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Cited by 177Sexual Differentiation and DisordersHormonal and reproductive studiesSperm and Testicular Function

Alternative (backdoor) androgen production and masculinization in the human fetus

Open access
O’Shaughnessy, Peter J., Antignac, Jean Philippe, Le Bizec, Bruno, Morvan, Marie-Line, Svechnikov, Konstantin, Söder, Olle, Savchuk, Iuliia, Monteiro, Ana +7 more·2019·PLOS Biology
DOI 10.1371/journal.pbio.3000002PMC PMC6375548

Published version of a preprint

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Abstract

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

AI snapshot

Written from the paper’s full text.

TL;DR

In second-trimester human fetuses, androsterone is the principal circulating backdoor androgen in males, while DHT is undetectable (<1 ng/mL) in male plasma. The data indicate that backdoor androgen synthesis occurs mainly in nongonadal tissues, especially placenta and fetal liver, rather than in the fetal testis.

Key findings

  • In 42 male fetuses, plasma DHT was undetectable (<1 ng/mL); the main circulating backdoor androgen was androsterone (male mean about 3 ng/mL), with 5αDHP and allopregnanolone present at means of 135 ng/mL and 243 ng/mL, respectively, compared with progesterone at 258 ng/mL.
  • Female fetuses (n = 16) had significantly lower plasma androsterone and testosterone than males, while Δ5 steroids and progesterone did not differ; allopregnanolone was higher in females, but 17α-hydroxyallopregnanolone and androsterone were lower.
  • Backdoor intermediates were found primarily in placenta and fetal liver, with significant androsterone also in fetal adrenal; fetal testes contained only very low/undetectable androsterone, and no backdoor androgens or intermediates were detectable in testicular cell cultures.
  • Transcript data showed SRD5A1 highest in liver, with liver and placenta having about 1,000 times greater total SRD5A1 expression than testis; liver and placenta each had about 200 times more total AKR1C2 transcript than fetal testes, and adrenal CYP17A1 mean was about 6 times that of testis.

Methods

  • Endogenous steroids were measured in second-trimester human fetal plasma and tissues using GC-MS/MS and LC-HRMS; plasma came from 42 male and 16 female fetuses, and tissues included placenta/liver (n = 20), adrenal (n = 30), and testis (n = 10 for DHEA/androsterone or 25 for other steroids).
  • Transcript levels of steroidogenic enzymes were measured by quantitative PCR in testis (n = 22), adrenal (n = 21), liver (n = 44–50), genital tubercle (n = 10), and placenta (n = 20), normalized to TBP.
  • Dispersed fetal testicular cells were incubated with or without hCG for 24 hours, and steroids were measured by GC-MS/MS; plasma steroid data were analyzed by two-factor ANOVA with fetal age and maternal smoking as factors, with t tests and Cohen correction for nondetectable samples.

Limitations

  • Stated in the paper: it remains to be shown whether the genital tubercle can convert androsterone to DHT, and it remains to be established how sex differences in plasma androsterone levels arise.
  • Stated in the paper: whether placental AKR1C2 is of fetal or maternal origin is unknown.
  • Evident from the text: tissue steroid measurements were limited by LOD/LOQ and matrix effects, some values were above LOD but below LOQ and therefore less reliable, 17α-hydroxylated intermediates were not measured in the tissue study, 5αDHP was not measured in the testis/ovary GC-MS/MS comparison, and testicular DHEA/androsterone data were reported for only 10 samples because recovery of steroid sulfates was poor in the first batch.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pbio.3000002

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 55 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes.

    “Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes.”
    • Also stated in: § Backdoor steroids are present primarily in nongonadal tissues in the male fetus · plain
      “The backdoor intermediates 5αDHP and allopregnanolone were detectable in 6 and 9 testes, respectively (out of 25), but androsterone was not detectable, and androstanediol was only detectable in one testis.”
    Quote in the paperSubstantial claim
  • Findingown data

    The principal Δ4 and backdoor androgens detectable in most male fetal plasma samples were androstenedione, testosterone, and androsterone.

