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Anogenital distance

Anogenital distance (AGD) is an anthropometric defined as the distance from the center of the to the base of the genitals, specifically the perineoscrotal junction in males and the posterior in females. This trait is sexually dimorphic, with males exhibiting an AGD approximately 1.5 to 2.5 times longer than females due to the masculinizing effects of prenatal androgens during fetal development around weeks 8–14 of . AGD serves as a noninvasive reflecting in utero exposure to androgens or potential endocrine disruptors, and it remains relatively stable from infancy through adulthood. In humans, AGD was first systematically studied in the early as an extension of its established use in rodent toxicology, where it reliably indicates anti-androgenic effects from chemicals like and pesticides. Shorter AGD in male newborns and adults correlates with impaired reproductive health, including reduced count, lower , and higher incidence of congenital disorders such as and . In females, AGD variations have been linked to and other androgen-related conditions, though research is less extensive. Measurement protocols involve prone or positioning with for precision, typically yielding values of 2–4 cm in adult females and 3–6 cm in adult males, though population norms vary by age, ethnicity, and environmental factors. Ongoing studies explore AGD's role in assessing environmental exposures to persistent organic pollutants and stress, underscoring its value in and .

Definition and Anatomy

Definition

Anogenital distance (AGD) is defined as the linear distance from the center of the to the base of the external genitalia, specifically the posterior in females and the posterior aspect of the in males. This measurement serves as a sexually dimorphic , with the distance being notably longer in males due to the masculinizing effects of prenatal androgens on genital . AGD is typically expressed in millimeters (mm) and can be assessed using precise tools such as for accurate endpoint identification. The trait was first documented in animal research over a century ago, with early measurements in rats used to differentiate sexes based on this anatomical feature, as described by Jackson in 1912. Its application extended to human studies in the early , where it emerged as a potential for assessing prenatal endocrine influences on . At birth, average AGD values exhibit clear sex differences, with males typically measuring 19-22 mm compared to 9-11 mm in females, a dimorphism that underscores the role of exposure during critical fetal periods. These baseline differences persist across , though proportions vary, establishing AGD as a conserved marker of reproductive development.

Anatomical Variations

Anogenital distance (AGD) exhibits distinct anatomical landmarks that differ between sexes, reflecting the developmental divergence of external genitalia during embryogenesis. In males, AGD is typically measured from the center of the to the posterior base of the (perineoscrotal junction), encompassing the perineal region between the anal opening and the scrotal attachment. In females, the measurement extends from the to the of the (), highlighting the shorter perineal span due to the absence of scrotal development. These sex-specific features establish AGD as a dimorphic , with male distances generally 1.5 to 2 times longer than female equivalents across populations. Age-related changes in AGD occur primarily during early postnatal development, after which the distance stabilizes. At birth, mean AGD measures approximately 20 mm in males and 9-11 mm in females, increasing progressively through the first year of life due to perineal tissue growth and reaches a plateau by 6-12 months, maintaining relative consistency into adulthood. Studies indicate that adult AGD remains stable compared to infancy values, with no significant decline observed across age groups in healthy individuals. These patterns underscore AGD's role as a marker fixed largely by prenatal factors, with postnatal variations influenced minimally by physiological growth. Population norms for adult AGD show and subtle ethnic variations, providing benchmarks for anatomical assessment. In adults, average male AGD ( to scrotal base) ranges from 40-50 mm, while AGD ( to ) averages around 40 mm, based on studies of diverse cohorts. Ethnic differences exist, with some reports indicating slightly longer distances in certain non-Caucasian groups; for instance, populations exhibit extended AGD compared to or North references in fetal and early life measurements. These norms vary by measurement subtype (e.g., shorter for anus-to- versus longer for anus-to-clitoris in s) and highlight the need for population-specific references to account for genetic and environmental influences. Rare congenital anomalies can alter AGD morphology, affecting the effective distance through structural deviations. Conditions such as bifid scrotum, a midline fusion defect of the scrotal , result in a split scrotal sac that shortens the apparent perineoscrotal distance in males by disrupting the normal posterior landmark. This anomaly, occurring in approximately 1 in 200-300 cases of severe or as an isolated trait, exemplifies how embryological disruptions in signaling lead to atypical AGD configurations without necessarily impacting overall perineal length. Such variations are typically identified at birth and may require surgical correction, but they represent exceptions to standard anatomical norms.

