Linn Salto Mamsen

Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

BACKGROUND Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimester gonads in relation to maternal smoking. METHODS The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated stereological methods were used to estimate gonadal cell numbers in histological sections. Results were also evaluated in the context of previously published data on ovaries from our laboratory. RESULTS A significant reduction in the number of germ cells by 55% [95% confidence interval (CI) 74-21% reduction, P = 0.004] and somatic cells by 37% (95% CI 59-3%, P = 0.023) was observed in testes prenatally exposed to maternal cigarette smoking, compared with unexposed. The effect of maternal smoking was dose-dependent being higher in the heavy smokers and remained consistent after adjusting for possible confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS Prenatal exposure to maternal cigarette smoke reduces the number of germ and somatic cells in embryonic male and female gonads. This effect may have long-term consequences on the future fertility of exposed offspring. These findings may provide one potential cause of the reduced fertility observed during recent years.

Concentration of perfluorinated compounds and cotinine in human foetal organs, placenta, and maternal plasma

BACKGROUND Perfluoroalkyl substances (PFASs) are bio-accumulative pollutants, and prenatal exposure to PFASs is believed to impact human foetal development and may have long-term adverse health effects later in life. Additionally, maternal cigarette smoking may be associated with PFAS levels. Foetal exposure has previously been estimated from umbilical cord plasma, but the actual concentration in foetal organs has never been measured. OBJECTIVES The concentrations of 5 PFASs and cotinine - the primary metabolite of nicotine - were measured in human foetuses, placentas, and maternal plasma to evaluate to what extent these compounds were transferred from mother to foetus and to determine if the PFAS concentrations were associated with maternal cigarette smoking. METHODS Thirty-nine Danish women who underwent legal termination of pregnancy before gestational week 12 were included; 24 maternal blood samples were obtained together with 34 placental samples and 108 foetal organs. PFASs and cotinine were assayed by liquid chromatography/triple quadrupole mass spectrometry. RESULTS In foetal organs, the average concentrations of perfluorooctanesulphonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDa), and perfluorodecanoic acid (PFDA) were 0.6ng/g, 0.2ng/g, 0.1ng/g, 0.1ng/g, and 0.1ng/g, respectively. A significant positive correlation was found between the exposure duration, defined as foetal age, and foetal to maternal ratio for all five PFASs and cotinine. Smokers presented 99ng/g cotinine in plasma, 108ng/g in placenta, and 61ng/g in foetal organs. No correlation between the maternal cotinine concentrations and PFAS concentrations was found. CONCLUSIONS PFASs were transferred from mother to foetus, however, with different efficiencies. The concentrations of PFOS, PFOA, PFNA, PFUnDA, and PFDA in foetal organs were much lower than the maternal concentrations. Furthermore, a significant correlation between the exposure duration and all of the evaluated PFASs was found. The health-compromising concentrations of these substances during foetal development are unknown.

Proteolytic processing of anti-Müllerian hormone differs between human fetal testes and adult ovaries

From early embryonic life, anti-Müllerian hormone (AMH) is produced by Sertoli cells and is essential for male sex differentiation. In females, AMH is produced by immature granulosa cells (GCs) but a definitive function in females is uncertain. We have assessed the cellular localization and specificity of a panel of five novel high-affinity AMH monoclonal antibodies. Two recognize the mature C-terminal form of AMH, whereas three recognize the active pro-mature form of AMH in human tissue. The antibodies were tested on fetal male testicular and mesonephric tissue aged 8-19 weeks post conception (pc), fetal male serum aged 16-26 weeks pc and human immature GCs by immunofluorescence, immunohistochemistry, ELISA and western blotting. The active pro-mature forms of AMH were expressed in both Sertoli cells from human fetal testis and human immature GCs. In contrast, the mature C-terminal form of AMH was hardly detected in Sertoli cells, but was readily detected in GCs. This particular form was also located to the nucleus in GCs, whereas the other investigated AMH forms remained in the cytoplasm. Interestingly, the distribution of the AMH forms in the fetal serum of boys showed that the fraction of inactive precursor AMH only accounted for 4.5% ± 0.6 (mean ± SD) of the total AMH measured, and the remaining AMH was the active pro-mature form. Furthermore, western blot analysis demonstrated a number of previously unrecognized molecular forms of AMH. The present findings suggest that processing of AMH is a tightly regulated process, which is likely to be important for the function of AMH and which differs between the two sexes.

