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Departments of Growth and Reproduction (K.B., N.E.S., A.-M.A.), of Fetal Medicine (C.J.), and of Clinical Genetics (A.M.L.), University Hospital of Copenhagen, Rigshospitalet, DK-2100 Copenhagen, Denmark; Department of Clinical Biochemistry (A.S.C.), Statens Serum Institute, DK-2300 Copenhagen, Denmark; and Department of Obstetrics and Gynecology (F.S.J.), Fetal Medicine Unit, University Hospital of Copenhagen, Hvidovre Hospital, DK-2650 Hvidovre, Denmark
Address all correspondence and requests for reprints to: Anna-Maria Andersson, University Department of Growth and Reproduction, Rigshospitalet, GR 5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: anna{at}rh.dk.
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Abstract |
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Methods: Amniotic fluid from 91 pregnant women undergoing amniocentesis was analyzed for INSL3 and testosterone (T) levels. Data were related to gestational age (15–25 wk) at amniocentesis and to sex (45 males and 48 females).
Results: INSL3 was present in amniotic fluid from all but one of the investigated male fetuses (range: <0.02–0.36 ng/ml; mean ± SD: 0.12 ± 0.07), whereas the hormone was undetectable in the female fetuses. T was significantly higher in male (range: 0.54–1.71 nmol/liter; mean ± SD: 1.04 ± 0.30) as compared with in female amniotic fluid (range: 0.19–0.50 nmol/liter; mean ± SD: 0.34 ± 0.06) (P < 0.001). In males there was no correlation between INSL3 and T. A statistically borderline negative association was found between INSL3 and gestational age (P = 0.07), whereas the corresponding association was not significant for T (P = 0.12). In contrast, T in females correlated positively with gestational age (P = 0.02).
Conclusions: INSL3 is clearly present in human male amniotic fluid in the second trimester, where abdominal testis translocation takes place. In contrast, the hormone is undetectable in female amniotic fluid. The prenatal presence of INSL3 supports the hypothesis that this hormone is essential for testicular descent in humans.
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Introduction |
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Human INSL3 serum levels have been described from early infancy throughout childhood, puberty, and adulthood (4, 5, 6, 7). However, even though the most well-characterized function of INSL3 is exerted in utero, human prenatal INSL3 levels have only been characterized in a single recent study (8). Therefore, we have taken the approach to analyze INSL3, along with testosterone (T), levels in amniotic fluid from pregnant women. Amniocentesis is normally performed in the early second trimester, a time period that overlaps substantially with testicular development and testicular descent in the male fetus (9).
Insl3 mediates the outgrowth of the ligament gubernaculum, thereby facilitating the anchoring of the testes to the abdominal wall, close to the inguinal canal (10). Therefore, Insl3 knockout mice appear with testes located intra-abdominally, due to an undifferentiated gubernaculum (2). After the abdominal translocation of the testes, which may be completed already at gestational wk 15 in humans, the subsequent transinguinal and inguinoscrotal migrations are responsible for the passage of the testes through the inguinal canal and further down to the bottom of the scrotum (1). These two processes have traditionally been regarded as a single inguinoscrotal phase mediated primarily by androgens. Before the transinguinal migration of the testes, a timely pause is present during which the part of the gubernaculum embedded in the abdominal wall undergoes extensive remodeling/enlargement, thereby dilating the inguinal canal, making room for the testes to pass through (1).
Incomplete descent of the testes, cryptorchidism, is one of the most common congenital malformations in infant boys (11), and a risk factor for reduced semen quality and the development of testicular cancer in adult life (12). It has been hypothesized that these male reproductive health problems often share a common fetal origin and may be related to prenatal exposure to endocrine disrupting chemicals, e.g. phthalate esters (13). Interestingly, rodent studies have shown that gubernacular lesions and cryptorchidism, induced by prenatal exposure to phthalate esters, are associated with reduced Insl3 expression (14).
In addition, adult female ovarian theca cells produce INSL3 (15), although in minimal amounts compared with male INSL3 production. The hormone is undetectable in amniotic fluid from human female fetuses (8).