Measurement Techniques

Methods in Humans

In humans, anogenital distance (AGD) is primarily measured as the ano-scrotal distance (AGD-AS) in males, from the center of the to the posterior base of the , and the ano-fourchette distance (AGD-AF) in females, from the center of the to the posterior . An alternative measure, the ano-penile distance (AGD-AP), is sometimes used in males, extending from the center of the to the anterior base of the . These dimensions reflect sexually dimorphic traits, with male AGD typically approximately twice that of females in newborns. Standardized protocols employ rigid plastic to minimize compression. The subject is positioned prone with legs extended and slightly separated to ensure consistent landmark identification, and measurements are taken three times per dimension, using the value to enhance accuracy. Intra-observer reliability exceeds 0.9 in validated studies, with intra-rater agreement reaching 96% in males and 92% in females when performed by trained examiners. Measurements are optimally conducted at birth, often within the first 48 hours while the umbilical cord remains attached to facilitate infant stability and reduce movement artifacts, though they are repeatable in adults via similar prone positioning. Each assessment typically requires 1-2 minutes, making it feasible in both research and clinical environments. Challenges include variability from perineal body fat, which can obscure landmarks and necessitate gentle palpation, particularly in obese individuals. In specific clinical contexts, such as fetal assessment or adult diagnostics for conditions like endometriosis, ultrasound or MRI has been used to measure AGD. Standardization efforts intensified in the 2010s through seminal anthropometric protocols developed in cohort studies, providing reproducible benchmarks for endocrinological research without formal society-specific guidelines. Recent studies as of 2025 have validated self-measurement techniques by individuals, potentially increasing accessibility. These human methods differ from animal adaptations, which often involve sedation for larger species.

Methods in Animals

In rodents such as rats and mice, anogenital distance (AGD) is measured from the center of the anus to the proximal penis in males or the clitoris in females, typically using vernier or digital calipers for postnatal assessments to capture sexually dimorphic differences established prenatally. For late fetuses and early postnatal pups, a microscopic method employing a stereomicroscope with a micrometer eyepiece is recommended to achieve the necessary precision, with measurements adjusted by dividing AGD by the cube root of body weight for size normalization. These protocols are standardized in OECD Test Guideline 414 for prenatal developmental toxicity studies, adopted in 2018 (originally 1981, revised 2001) and building on reproductive toxicity guidelines from the 1990s, where AGD evaluation is mandatory for detecting endocrine disruption in live rodent fetuses. In livestock such as and sheep, AGD is assessed post-restraint using digital , measuring from the to the base of the in females or the in males, with multiple readings averaged for reliability. A 2022 study on dairy cows reported high repeatability of these measurements across stages, , and , with intra-class correlation coefficients exceeding 0.85 except at late , underscoring the method's consistency in larger animals. Similar digital caliper approaches are applied in sheep, often following tail restraint to facilitate access. AGD lengths vary markedly across species, requiring methodological adjustments like parting fur or hair to expose landmarks accurately. In ungulates such as and sheep, values are substantially longer at to 100 mm or more, with mean female AGD around 132 mm in lactating cows. These differences highlight the need for species-tailored normalization, such as relative to in smaller mammals. Emerging trends since 2023 involve non-contact software for AGD in lab animals, enabling precise analysis of high-resolution photographs to minimize handling and improve reproducibility. For example, image analysis tools have been used in sheep to quantify AGD from photos, offering advantages over manual in controlled settings.