    “The principal Δ4 and backdoor androgens detectable in most samples were androstenedione, testosterone, and androsterone, all potential substrates for DHT synthesis.”
    Quote in the paperSubstantial claim
  • Findingown data

    Maternal smoking had no significant effect on fetal plasma steroid levels.

    “Maternal smoking had no significant effect on fetal plasma steroid levels.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Androsterone is the principal backdoor androgen in the male fetal circulation.

    “Results show that androsterone is the principal backdoor androgen in the male fetal circulation”
    • Also stated in: § Introduction · plain
      “Our results show that high levels of intermediates in the backdoor pathway are present in the human fetal circulation, that androsterone is the major circulating backdoor androgen”
    • Also stated in: § Discussion · plain
      “In this study, we now show that androsterone is the major circulating backdoor androgen in the human male fetus”
    Quote in the paperSubstantial claim
  • Findingown data

    Female fetuses have significantly lower levels of androsterone and testosterone than male fetuses.

    “in female fetuses, there are significantly lower levels of androsterone and testosterone.”
    • Also stated in: § Levels of backdoor androgens and testosterone are lower in human female fetal plasma · plain
      “17α-hydroxyallopregnanolone and androsterone levels were significantly lower”
    Quote in the paperSubstantial claim
  • May combine two claims. 51
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    The placenta contained high levels of progesterone, with lower amounts of 5αDHP and allopregnanolone.

    “The placenta contained high levels of progesterone, with lower amounts of 5αDHP and allopregnanolone.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The fetal testes contained high levels of pregnenolone and testosterone, with lower levels of progesterone and androstenedione.

    “The fetal testes contained high levels of pregnenolone and testosterone, with lower levels of progesterone and androstenedione.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In female fetal plasma, progesterone levels were similar to males, but 17α-hydroxyprogesterone and androstenedione were significantly higher in females, while testosterone was significantly lower.

    “Progesterone levels were also similar, but the Δ4 steroids 17α-hydroxyprogesterone and androstenedione were significantly higher in females, while testosterone was significantly lower (although there was some overlap between sexes).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Testosterone was present in about half the fetal adrenals, but other steroids were not detectable.

    “Testosterone was present in about half the adrenals but other steroids were not detectable (Fig 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the fetal testes, 5αDHP and allopregnanolone were detectable in 6 and 9 out of 25 testes, respectively, but androsterone was not detectable, and androstanediol was only detectable in one testis.

    “The backdoor intermediates 5αDHP and allopregnanolone were detectable in 6 and 9 testes, respectively (out of 25), but androsterone was not detectable, and androstanediol was only detectable in one testis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The fetal adrenals contained high levels of pregnenolone, progesterone, and DHEA, and androsterone was present in most adrenals.

    “The fetal adrenals contained high levels of pregnenolone, progesterone, and DHEA, and androsterone was present in most adrenals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most forms of 5α-androstanediol were undetectable in fetal plasma, although 5α-androstan-3α,17β-diol was detectable in 10/42 male samples.

    “Most forms of 5α-androstanediol were undetectable in fetal plasma (including 5α-androstane-3β,17β-diol), although 5α-androstan-3α,17β-diol (labeled androstanediol in Figs 1 and 2), which is a potential substrate for DHT synthesis, was detectable in 10/42 samples (Fig 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The androsterone and DHEA in the fetal adrenals were both sulfated.

    “The androsterone and DHEA in the adrenals were both sulfated, which is likely to be a reflection of the high levels of sulfotransferase type 2A1 (SULT2A1) in this tissue [21].”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In male fetal plasma, intermediates in the backdoor pathway were present at significant levels, with the pathway apparently going from progesterone through 5αDHP, allopregnanolone, 17α-hydroxyallopregnanolone, to androsterone.