Assessment of global DNA methylation in the first trimester fetal tissues exposed to maternal cigarette smoking

AimsMaternal cigarette smoking during pregnancy increases the risk of negative health consequences for the exposed child. Epigenetic mechanisms constitute a likely link between the prenatal exposure to maternal cigarette smoking and the increased risk in later life for diverse pathologies. Maternal smoking induces gene-specific DNA methylation alterations as well as global DNA hypermethylation in the term placentas and hypomethylation in the cord blood. Early pregnancy represents a developmental time where the fetal epigenome is remodeled and accordingly can be expected to be highly prone to exposures with an epigenetic impact. We have assessed the influence of maternal cigarette smoking during the first trimester for fetal global DNA methylation.Methods and resultsWe analyzed the human fetal intestines and livers as well as the placentas from the first trimester pregnancies. Global DNA methylation levels were quantified with ELISA using a methylcytosine antibody as well as with the bisulfite pyrosequencing of surrogate markers for global methylation status, LINE-1, and AluYb8. We identified gender-specific differences in global DNA methylation levels, but no significant DNA methylation changes in exposure responses to the first trimester maternal cigarette smoking.ConclusionsAcknowledging that only examining subsets of global DNA methylation markers and fetal sample availability represents possible limitations for the analyses, our presented results indicate that the first trimester maternal cigarette smoking is not manifested in immediate aberrations of fetal global DNA methylation.

Hallmarks of Human Small Antral Follicle Development: Implications for Regulation of Ovarian Steroidogenesis and Selection of the Dominant Follicle

Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected in connection with cryopreservation of ovarian tissue for fertility preservation. The antral follicles (ranging from 3 to 13 mm) were generally aspirated from one ovary surgically removed during the natural cycle, and the follicular fluid (FF) and the granulosa cells (GC) were isolated and snap-frozen. In FF, the following hormones were measured: inhibin-B, inhibin-A, AMH, follistatin, PAPP-A, estradiol, progesterone, testosterone, and androstenedione. In GC, mRNA gene expressions using q-PCR were measured for the following genes: FSHR, AMH, CYP19, and AR. All samples in which one of the abovementioned parameters was measured were included, but typically multiple parameters were measured. Highly significant differences in concentration and follicular content in relation to follicular diameter were found for all measured hormones despite massive variability in-between follicles for any given diameter. The results demonstrate that profound changes take place in the hormonal microenvironment around follicular diameters of 8–11 mm corresponding to when follicular selection occurs. At this point, inhibin-B and inhibin-A showed distinct peaks concomitant with a significant reduction in both AMH protein and mRNA expression. Concentrations of inhibins, androgens, FSHR, and AR were intimately associated, and it is suggested that inhibin-B in combination with PAPP-A and thereby IGF2 activity exerts important paracrine signaling at follicular selection. At the same time upregulation of estradiol synthesis and CYP19 mRNA expression increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation of steroidogenesis differs. The profound changes taking place around follicular selection highlight important paracrine actions of TGF-β family members and IGFs for securing dominance of the selected follicle.

Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking

Prenatal exposure to maternal cigarette smoking increases the risk of intrauterine growth retardation, adverse pregnancy outcomes, and diseases later in life. Exposure can result in postnatal global and gene-specific DNA methylation changes, with the latter well documented for the CYP1A1 and AHRR genes involved in the detoxification of xenobiotic substances. This study assessed the impact of exposure to maternal smoking on first trimester fetal CYP1A1 and AHRR mRNA expression and DNA methylation for CpG-sites displaying maternal smoking during pregnancy-mediated methylation changes at birth. The analyses included first trimester (6-12 weeks) placentas (N=39) and livers (N=43). For AHRR, exposure to maternal smoking was associated with increased DNA methylation in the placentas of female fetuses; mRNA expression, however, was unchanged. While exposure to maternal smoking was not associated with AHRR DNA methylation changes in fetal livers; mRNA expression was increased. For CYP1A1, exposure to maternal smoking was not associated with fetal DNA methylation changes whereas mRNA expression increased in placentas and male fetal livers. These results show that first trimester exposure to maternal smoking is associated with CYP1A1 and AHRR DNA methylation and mRNA expression changes. However, the results also indicate that maternal smoking during pregnancy-mediated postnatal CYP1A1 and AHRR DNA methylation changes are not imprinted during the first trimester.