Biological and Developmental Role

Prenatal Determination

The anogenital distance (AGD) differentiates during early human , with emerging between weeks 8 and 14, corresponding to the masculinization programming window. A of newborns (gestational ages ≥22 weeks) demonstrated consistent sex-specific AGD lengths unaffected by circulating androgens at birth, indicating that AGD is established and fixed prenatally by the mid-second . In males, AGD measures approximately 28 mm from to and 42 mm from to base of the , while in females, it is shorter at 20 mm from to and 34 mm from to , respectively, with minimal overlap between sexes. Embryologically, AGD forms as the urogenital folds and swellings develop from the cloacal region around week 6, with the distance between the anus and becoming sexually dimorphic under influence. In the female default pathway, lacking significant exposure, the forms the and , resulting in a shorter AGD due to limited perineal expansion. Masculinization in males occurs via testosterone produced by fetal testes, converted locally to (DHT), which activates receptors in perineal to stimulate elongation of the into the and fusion of urogenital folds, thereby increasing AGD through enhanced perineal growth. This -driven process, peaking during the critical of weeks 8-12, is essential for establishing the longer male AGD. In animal models, similar timelines apply, with AGD set by embryonic days 15-18 in rats through receptor-dependent perineal masculinization, providing a comparative framework for understanding development. Disruptions to signaling within these windows, such as from anti-androgenic exposures, can permanently shorten AGD or lengthen AGD. Longitudinal population studies up to 2024 show that birth or infant AGD significantly predicts childhood AGD, with correlation coefficients of 0.54-0.69 in boys from 3 months to 9 years, indicating relative stability postnatally. These prenatal processes are primarily governed by endocrine mechanisms involving fetal gonadal hormones.

Endocrine Mechanisms

The development of anogenital distance (AGD) is primarily driven by androgens during critical windows of fetal masculinization, with testosterone produced by the fetal testes serving as the key that promotes elongation of the and differentiation of perineal tissues. Testosterone is converted locally to the more potent (DHT) by the enzyme , which binds to the () to activate genes responsible for external genitalia formation, including the structures that determine AGD length. This androgen action is essential for establishing a longer AGD in males compared to females, as disruptions in DHT-mediated signaling lead to shortened AGD and undermasculinized genitalia. Variations in sensitivity, influenced by polymorphisms such as the repeat length in the gene, further modulate AGD outcomes; longer CAG repeats are associated with reduced androgen responsiveness and shorter AGD in males. Fetal Leydig cells are the primary source of testosterone for AGD development, beginning production around week 8 of and peaking between weeks 11 and 14, which coincides with the masculinization programming window when androgen-dependent perineal growth occurs. These cells respond to chorionic to synthesize testosterone, which diffuses to target tissues without significant reliance on maternal or contributions, as the placenta acts as a barrier limiting transplacental transfer of maternal to the . Minimal maternal/placental testosterone input ensures that AGD patterning reflects endogenous fetal androgen exposure rather than external hormonal influences. Genetic regulation of AGD involves upstream factors that establish gonadal identity and downstream pathways. The SRY on the initiates testis around week 7, triggering development and subsequent testosterone production essential for AGD elongation. , particularly those in the Hoxa and Hoxd clusters, contribute to urogenital tract patterning by specifying segmental identity along the anterior-posterior axis, indirectly influencing the positional development of perineal structures responsive to . Although genome-wide association studies (GWAS) have not yet identified specific loci directly linked to human AGD variation, animal models highlight how disruptions in these genetic pathways, combined with signaling, underlie AGD phenotypes. Endocrine disruption models demonstrate the sensitivity of AGD to anti-androgen interference, providing mechanistic insights into hormonal regulation. In rodent studies, exposure to anti-androgens like during the masculinization window suppresses fetal testosterone synthesis and activation, resulting in shortened male AGD by approximately 20-40%, alongside reduced growth. These effects mimic partial androgen insensitivity, with inhibiting steroidogenesis and DHT formation. In humans, correlations between levels and newborn AGD support these mechanisms, as lower prenatal testosterone or elevated anti-androgenic metabolites in are associated with reduced AGD, indicating disrupted fetal endocrine signaling.