In this paper we present INSL3 levels in human second-trimester amniotic fluid, which provide information of the potential importance of this hormone in human testicular descent.
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Subjects and Methods |
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Pregnant women undergoing amniocentesis at the University Hospitals of Copenhagen (Rigshospitalet and Hvidovre Hospital) in 2007 were invited to participate. In Denmark, pregnant women are offered a first-trimester nuchal translucency scanning, including biochemistry for detection of chromosomal abnormalities. In cases of increased risk, an amniocentesis is offered. Karyotyping on amniotic fluid cells was performed at Rigshospitalet. For the present study, only redundant supernatant not necessary for karyotyping was used. Available information included maternal age, gestational age at amniocentesis (based on ultrasonography), and genetic test outcome. A total of 91 women participated in our study; of these, three had a dichorionic twin pregnancy, giving a total of 94 fetuses (46 males, 48 females). A single male fetus had a trisomy 18 and was excluded from this study. Twin fetuses were included. All women had their amniocentesis performed in gestational wk 15–25 (wk 15–21 for male fetuses and 15–25 for female fetuses). Written informed consent was given by all participants. The study was approved by the local ethical committee (KF-01-259468, KF-11-2006-4093).
Hormone analysis
Amniotic fluid was stored at –20 C. INSL3 concentrations in amniotic fluid were determined using a human INSL3-specific fluorescence immunoassay (for details, see Ref. 4). To adapt to amniotic fluid matrix, female amniotic fluid was used as new blank [female amniotic fluid is reported not to contain detectable amounts of INSL3 (8), and the female amniotic fluid used as blank in the present study was undetectable when analyzed under traditional assay conditions]. Standards consisted of 100 µl blank spiked with defined concentrations of synthetic human INSL3 (0.02–1.60 ng/ml). The detection limit of this set up was 0.02 ng/ml. Intraassay coefficient of variance was 0.9–48.8% in the very low range (0.2–0.8 ng/ml) and 0.1–18.9% in the higher range (0.9–1.6 ng/ml). All samples were analyzed in a single assay run.
T was measured by liquid-chromatography mass-spectrometry (16) at Statens Serum Institute using an accredited method. The limit of detection was 0.01 nmol/liter.
Statistical analyses
Data are presented as range and mean ± SD. In male amniotic fluid, INSL3 and T levels adhered to normal distribution. INSL3 and T correlation was investigated by Pearson’s correlations. The influence of gestational age and maternal age on INSL3 and T was tested using linear regression. In female amniotic fluid, T levels adhered to normal distribution after Ln transformation. In females the influence of gestational age and maternal age on natural logarithm (Ln) T was tested using linear regression. Gender differences in T levels were examined using the t test.
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Results |
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All but one male amniotic fluid samples had measurable INSL3 levels [(Fig. 1A
) (range: <0.02–0.36 ng/ml; mean ± SD: 0.12 ± 0.07)], and all had measurable T levels [(Fig. 1B) (range: 0.54–1.71 nmol/ liter; mean ± SD: 1.04 ± 0.30)]. There was no correlation between INSL3 and T (P = 0.98). In the linear regression analyses, there was a borderline negative association between INSL3 and gestational age (P = 0.07), whereas the negative association was not significant between T and gestational age (P = 0.12). Neither of the Leydig cell hormones was associated with maternal age at amniocentesis (P = 0.11 for INSL3 and P = 0.28 for T) (Fig. 1).
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INSL3 was unmeasurable (<0.02 ng/ml), whereas T was measurable [(Fig. 2) (range: 0.19–0.50 nmol/liter; mean ± SD: 0.34 ± 0.06)] in all female amniotic fluid samples. A significant positive association was found between Ln T and gestational age (P = 0.02). There was no association between Ln T and maternal age (P = 0.35). T levels were significantly higher in male compared with in female amniotic fluid (P < 0.001) (Fig. 2).