    “Noticeably, the data also show that intermediates in the backdoor pathway were present at significant levels, with the backdoor pathway apparently going from progesterone through 5α-dihydroprogesterone (5αDHP), allopregnanolone, 17α-hydroxyallopregnanolone, to androsterone (Fig 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The backdoor androgens androsterone and androstanediol were detectable in about half the placentas, while the Δ4 steroids androstenedione and testosterone were detectable in most placentas.

    “The backdoor androgens, androsterone, and androstanediol were detectable in about half the placentas, while the Δ4 steroids androstenedione and testosterone were detectable in most placentas.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the backdoor pathway, female fetal plasma allopregnanolone levels were higher than males, but 17α-hydroxyallopregnanolone and androsterone levels were significantly lower.

    “In the backdoor pathway, plasma allopregnanolone levels were higher in female fetuses, but 17α-hydroxyallopregnanolone and androsterone levels were significantly lower (Fig 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Levels of most steroids did not change over the course of the second trimester in male fetal plasma, with the exception of testosterone, which declined significantly (P < 0.048), and androstenediol, which increased (P < 0.004).

    “Levels of most steroids did not change over the course of the second trimester, with the exception of testosterone, which declined significantly (P < 0.048), and androstenediol, which increased (P < 0.004) over the same period (S2 Fig).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There were no significant age-specific changes in any of the female plasma steroids measured over the course of the second trimester, although DHEA and androstenedione showed an interaction between age and maternal smoking.

    “There were no significant age-specific changes in any of the female plasma steroids measured over the course of the second trimester, although DHEA and androstenedione showed an interaction between age and maternal smoking (S3 Fig).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the male fetus, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal.

    “In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Low levels of androsterone and DHT were detectable in most fetal livers, while androstanediol was detectable in about half the samples.

    “Low levels of androsterone and DHT were also detectable in most fetal livers, while androstanediol was detectable in about half the samples (Fig 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In testicular extracts from a further 6 fetuses measured by GC-MS/MS, all backdoor steroids were detectable, but levels of 17α-hydroxyallopregnanolone and androsterone were very low.

    “In samples measured by GC-MS/MS, all of the backdoor steroids were detectable, but levels of 17α-hydroxyallopregnanolone and androsterone were very low.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Tissue levels of 5αDHP are highest in the placenta, which probably reflects the high concentration of progesterone substrate in this tissue.

    “Tissue levels of 5αDHP are highest in the placenta, however, which probably reflects the high concentration of progesterone substrate in this tissue.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    HSD3B2 was expressed predominantly in the testis and adrenal, although the placenta also contained HSD3B2 transcripts, and some low-level expression was seen in the fetal liver.

    “HSD3B2 was expressed predominantly in the testis and adrenal, although the placenta also contained HSD3B2 transcripts, and some low-level expression was seen in the fetal liver (Fig 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ovarian samples contained similar levels of progesterone and allopregnanolone compared with testes, but all other steroids were reduced, and many were undetectable.

    “Ovarian samples contained similar levels of progesterone and allopregnanolone compared with the testes, but all other steroids were reduced, and many were undetectable (Fig 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    In female fetuses, allopregnanolone plasma levels are higher than in males, but downstream metabolites in the backdoor pathway are all reduced.

    “In female fetuses, allopregnanolone plasma levels are higher than in males, but downstream metabolites in the backdoor pathway are all reduced.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The mean progesterone concentrations in the placenta and liver are 6 ng/mg and 0.4 ng/mg, which means that, in most samples, they exceed the enzyme Km.

    “The mean progesterone concentrations in the placenta and liver are 6 ng/mg and 0.4 ng/mg, which means that, in most samples, they exceed the enzyme Km.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transcripts encoding HSD17B3 were expressed at similar levels in the fetal testis and liver, with very low or undetectable levels in the placenta, adrenal, and genital tubercle.