Clinical and Health Implications in Humans

Associated Conditions

Boys with and exhibit significantly shorter anogenital distance (AGD) compared to healthy controls, with meta-analyses confirming mean reductions in affected individuals. These conditions are manifestations of (TDS), a spectrum of reproductive disorders linked to prenatal disruption, where short AGD serves as a reflecting reduced fetal testosterone action. Short AGD, particularly values more than 2 standard deviations below the population mean, correlates with increased odds of and , with studies reporting odds ratios indicating 2- to 4-fold elevated risk in cohorts of newborns. Recent research as of 2025 has shown that fetal AGD correlates with hypospadias severity, suggesting potential for prenatal screening. In (DSD), AGD measurements provide insight into prenatal exposure and help differentiate underlying etiologies in cases of ambiguous genitalia. Affected infants often present with intermediate AGD values, typically ranging from 10 to 20 mm, overlapping male (mean ~22 mm) and female (mean ~11 mm) norms and reflecting partial or undervirilization. For instance, in 46,XX DSD due to (CAH), excess prenatal androgens result in lengthened AGD in females, promoting masculinization of external genitalia and distinguishing it from other DSD forms with . AGD assessment thus aids in initial evaluation and management of DSD, complementing karyotyping and hormone assays. Prenatal exposure to endocrine-disrupting chemicals such as bisphenol A (BPA) and phthalates is associated with shortened AGD in male offspring, as evidenced by cohort studies and meta-analyses from 2015 to 2023. Systematic reviews of human data indicate that high maternal urinary levels of phthalate metabolites, particularly di(2-ethylhexyl) phthalate (DEHP), correlate with AGD reductions in newborns, mimicking anti-androgenic effects observed in animal models. Similar associations hold for BPA, with cord blood concentrations linked to decreased anoscrotal distance in males, supporting a role in TDS pathogenesis. A 2025 study found that maternal phthalate exposure is linked to shorter AGD in 3-year-old boys. Prenatal exposure to per- and polyfluoroalkyl substances (PFAS) has also been associated with altered AGD growth trajectories in infants. These findings underscore the utility of AGD as a noninvasive indicator of environmental endocrine disruption during critical gestational windows. Clinically, AGD measurement in newborns serves as a diagnostic tool for identifying genital anomalies, with values below 16 mm signaling elevated risk for conditions like or —approximately 50% higher than in those with normal AGD. Short AGD prompts further evaluation, including and endocrine testing, to detect TDS components early. While not yet formalized in universal , AGD can be incorporated in genital examinations where indicated to flag potential reproductive risks. This approach facilitates timely intervention and monitoring for long-term health implications. Recent 2025 research also links shortened AGD to , a condition associated with impaired fertility.

Fertility and Reproductive Health

In men, longer anogenital distance (AGD) has been consistently associated with higher concentration, total count, and , reflecting its role as a of prenatal exposure and adult reproductive potential. Studies from the , including cohort analyses of over 1,000 men, report positive correlations between AGD and parameters, with standardized coefficients indicating modest but significant relationships (e.g., β ≈ 0.2–0.4 for concentration after adjustment for confounders like age and ). Shorter AGD, particularly below the population median of approximately 50 mm, is linked to reduced odds; for instance, men with AGD shorter than this threshold exhibit up to sevenfold higher risk of subfertility compared to those with longer AGD, based on analyses from fertility clinics in the . In women, AGD variations similarly influence reproductive health outcomes, with shorter distances associated with diminished and poorer response to treatments. on women undergoing IVF demonstrates that AGD positively correlates with antral follicle count and the number of oocytes retrieved during controlled ovarian stimulation, where each 1 cm increase in AGD predicts approximately 1–2 additional oocytes, underscoring its utility in assessing ovarian function. Conversely, longer AGD is a marker of elevated prenatal exposure and is strongly linked to (PCOS); women in the highest AGD tertile (>43 mm) have over sixfold odds of PCOS diagnosis compared to the lowest tertile, based on multicenter studies from the late . A 2025 study confirmed longer AGD as a potential for PCOS. Additionally, shorter AGD (<36 mm) in reproductive-age women is tied to challenges, including extended time to natural conception and lower success rates in assisted reproduction. AGD also serves as a predictor of reproductive lifespan and treatment outcomes in clinical settings. Longitudinal data indicate that longer AGD in both sexes is associated with shorter time to pregnancy among couples attempting natural conception, with 2022 analyses showing that a 1 cm increase in male AGD reduces the odds of prolonged time to pregnancy (>3 months) by about 8%, independent of . In IVF models, AGD explains up to 15% of variance in success metrics like embryo yield and implantation rates, particularly when integrated with hormonal profiles, as evidenced by prospective studies integrating AGD measurements into predictive algorithms. From a perspective, increasing incidence of reproductive disorders has been attributed to rising exposure to endocrine-disrupting chemicals like , mirroring parallel drops in and rates. These trends highlight AGD's value as a noninvasive screening tool in clinics, where its integration could identify at-risk individuals early and inform interventions to mitigate reproductive health declines.

