This is the first study to measure AGD in adult women and examine the relationships between AGD and female reproductive system characteristics. Both AGD measures were positively and strongly associated with the presence of greater ovarian follicular number. Moreover, a woman with AGDAC or AGDAF in the highest tertile of the distribution was 3 and 6 times, respectively, as likely to have ≥ 6 ovarian follicles as a woman with an AGDAC or AGDAF in the lowest tertile. This underscores the possible clinical implications of the associations that we are reporting here.
Although we typically find that in men AGD measurements are not sensitive to physiologic and lifestyle factors
, in the current study population the use of hormonal contraception was associated with shorter measures in both AGD measurements (p values <0.05). One immediate consequence of that finding is that, in females, it may be necessary to control not only for body size (height or BMI) as in men, but also for hormonal status in studies seeking associations with AGD measurements. In our study population, 74% (n = 29) of the young women taking hormonal contraception reported irregular menstrual cycles in the previous three months. However, no association was found between AGD measurements and irregular menstrual cycle. Therefore we could speculate that hormonal intake might be related to the AGD measurements. To our knowledge, this is the first time this association has been reported in humans.
Recently, Dusek and Bartos
 examined the effect of the stage of the oestrous cycle on the AGD in female mice and showed that AGD varied during the oestrous cycle, and suggesting that female genital morphology systematically varied within the oestrous cycle. Unfortunately, all the AGD measures in our study were taken during the early follicular phase, so that we cannot assess that hypothesis. Nonetheless, it opens a door to the possibility that hormonal changes (including hormonal contraception) could result in small fluctuations in the morphology of the female external genitalia. Longitudinal study measuring AGD within women at multiple points in the cycles is needed to address this possibility.
Alternatively, it has been suggested that women using hormonal contraception are somewhat more fertile than women who do not
. In many mammalian species females with shorter AGD are more fertile
[33, 34]. Therefore, it is possible that the shorter AGD we observed in women using hormonal contraceptives reflects increased fertility, rather than a direct effect of hormone use on AGD.
As multiple animal studies have shown, the female reproductive tract is susceptible to virilization by exogenous androgen exposure prior to, as well as during, the in utero masculinization programming window (MPW)
[5–7]. This prenatal hyperandrogenism results in enlarged cystic ovaries, anovulation or increases ovarian follicular recruitment in female offspring
[3, 4, 7, 35]. Excessive prenatal androgens (either endogenous or exogenous) can produce a polycystic ovary syndrome (PCOS)-like phenotype in animal models, furthermore
[35, 36]. As such, it has been widely hypothesized that the etiology of PCOS in humans may include excess prenatal androgen exposure
. Pregnant women with PCOS have elevated circulating concentrations of androgens at mid-gestation, which may increase fetal androgen exposure, and female offspring of PCOS mothers are at increased risk of altered ovarian development and function
[38, 39]. Long-term follow-up studies are required to determine whether this excessive exposure resulted in long-term changes in ovarian function and/or alterations in reproductive function. Here, we do not examine PCOS per se, however we do demonstrate that AGD, a purported biomarker of prenatal androgen exposure, is associated with increased ovarian follicle number.
With regard to reproducibility of follicle count estimated by transvaginal ultrasound, it has been shown that determination of follicle count by transvaginal ultrasound results in acceptable intra- and inter- observer variability
. While there is undoubtedly some inter-cycle variability of follicle count, it is considered to be of little clinical significance, for example, in predicting the response in in vitro fertilization
. Several articles have previously defined having ≥ 6 or > 12 ovarian follicles as multifollicular
[28, 29] or polycystic ovaries
, respectively. We were unable to look at women with polycystic ovaries separately due to small numbers; only 10 women met this criterion. We chose instead to use the cutoff for multifollicular ovary (≥ 6), a cutoff which several studies suggest may have clinical relevance
We found significant positive associations between both AGDAF and AGDAC and the presence of greater ovarian follicle number, which could indicate a common fetal origin between longer AGD and greater follicular recruitment. In fact, in animal models, an association between prenatal exposures to androgen excess and an increase in ovarian follicular recruitment
[3, 4, 7] and longer AGD measurement
 has been reported.
All previous published studies on human female infants have measured both AGDAF and AGDAC[19, 20, 23, 25–27]. Callegari et al.
 also calculated the ratio (i.e.: AGDAF/AGDAC) both, in premature/full-term newborn infants and newborn infant girls with congenital adrenal hyperplasia, reporting a relatively higher ratio among subjects with that condition. This result supports the hypothesis that androgen exposure in utero may affect measures of the anogenital region in human females. Callegari et al.
 also took both AGD measurements on a small number of pregnant women (n = 10), reporting the first such data, to the best of our knowledge. With all the possible limitations regarding study population and methodologies, our young women presented longer AGDAF and shorter AGDAC compared to those ten pregnant women.
We had only a few cases of clinically diagnosed PCOS in our study population; therefore we were unable to explore the relationship between that condition and the AGD measures. A larger and more diverse population or another type of study design (case–control) would add much more information on whether androgen action during early fetal life exerts a fundamental influence on the female reproductive tract in humans, as has been demonstrated in rodents.
Our population was small and limited in age and ethnicity, and thus cannot provide normative values for human female AGD measurements. AGD measures were well tolerated by all subjects, quick to perform, with acceptable intra- and inter-examiner reliability. We plan to assess reproductive hormones in a future publication. A finding of higher FSH and/or low inhibin-B, estradiol or free testosterone with shorter or longer AGD would lend support to the association between AGD and female reproductive system characteristics reported here.
In human males, shorter AGD in adulthood has been associated with poorer semen quality
[21, 22] and infertility
 suggesting a common origin, including a disruption of testicular development in utero, as suggested by the testicular dysgenesis syndrome hypothesis
[42, 43]. As hypothesized, this syndrome, although potentially multifactorial, may be caused by exposure to EDC during the MPW
Recently, Buck Louis et al.
 suggested a similar paradigm for human females, the ovarian dysgenesis syndrome, which the authors define as alterations in ovarian structure or function that may manifest as fecundity impairments, gynecologic disorders, gravid diseases or later onset of adult diseases. Environmental exposures, particularly EDC, may be related to in utero origin of gynecologic outcomes, which in turn would be associated with later onset of adult diseases.