    “Transcripts encoding 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) were expressed at similar levels in the fetal testis and liver, with very low or undetectable levels in the placenta, adrenal, and genital tubercle (Fig 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CYP17A1 expression was high in both fetal adrenal and fetal testis, with the mean adrenal level about 6 times that of the testis.

    “Predictably, expression was high in both fetal adrenal and fetal testis, with the mean adrenal level about 6 times that of the testis (Fig 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Allopregnanolone levels in human fetal plasma are relatively high (male, about 200 ng/mL) but androsterone levels are significantly lower (male, about 3 ng/mL), which suggests that this is the limiting step in the pathway, probably reflecting the low affinity of human CYP17A1 for allopregnanolone.

    “Allopregnanolone levels in human fetal plasma are relatively high (male, about 200 ng/mL) but androsterone levels are significantly lower in comparison (male, about 3 ng/mL), which suggests that this is the limiting step in the pathway, probably reflecting the low affinity of human CYP17A1 for allopregnanolone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In males, tissue androsterone levels are highest in the adrenals, which is consistent with high CYP17A1 transcript levels and with a previous study showing androsterone present in the fetal adrenal at the end of the first trimester.

    “In males, tissue androsterone levels are highest in the adrenals, which is consistent with high CYP17A1 transcript levels and with a previous study showing androsterone present in the fetal adrenal at the end of the first trimester [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] I Savchuk, 2017, Androgenic potential of human fetal adreals at the end of the first trimester, Endocr Connect source

      Not checked in the free run.

  • The quote only partly states this claim.

    Findingown data

    Androsterone is significantly higher in male plasma than female plasma.

    “is significantly higher in male plasma than female plasma”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The genital tubercle expresses high levels of AKR1C2 and detectable AKR1C3.

    “the tissue expresses high levels of AKR1C2, as well as detectable AKR1C3”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Normal masculinization of the fetus is dependent upon androgen secretion by the testis, and androgens act initially during a critical masculinization programming window.

    “Normal masculinization of the fetus is dependent upon androgen secretion by the testis, and androgens act initially during a critical masculinization programming window to ensure normal male reproductive development [3–5].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3] G Shaw, 2000, Prostate formation in a marsupial is mediated by the testicular androgen 5α-androstane-3α,17β-diol, Proc Natl Acad Sci USA source

      Not checked in the free run.

    • cites [4] MW Leihy, 2004, Penile development is initiated in the tammar wallaby pouch young during the period when 5α-androstane-3α,17β-diol is secreted by the testes, Endocrinology source

      Not checked in the free run.

    • cites [5] M Welsh, 2008, Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism, J Clin Invest source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Masculinization of the human fetus depends on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle.

    “Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Disruption of the backdoor pathway causes disordered sex development (DSD) and failure of normal masculinization.

    “studies by Flück and colleagues [15] have shown that disordered sex development (DSD) will arise if the pathway is disrupted. In one individual with mutations in aldo-keto reductase type 1C2 (AKR1C2) and in another family with an added mutation in aldo-keto reductase type 1C4 (AKR1C4), there was failure of normal masculinization.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] CE Flück, 2011, Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation, Am J Hum Genet source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues.

    “Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In humans, male-specific morphological differentiation of the genital tubercle/penis begins around 10 weeks of gestation, with closure of the urethral groove, and is complete by about gestation weeks 15-16.5.

    “In humans, male-specific morphological differentiation of the genital tubercle/penis begins around 10 weeks of gestation (i.e., 8 weeks postconception), with closure of the urethral groove [6,7]. The process is complete by about gestation weeks 15–16.5 [6,7]”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] M Overland, 2016, Canalization of the vestibular plate in the absence of urethral fusion characterizes development of the human clitoris: the single zipper hypothesis, J Urol source

      Not checked in the free run.

    • cites [7] Y Li, 2015, Canalization of the urethral plate precedes fusion of the urethral folds during male penile urethral development: the double zipper hypothesis, J Urol source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The currently accepted model for masculinization is that circulating DHT, formed via the backdoor pathway in the fetal testis, is important for virilization alongside circulating testosterone.