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry (IHC). SRY/SOX9 expression initiated in testis around day 40 pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53 pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1 was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human sex-differentiation is defined, with identification of new genes of potential importance.

Reply: Methodological considerations in measuring different AMH splice forms using ELISA: validity of proAMH ELISA

Sir, We would like to thank McLennan and Pankhurst for raising the issue of how different forms of AMH are measured using ELISA-based methods (McLennan and Pankhurst, 2016). The main focus of our paper (Mamsen et al., 2015) was to detect different AMH forms in human fetal testis tissue and in granulosa cells from human small antral follicles using immunohistochemistry, immunofluorescence and western blot techniques. In addition, we included data on the distribution of AMH forms in four fetal male serum samples. As we understand McLennan and Pankhurst question the ELISA method we used to detect the two different forms of AMH, in the four fetal male serum samples. AMH is secreted as an inactive full-length precursor (proAMH) and is proteolytically cleaved into a non-covalent complex, AMH-N,C, which can dissociate into a pro-fragment (N-terminal) and a mature fragment (C-terminal) (di Clemente et al., 2010). The ELISA method we used was previously validated in the methodological article by Robertson et al. (2014). The methodology is straightforward used in many ELISA kits. This assay is based on monoclonal antibodies with epitopes mapped to either the N-terminal (mAb-24) or the C-terminal (mAb-32) of AMH. Thus this assay recognizes both proAMH and AMH-N,C but not the dissociated pro-fragment or the mature fragment alone. The used ELISAwas validated using proAMH (including a point-mutated non-cleavable site in the recombinant AMH), AMH-N,C, pro-fragment and mature fragment. As shown in Table I, this assay selectively recognize proAMH and AMH-N,C. By pretreating the samples with SDS (0.066% final concentration) and thereby disrupting the AMH-N,C complex only full-length proAMH is detected thus enabling discrimination of the pro-fragment and AMH-N,C (Table I). In the paper we specifically state that SDS was used to split the noncovalent complex of the Nand C-terminal part of AMH prior to measurement. However, McLennan and Pankhurst show that they are unable to detect AMH in ELISAs, when even low concentrations of SDS are present. It is well known that small SDS concentrations, between 0.005 and 0.05%, can be used to denature the proteins to a liner form without disrupting the ELISA detection system (Winkler et al., 1987; Morrison et al., 2007). As we demonstrate in our study and in Table I the SDS concentrations used with the AnshLabs picoAMH ELISA allow detection to takeplace.A slight impairment of the assay is expected when adding even these small amounts of SDS to the analyte (31% reduction in the AnshLabs picoAMH ELISA assay). The loss in the observed signal in presence of SDS was compensated for in the standards and thereby in the calculation of the results. McLennan and Pankhurst report a low detection of AMH in male serum in the AL-124 picoAMH ELISA with the addition of SDS in low concentrations. Since the standard used in the picoAMH Ansh ELISA kit does not contain proAMH only AMH-N,C no signal would be expected when treated with SDS. Further, McLennan and Pankhurst claim that male serum is known to contain high levels of proAMH (Pankhurst and McLennan, 2013, 2016). We have therefore included data from adult male and female serum using the same picoAMH ELISA kit from Ansh lab. AL-124 as used in this article (Mamsen et al., 2015) (Table II). In adult serum of both gender we detected only low concentrations of the proAMH — between 3 and 4% in both genders supporting the measurements we did on fetal male serum, which were 4.7+0.5% (mean+SD). We are therefore unable to confirm the results from McLennan and Pankhurst.