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Discussion |
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Our findings are in accordance with data presented in the recent study of INSL3 levels in human amniotic fluid (8), in that it also found INSL3 levels to decline with increasing gestational age in males. However, despite usage of the same antibodies and detection limits, Anand-Ivell et al. (8) reported INSL3 undetectable in 32% of male amniotic fluid samples, whereas only a single sample was undetectable in the present study. This discrepancy is most likely due to our adaptation of the assay to amniotic fluid rather than biological differences in the samples analyzed.
Importantly, our data indicate high INSL3 production also after wk 15, where the testes may already be anchored close to the inguinal canal (1). This renders it possible that INSL3 is also involved in the subsequent process of dilating the inguinal canal via gubernacular enlargement, thereby facilitating the transinguinal testis migration. This theory is further supported by the phenotype of transgenic female mice overexpressing Insl3. Such mice are characterized not only by ovary descent, but also the development of the processus vaginalis (17). In this regard, analysis of INSL3 amniotic fluid levels throughout embryogenesis would have been optimal. However, of note is the presence of INSL3 in male cord blood (7), indicating a continued production of INSL3 throughout the remaining fetal development.
Cryptorchidism in humans is predominantly of the mild type, involving only the latter, androgen dependent, inguinoscrotal part of testicular descent (11). However, reduced INSL3 expression could theoretically cause a delay in the entire process of gubernacular outgrowth and testicular descent, thereby also affecting the final inguinoscrotal phase. Indeed, many boys born with cryptorchidism have spontaneous testis descent within the first 3 months of life (11), suggesting that process delay could be a contributing factor.
Maximal gender differences in amniotic T levels were reported to occur around gestational wk 12–18 (18). In agreement with this, we found markedly higher T levels in male compared with female amniotic fluid. Amniotic fluid T concentrations in the two sexes should become similar with increasing age because testicular T biosynthesis in the male decreases, and T synthesis from nontesticular sources increases in both male and female fetuses. Indeed, we found a significant positive correlation between amniotic T levels and gestational age in female and a tendency toward a negative corresponding association in male amniotic fluid, resulting in T levels approaching each other in the two sexes toward gestational wk 20–25.
Numerous factors influence hormone levels in amniotic fluid, including fluid volume, hormone stability, and fetal growth relative to the growth of the hormone-producing organ. In a recent study of developmental changes in human fetal testes (9), the exponential increase in Leydig cell numbers registered in the first part of second trimester slowed down after gestational wk 15. Gene expression related to INSL3 and T production was constant during the second trimester. In comparison, Sertoli and germ cell numbers and testis volume in general increased exponentially through the entire second trimester (9). Thus, the negative association between gestational age and INSL3 (and T) levels in amniotic fluid reported here may reflect constant levels of expression per Leydig cell combined with a decreased Leydig cell to testis volume ratio. Furthermore, amniotic fluid volume increases approximately 3-fold between gestational wk 15 and 22 (19). No exact data are available on the stability of INSL3 in amniotic fluid. However, amniotic fluid concentrations of the structurally related hormones insulin-like growth factor I and II represent very well fetal serum levels of the same hormones (20).
In conclusion, INSL3 was clearly present in human second-trimester male amniotic fluid. In contrast, the hormone was absent from female amniotic fluid. The presence of INSL3 in male fetuses suggests that the hormone is involved in the abdominal testis translocation in humans. Continued production of INSL3 after gestational wk 15 could suggest an involvement of the hormone in the modification of the inguinal canal through continued enlargement of the gubernaculum, thereby facilitating also the subsequent transinguinal testis migration.
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Footnotes |
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First Published Online July 8, 2008
Abbreviations: INSL3, Insulin-like factor 3; Ln, natural logarithm; T, testosterone.
Received February 13, 2008.
Accepted June 30, 2008.
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This article has been cited by other articles:
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  R. Ivell and R. Anand-Ivell Biology of insulin-like factor 3 in human reproduction Hum. Reprod. Update, July 1, 2009; 15(4): 463 - 476. [Abstract] [Full Text] [PDF]
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