    “Currently, the accepted model for masculinization is that circulating DHT, formed via the backdoor pathway in the fetal testis [15,16], is important for virilization alongside circulating testosterone.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] CE Flück, 2011, Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation, Am J Hum Genet source

      Not checked in the free run.

    • cites [16] A Biason-Lauber, 2013, Of marsupials and men: &quot;backdoor&quot; dihydrotestosterone synthesis in male sexual differentiation, Mol Cell Endocrinol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Levels of progesterone were high in the fetal male circulation and were likely derived principally from the placenta.

    “Levels of progesterone were also high and were likely to be derived principally from the placenta [22] (and see below).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] RC Tuckey, 2005, Progesterone synthesis by the human placenta, Placenta source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Data in Fig 2 indicate that in the fetal human, the backdoor pathway of androgen synthesis appears to depend initially on progesterone formed from pregnenolone, and in the fetus, the major de novo sources of pregnenolone are the adrenal and the testis.

    “Data in Fig 2 indicate that in the fetal human, the backdoor pathway of androgen synthesis appears to depend initially on progesterone formed from pregnenolone, and in the fetus, the major de novo sources of pregnenolone are the adrenal and the testis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The process of masculinization at the external genitalia starts in the late first/early second trimester, and the most intense phase of penile growth occurs later in the second trimester.

    “The process of masculinization at the external genitalia starts in the late first/early second trimester, and the most intense phase of penile growth occurs later in the second trimester [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] CB Gallo, 2013, Development of the penis during the human fetal period (13 to 36 weeks after conception), J Urol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Several other human fetal tissues express CYP11A1 and may be capable of pregnenolone synthesis, but based on transcript levels, they are likely to make a minor overall contribution to plasma levels.

    “Several other human fetal tissues express CYP11A1 and may be capable of pregnenolone synthesis [30] but, based on transcript levels, they are likely to make a minor overall contribution to plasma levels.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] V Pezzi, 2003, Profiling transcript levels for steroidogenic enzymes in fetal tissues, J Steroid Biochem Mol Biol source

      Not checked in the free run.

    • Rests on an assumption: Transcript levels of steroidogenic enzymes reflect their enzymatic activity in fetal tissues.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Masculinization of the fetus is dependent on the action of testosterone at the Wolffian ducts and on the action of DHT at the external genitalia.

    “Masculinization of the fetus is dependent on the action of testosterone at the Wolffian ducts and on the action of DHT at the external genitalia [25].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] JD Wilson, 1993, Steroid 5α-reductase 2 deficiency, Endocr Rev

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The substrate for adrenal androsterone synthesis must come from circulating allopregnanolone, because the adrenals lack SRD5A1 and appear unable to make 5α-reduced steroids.

    “The substrate for this reaction must, however, come from circulating allopregnanolone, because the adrenals lack SRD5A1 and appear unable to make 5α-reduced steroids.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Only the testes consistently express transcripts encoding all three enzymes SRD5A1, AKR1C2, and CYP17A1, which would suggest that they are a likely source of backdoor androgens.

    “Only the testes consistently express transcripts encoding all three enzymes, which would suggest that they are a likely source of backdoor androgens.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    In tissues that lack CYP17A1 but express SRD5A1 and AKR1C2, progesterone will be metabolized to allopregnanolone and potentially become available for metabolism by other tissues that express CYP17A1.

    “In tissues that lack CYP17A1 but express SRD5A1 and AKR1C2, progesterone will be metabolized to allopregnanolone and potentially become available for metabolism by other tissues that express CYP17A1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The fetal liver and placenta are unlikely to be responsible for sex differences in circulating androsterone, as they express little or no CYP17A1 and so will not metabolize allopregnanolone.

