Amniotic fluid and serum biomarkers from women with neural tube defect–affected pregnancies: a case study for myelomeningocele and anencephaly

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The authors sought to identify novel biomarkers for early detection of neural tube defects (NTDs) in human fetuses.


Amniotic fluid and serum were drawn from women in the second trimester of pregnancy. The study group included 2 women pregnant with normal fetuses and 4 with fetuses displaying myelomeningocele (n = 1), anencephaly (n = 1), holoprosencephaly (n = 1), or encephalocele (n = 1). Amniotic fluid stem cells (AFSCs) were isolated and cultured. The cells were immunostained for the stem cell markers Oct4, CD133, and Sox2; the epigenetic biomarkers H3K4me2, H3K4me3, H3K27me2, H3K27me3, H3K9Ac, and H3K18Ac; and the histone modifiers KDM6B (a histone H3K27 demethylase) and Gcn5 (a histone acetyltransferase). The levels of 2 markers for neural tube development, bone morphogenetic protein–4 (BMP4) and sonic hedgehog (Shh), were measured in amniotic fluid and serum using an enzyme-linked immunosorbent assay.


The AFSCs from the woman pregnant with a fetus affected by myelomeningocele had higher levels of H3K4me2, H3K4me3, H3K27me2, and H3K27me3 and lower levels of KDM6B than the AFSCs from the women with healthy fetuses. The levels of H3K9ac, H3K18ac, and Gcn5 were also decreased in the woman with the fetus exhibiting myelomeningocele. In AFSCs from the woman carrying an anencephalic fetus, levels of H3K27me3, along with those of H3K9Ac, H3K18ac, and Gcn5, were increased, while that of KDM6B was decreased. Compared with the normal controls, the levels of BMP4 in amniotic fluid and serum from the woman with a fetus with myelomeningocele were increased, whereas levels of Shh were increased in the woman pregnant with a fetus displaying anencephaly.


The levels of epigenetic marks, such as H3K4me, H3K27me3, H3K9Ac, and H3K18A, in cultured AFSCs in combination with levels of key developmental proteins, such as BMP4 and Shh, are potential biomarkers for early detection and identification of NTDs in amniotic fluid and maternal serum.

Abbreviations used in this paper:AFSC = amniotic fluid stem cell; BMP4 = bone morphogenetic protein–4; ELISA = enzymelinked immunosorbent assay; NTD = neural tube defect; Shh = sonic hedgehog.

Article Information

* Drs. Tsurubuchi, Ichi, and Shim contributed equally to this work.

Address correspondence to: C. Shekhar Mayanil, Ph.D., M/C 204, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, 2430 N. Halsted St., Chicago, IL 60614. email:

Please include this information when citing this paper: published online August 23, 2013; DOI: 10.3171/2013.7.PEDS12636.

© AANS, except where prohibited by US copyright law.



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    Amniotic fluid stem cells express stem cell markers. Neurospheres were grown from total AFSCs as described in Methods. Maternal cells do not survive in neurosphere culture medium (Neurobasal Media with basic fibroblast growth factor and epidermal growth factor). Embryonic stem cells grew in Neurobasal Media with these 2 growth factors and formed neurosphere colonies and represented AFSCs. Colonies were immunostained for Sox2, Oct4, CD133, and nestin. The DAPI stain was used to stain cell nuclei. The Sox2, Oct4, CD133, and nestin markers were present in AFSCs from tissues recovered from normal (A), myelomeningocele (MM)- (B), and anencephaly- (C) affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

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    Differentiated amniotic fluid stem cells express different markers for cell differentiation. Neurospheres were grown in Neurobasal Media without growth factors for 7 days and were immunostained for astrocyte (GFAP), neuron (TuJ1), and oligodendrocyte (O4) markers. The DAPI stain was used to stain the cell nuclei. The levels of GFAP and TuJ1 were similar in AFSCs obtained from normal (A), myelomeningocele (MM)- (B), and anencephaly- (C) affected pregnancies. The O4 staining intensity was MM > anencephaly > normal. Each experiment was performed at least 4 times and in 3 technical replicates.

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    Methylation of H3K4 assessed through immunostaining of cultured AFSCs from normal and from MM- and anencephaly-affected pregnancies. A: Staining for H3K4me2 and Oct4. B: Staining for H3K4me3 and Oct4. All cells were counterstained with DAPI nuclear stain. Presence of Oct4, a stem cell marker, demonstrated that stained cells were of embryonic and not of maternal origin. Compared with the cells from normal pregnancies, both H3K4me2 and H3K4me3 immunostaining were increased in cells from MM- but not in cells from anencephaly-affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

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    Methylation of H3K27 and KDM6B assessed through immunostaining of cultured AFSCs from normal, myelomeningocele (MM)-, and anencephaly-affected pregnancies. A: Staining for H3K27me2 and Oct4. B: Staining for H3K27me3 and Oct4. C: Staining for KDM6B and Oct4. All cells were counterstained with DAPI nuclear stain. The immunostaining of H3K27me2 and H3K4me3 was increased in the MM-affected pregnancy, whereas in the anencephaly-affected pregnancy, only H3K27me3 staining was increased. The immunostaining of KDM6B was decreased in both MM- and anencephaly-affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

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    Acetylation of H3K9, H3K18, and Gcn5 assessed through immunostaining of AFSCs from normal and MM- and anencephaly-affected pregnancies. A: Staining for H3K9Ac and Gcn5. B: Staining for H3K18Ac and Gcn5. All cells were counterstained with DAPI nuclear stain. In the AFSCs cultured from the woman pregnant with an anencephalic fetus, the staining intensity of H3K9Ac and H3K18Ac was increased. Each experiment was performed at least 4 times and in 3 technical replicates.

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    Summary of the densitometry results for the epigenetic markers. A minimum of 6 different fields of immunostained cells were measured by densitometry, and arbitrary densitometry units with that for Oct4 set to 1 are shown. Compared with immunostaining intensities in cells from women with normal pregnancies, the staining intensities for H3K4me2, H3K4me3, H3K27me2, and H3K27me3 were high in the cells from the woman with the myelomeningocele-affected pregnancy. Staining intensities for H3K27me3, H3K9Ac, H3K18Ac, and Gcn5 were high in AFSCs derived from anencephaly-affected pregnancy, and KDM6B levels were low in both myelomeningocele and anencephalic samples relative to its levels in the normal sample. The error bars indicate the SEM.

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    Amniotic fluid and serum levels of BMP4 and Shh in normal and NTD-affected pregnancies. Serum and amniotic fluid showed similar trends in these levels. A: The levels of BMP4 in amniotic fluid were higher in myelomeningocele-affected and lower in anencephaly- and encephalocele-affected pregnancies. In the holoprosencephaly-affected pregnancy, BMP4 levels were similar to those in the normal pregnancies. B: The levels of serum BMP4 were high in the myelomeningocele-affected pregnancy but nondetectable in the anencephaly-affected pregnancy. C: The Shh levels in amniotic fluid were higher in the anencephaly-affected pregnancy. D: Serum Shh levels were highest in the anencephaly-affected pregnancy and lowest in the myelomeningocele-affected pregnancy. Each experiment was done in triplicate.



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