Research and Applications in Animals

Toxicology and Endocrine Disruption

Anogenital distance (AGD) is utilized as a sensitive biomarker in animal toxicity studies to detect endocrine-disrupting chemicals (EDCs), particularly those with anti-androgenic effects, by measuring reductions in male offspring that indicate disrupted sexual differentiation. In regulatory toxicology, AGD assessment is incorporated into several OECD Test Guidelines for reproductive and developmental toxicity, including TG 421 (Reproduction/Developmental Toxicity Screening Test) and TG 422 (Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening), where shortening of male AGD alongside nipple retention serves as a key endpoint for identifying potential EDCs. Similarly, OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) requires AGD measurement in pups from postnatal day 0 to 4 to evaluate EDC-related impacts on androgen-dependent development. A significant reduction in male AGD, typically exceeding 20% compared to controls, signals anti-androgenic activity warranting further investigation, as established in validation studies for these guidelines. In model organisms, such as rats, gestational exposure to the vinclozolin demonstrates AGD's utility in testing, with doses of 100-200 mg/kg/day resulting in approximately 30% shortening of male AGD in F1 due to androgen antagonism during critical prenatal windows. Multigenerational studies reveal persistent effects, where vinclozolin exposure in pregnant F0 dams leads to reduced AGD in male F1, F2, and F3 generations, highlighting heritable EDC impacts on reproductive development. Phthalates, common plasticizers, exert anti-androgenic effects by inhibiting fetal testosterone synthesis in the testes rather than direct antagonism, leading to dose-dependent AGD reductions in male rat offspring; EPA-sponsored studies from the early 2000s established nonlinear dose-response curves, showing significant shortening at exposures as low as 100-500 mg/kg/day during gestation days 8-19.

Breeding and Phenotypic Selection

In breeding programs, anogenital distance (AGD) serves as a phenotypic marker for selecting animals with enhanced reproductive performance. Shorter AGD in heifers and cows is associated with earlier onset, higher pregnancy rates per (P/AI), and reduced days open. For instance, a 2022 validation study of 4,709 lactating cows found that those with short AGD (≤129 mm) achieved a P/AI of 36%, compared to 30% for long AGD (>129 mm) animals, alongside fewer inseminations per (2.3 vs. 2.4) and shorter intervals to (137 vs. 142 days). Similarly, in nulliparous heifers, AGD thresholds around 110 mm distinguish high-fertility groups, with shorter distances predicting fewer services per and earlier breeding eligibility. These traits enable early-life selection to improve herd fertility, with estimates for AGD at approximately 0.37, supporting its integration into genetic evaluations. In and sheep , AGD measurements inform genomic selection strategies to optimize size and reproductive efficiency. In pigs, longer AGD in gilts (e.g., >11.55 mm at 16 weeks) correlates with larger (born alive: 11.79 vs. 11.20 piglets), earlier (179.6 vs. 182.2 days), and higher success rates (91% vs. 83%), reflecting intrauterine masculinization effects that enhance female fertility. Single nucleotide polymorphisms (SNPs) linked to AGD are incorporated into genomic selection indices, alongside traditional metrics like total born and weights, to boost productivity in commercial herds. In sheep, AGD assessments at or pre- predict fertility and reproductive status, allowing earlier of low-potential animals and integration into value models for prolificacy traits. data from related reproductive phenotypes supports genomic tools that target AGD-influenced loci for improved lambing rates. AGD measurements in wildlife conservation monitor endocrine-disrupting chemical (EDC) exposure and its effects on reproductive health in species like amphibians, where deviations signal population-level declines. These applications complement toxicity screening but focus on long-term phenotypic selection for resilient lineages in managed wildlife programs. Economically, incorporating AGD into breeding programs reduces insemination costs and enhances overall herd profitability, with selected lines showing 10–20% improvements in reproductive efficiency. In dairy cattle, short-AGD selection minimizes services per conception, lowering artificial insemination expenses by up to 20% while integrating into best linear unbiased prediction (BLUP) models since the 2010s to balance fertility gains against milk yield. Similar benefits extend to swine and sheep operations, where AGD-guided genomic selection amplifies litter output, yielding net returns through reduced replacement costs and higher throughput.

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