    “However, the fetal liver and placenta are unlikely to be responsible for sex differences in circulating androsterone, as they express little or no CYP17A1 and so will not metabolize allopregnanolone (the step at which sex differences are first seen in the backdoor pathway).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The recent demonstration that the alternative, backdoor pathway to DHT synthesis is also required for normal human fetal male development has shown that the process involves a complex interaction between different steroidogenic pathways.

    “However, the recent demonstration that the alternative, backdoor pathway to DHT synthesis is also required for normal human fetal male development [15,16] has shown that the process involves a complex interaction between different steroidogenic pathways.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] CE Flück, 2011, Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation, Am J Hum Genet source

      Not checked in the free run.

    • cites [16] A Biason-Lauber, 2013, Of marsupials and men: &quot;backdoor&quot; dihydrotestosterone synthesis in male sexual differentiation, Mol Cell Endocrinol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    The fetal adrenals do not express SRD5A1/2 and would not be expected to contribute to fetal 5αDHP or allopregnanolone production.

    “The fetal adrenals do not express SRD5A1/2 and would not be expected to contribute to fetal 5αDHP or allopregnanolone production.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    CYP17A1 is expressed in many fetal tissues, but with the exception of the adrenal and testis, transcript levels are low, suggesting enzyme activity is also probably low.

    “CYP17A1 is expressed in many fetal tissues [30] but, with the exception of the adrenal and testis, transcript levels are low ([30] and this study), suggesting enzyme activity is also probably low.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] V Pezzi, 2003, Profiling transcript levels for steroidogenic enzymes in fetal tissues, J Steroid Biochem Mol Biol source

      Not checked in the free run.

    • Rests on an assumption: Transcript levels of steroidogenic enzymes reflect their enzymatic activity in fetal tissues.

  • May combine two claims.

    Interpretationreasoning

    The placenta does not contain high levels of AKR1C2 transcript, and AKR1C4 is absent, but high substrate levels and lack of further metabolism via CYP17A1 probably explains the high tissue levels of allopregnanolone.

    “The placenta does not contain high levels of AKR1C2 transcript, and AKR1C4 is absent, but high substrate levels and lack of further metabolism via CYP17A1 probably explains the high tissue levels of allopregnanolone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Most progesterone in the fetal circulation comes from the placenta, which secretes progesterone directly into the fetal circulation at high levels.

    “It is likely, therefore, that most progesterone in the fetal circulation comes from the placenta, which secretes progesterone directly into the fetal circulation at high levels [31,32], similar to those reported here.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [31] CJ Partsch, 1991, The steroid hormonal milieu of the undisturbed human fetus and mother at 16–20 weeks gestation, J Clin Endocrinol Metab source

      Not checked in the free run.

    • cites [32] JR Pasqualini, 2005, Enzymes involved in the formation and transformation of steroid hormones in the fetal and placental compartments, J Steroid Biochem Mol Biol source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    Collections of fetal material were approved by the relevant ethics committees and written informed maternal consent was received from participants prior to inclusion.

    “Collections of fetal material in Aberdeen and Stockholm and by the Human Developmental Biology Resource (HBDR) were respectively approved by the NHS Grampian Research Ethics Committees (REC 04/S0802/21 and REC 15/NS/0123), the Regional Ethics Committee of Stockholm (EPN dnr 2014/1022-32), and the relevant research ethics committees in accordance with the United Kingdom Human Tissue Authority (HTA;”
    • Also stated in: § Ethics statement · plain
      “Written, informed, maternal consent was received from participants prior to inclusion in the study.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The Cohen method of maximum likelihood estimation was used to account for nondetectable samples in the analysis.

    “The Cohen method of maximum likelihood estimation of the mean and variance was used to account for nondetectable samples in the analysis [76].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] AC Cohen Jr., 1959, Simplified estimators for the normal distribution when samples are singly censored or truncated, Technometrics source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The endocrine control of masculinization in the human fetus is mediated through circulating testosterone and androsterone and is dependent on a complex interaction and exchange between the testes and nongonadal tissues, particularly the placenta.

