AFC measurements by a single experienced examiner and using standardized immunoassay protocols—some degree of inter-assay and procedural variability cannot be excluded. The present findings may not be generalizable to younger populations or patients with PCOS. In PCOS, elevated AMH reflects increased antral follicle numbers rather than primordial follicle pool size, potentially altering the strength of correlations. In conclusion, this study reinforces and extends the current literature by providing histological confirmation that serum AMH levels and antral follicle count are reliable, non-invasive surrogate markers of true ovarian reserve. These findings support the routine application of AMH and AFC in clinical practice for fertility assessment, reproductive lifespan estimation, and individualized treatment planning. Future research should explore longitudinal validation in larger, more diverse populations and integrate emerging molecular markers to further refine ovarian reserve assessment strategies.

Female fertility is intrinsically linked to the size of the primordial follicle pool, which constitutes the biological basis of ovarian reserve. This finite pool undergoes a physiological decline with advancing age, a process that carries significant implications for both reproductive potential and the timing of menopause [ 1 ]. Given that direct histological quantification of primordial follicles in the human ovary is impractical in clinical settings, ovarian reserve is commonly assessed using indirect methodologies [ 2 ]. In recent decades, socioeconomic and demographic transformations have led to a global trend of delayed childbearing, often into the late 30s or early 40s—a period during which fertility undergoes a steep decline. This decline primarily results from the progressive depletion of the primordial follicle pool, a process that ultimately culminates in reproductive senescence [ 3 , 4 ]. Primordial follicles—also referred to as resting or primitive follicles—are the smallest and most immature follicular units within the human ovary. Established during fetal development, these follicles form a fixed, non-renewable population present at birth [ 5 ]. The number of oocytes is believed to reach its peak between 5 and 7 million during mid-gestation, around the 20th week of fetal life [ 6 ]. By the time of birth, this number drops to approximately 0.5–1 million. During childhood, substantial follicular loss occurs via atresia, and by menarche, only 300,000 to 400,000 follicles remain. This attrition continues throughout the reproductive lifespan, with an accelerated decline typically observed after the mid-30s; by the late 30s, the ovarian follicle pool generally falls below 50,000 [ 7 ]. Considering that the average number of ovulatory cycles over a woman’s reproductive life is approximately 450, it becomes evident that the vast majority of follicles are lost through atresia without ever reaching ovulation [ 8 ]. The primordial follicle pool thus functions as a biological reservoir throughout reproductive life. Beginning at puberty, a cohort of primordial follicles is periodically recruited to initiate folliculogenesis with each menstrual cycle. While approximately 20 follicles commence this process monthly, typically only one achieves ovulation; the remainder undergo atresia at various developmental stages. Therefore, cyclic recruitment contributes to the progressive depletion of the follicle reserve. The rate of primordial follicle activation is a key determinant of the timing of ovarian reserve exhaustion [ 9 ]. Anti-Mullerian hormone (AMH), secreted by growing preantral and small antral follicles, plays a pivotal regulatory role in this dynamic. AMH functions to inhibit premature recruitment of resting follicles into the growth phase, thereby preserving the primordial follicle pool and prolonging reproductive longevity [ 10 ]. These findings underscore the complexity of the regulatory mechanisms involved, which encompass both endocrine and paracrine factors. Notably, follicle depletion follows a nonlinear trajectory rather than a fixed, uniform rate [ 11 ]. Ovarian reserve is broadly defined as the quantity of nongrowing follicles present in the ovary at a given age [ 12 ]. More precisely, it should refer exclusively to the pool of primordial follicles [ 13 ]. However, the absence of a validated gold standard for direct quantification poses a persistent challenge. Primordial follicles are neither amenable to visualization through imaging modalities such as ultrasound, nor do they produce measurable hormonal outputs. Consequently, surrogate markers are utilized in clinical practice to approximate ovarian reserve. These include serum levels of follicle-stimulating hormone (FSH) and AMH, as well as transvaginal ultrasound assessment of antral follicle count (AFC) [ 14 ]. Although widely adopted, these tests do not offer a direct estimation of the primordial follicle pool. Rather, they provide indirect information based on the subset of follicles that have already entered the growth phase. For example, AMH is exclusively secreted by granulosa cells of preantral and small antral follicles and is absent in dormant primordial follicles. Nevertheless, both AMH and AFC demonstrate clinical utility in forecasting ovarian response in assisted reproductive technologies (ART) [ 15 , 16 ]. This study aimed to investigate the correlation between serum AMH levels and ultrasonographically measured AFC, and to evaluate their predictive relationship with histologically quantified primordial follicle counts.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1