    “the endocrine control of masculinization in the human fetus is mediated through circulating testosterone and androsterone and is dependent on a complex interaction and exchange between the testes and nongonadal tissues, particularly the placenta”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    If placental AKR1C2 is of maternal origin, then this would be expected to affect backdoor steroid production in the placenta.

    “if placental AKR1C2 is of maternal origin, then this would be expected to affect backdoor steroid production in the placenta”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Tissue steroid levels were quantified by LC-HRMS.

    “Extraction and quantification of tissue steroid levels by LC-HRMS have been described previously [21].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] ZC Johnston, 2018, The human fetal adrenal produces cortisol but no detectable aldosterone throughout the second trimester, BMC Med source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Steroid levels in culture media were measured by GC-MS/MS.

    “steroid levels in the culture media were measured by GC-MS/MS as described below”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisereasoning

    Testosterone from the fetal testes acts as an essential substrate for DHT synthesis at the external genitalia.

    “Testosterone from the fetal testes also acts as an essential substrate for DHT synthesis at the external genitalia”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    DHT can be metabolized to androstanediol by all AKR1 enzymes, and AKR1C2 in particular.

    “DHT can be metabolized to androstanediol by all AKR1 enzymes, and AKR1C2 in particular [12,50]”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [12] TL Rizner, 2014, Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism, Steroids source

      Not checked in the free run.

    • cites [50] S Steckelbroeck, 2004, Human cytosolic 3α-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3β-hydroxysteroid dehydrogenase activity: implications for steroid hormone metabolism and action, J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Data were checked for normality and heterogeneity of variance and normalized by log transformation as appropriate.

    “Data were checked for normality and heterogeneity of variance and normalized by log transformation as appropriate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Fetal sex was confirmed by PCR of ZFX and SRY using genomic DNA.

    “Fetal sex was confirmed by PCR of ZFX and SRY using genomic DNA and primers described in S4 Table [71].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71] S Fain, 1995, Gender indentification of human and mammalian wildlife species from PCR amplified sex linked genes, Proc Am Acad Forensic Sci

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Maternal progesterone levels are reduced during IUGR, suggesting that placental steroidogenesis is affected.

    “maternal progesterone levels are reported to be reduced during IUGR [62], suggesting that placental steroidogenesis is affected”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [62] U Pecks, 2016, Maternal serum lipid, estradiol, and progesterone levels in pregnancy, and the impact of placental and hepatic pathologies, Geburtshilfe Frauenheilkd source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    It is not surprising that testosterone is detectable in most female samples, as levels of both potential substrates (androstenedione and androstenediol) are present in the circulation, and the enzymes necessary to form testosterone (HSD3B and HSD17B3) are expressed in nongonadal tissues.

    “It is perhaps not surprising that testosterone is detectable in most female samples, as levels of both potential substrates (androstenedione and androstenediol) are present in the circulation, and the enzymes necessary to form testosterone (HSD3B and HSD17B3) are expressed in nongonadal tissues.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The pathway is driven by placental progesterone production, which acts as substrate for androsterone synthesis, primarily in nongonadal tissues.

    “the pathway is driven by placental progesterone production, which acts as substrate for androsterone synthesis, primarily in nongonadal tissues”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Most circulating progesterone in the fetal circulation comes from the placenta.

  • Classified as probably not a substantial claim.

    Premisecitation

    Androgen is required for masculinization of the external genitalia.

    “androgen is required for masculinization of the external genitalia (reviewed in [63])”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [63] A Jost, 1970, Hormonal factors in the sex differentiation of the mammalian foetus, Philos Trans R Soc Lond Biol source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Testicular testosterone must be converted to DHT at the target organ to induce masculinization.

    “testicular testosterone must be converted to DHT at the target organ to induce masculinization [64]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [64] JD Wilson, 2001, The role of 5α-reduction in steroid hormone physiology, Reprod Fertil Dev source

      Not checked in the free run.

62 of 62

  • The paper relies on this without support.

    Transcript levels of steroidogenic enzymes reflect their enzymatic activity in fetal tissues.

    “based on transcript levels (this study and [28–30])”
    “suggesting enzyme activity is also probably low”

  • The paper relies on this without support.

    Most circulating progesterone in the fetal circulation comes from the placenta.

    “Most circulating progesterone in the fetal circulation is likely to come from the placenta”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Claim that all backdoor steroids were detected includes an unmeasured steroid

    Minor

    The text says all backdoor steroids were detectable in the gonadal GC-MS/MS samples, whereas the accompanying figure caption says 5αDHP, a backdoor intermediate, was not measured.

  • Supplementary data described as having both five and six sheets

    Minor

    The methods state that S1 Data has five sheets, but the discussion cites Sheet 6 of that same file.

  • Androstenedione described as both detectable and undetectable in fetal plasma

    Minor

    The results say androstenedione was detectable in most male fetal plasma samples, but the discussion says it was undetectable in fetal plasma.

3 of 3

The citation lists haven’t been fetched yet

References 85 · Cited by 177

Research works printed but not linked: 2

  1. PJ O'Shaughnessy, 2007, Developmental changes in human fetal testicular cell numbers and messenger ribonucleic acid levels during the second trimester, J Clin Endocrinol Metab, 92, 4792, 10.1210/jc.2007-1690 · DOI
  2. S Fain, 1995, Gender indentification of human and mammalian wildlife species from PCR amplified sex linked genes, Proc Am Acad Forensic Sci, 1, 34

Authors
O’Shaughnessy, P. J., Antignac, J. P., Le Bizec, B., Morvan, M.-L., Svechnikov, K., Söder, O., Savchuk, I., Monteiro, A., Soffientini, U., Johnston, Z. C., Bellingham, M., Hough, D., Walker, N., Filis, P., & Fowler, P. A.
Affiliations
  • University of Glasgow (GB)
  • Laboratoire D'étude des Résidus et Contaminants Dans les Aliments (FR)
  • Karolinska University Hospital (SE)
  • University of Aberdeen (GB)
ORCID
Venue
PLOS Biology
Publisher
Public Library of Science (PLoS)
Published
2019-02-14
Volume
17
Issue
2
Pages
e3000002
ISSN
  • 1544-9173 (ISSN-L)
  • 1545-7885
DOI
10.1371/journal.pbio.3000002
PMC
PMC6375548
OpenAlex
W2843507938
Cited by
177
References
85
Citations by year
2026: 7 · 2025: 17 · 2024: 39 · 2023: 18 · 2022: 29 · 2021: 22 · 2020: 31 · 2019: 13 · 2018: 1
FWCI
7.86
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (acceptedVersion, cc-by-sa)
  • Aberdeen University Research Archive (Aberdeen University) (submittedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Chief Scientist Office (CZG/4/742)
  • NHS Grampian Endowments (08/02 and 15/1/010)
  • Glasgow Children’s Hospital Charity (YRSS/PHD/2016/05)
  • FP7 Ideas: European Research Council (212885)
  • Medical Research Council (MR/L010011/1, MR/K501335/1)
  • H.K.H. Kronprinsessan Lovisas Förening för Barnasjukvård
  • Stiftelsen Gunvor och Josef Aners
  • Stiftersen Jane och Dan Olssons
  • Stiftelsen Tornspiran
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
  • Health Sciences › Medicine › Reproductive Medicine › Sperm and Testicular Function
Keywords
androsterone, masculinization, dihydrotestosterone, fetal testis, placenta, fetal liver, aldo-keto reductase, CYP17A1, placental insufficiency, disorders of sex development, second trimester fetus
MeSH terms
Androgens, Female, Fetus, Humans, Male, Ovary, Pregnancy, Pregnancy Trimester, Second, RNA, Messenger, Dihydrotestosterone, Testis, Metabolic Networks and Pathways, Masculinity

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

Statistics and arithmetic are recomputed, not judged.

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