Design
A genomic study.

Time and setting
This study was performed at the Department of Medical Biology and Forensic Medicine, Faculty of Medicine, Çukurova University, Turkey between April 2009 and March 2010.
Materials

This study was permitted by the Ministry of Forensic Medicine in Adana, Turkey. Review of the prior pregnancy and life histories of the 13 deceased women are summarized in Table 1. Patients were selected for this study on the basis of the following criteria: pregnancy with at least one male offspring, and no history of abortion and blood transfusion. All deceased women who had no sign of putridity were included in this study without looking to the cause of death. Informed consents were taken from executors of the cases.

Methods
Sample selection
In the included cases, autopsy was performed within 24 hours and the dead bodies were kept at 4°C during the period of procedures accomplished in the Ministry of Forensic Medicine in Adana. The brain was dissected and coronal cross-sections were taken with 1 cm intervals. Brain tissue samples (approximately 1 cm³) were taken from hippocampal dentate gyrus and the subventricular zone. Neurogenesis occurs primarily in the hippocampal dentate gyrus and the subventricular zone of the adult mammalian brain, as in rodents and non-human primates (Figure 1)^[12]. In the dentate gyrus, newly generated neuronal cells in the subgranular zone migrate to the granule cell layer^{[8, 31-33]}. Newly generated neuronal cells in the subventricular zone migrate to the olfactory bulb, through the rostro-migratory stream, where they differentiate into olfactory bulb interneurons^[34-36]. All tissue samples were put into labeled tubes containing 70% ethanol. Male babies' stem cells were detected by quantitative fluorescent-PCR amplification of Y, 13, 18, 21 and X chromosome-specific sequences.

Quantitative fluorescent-PCR analysis
Quantitative fluorescent-PCR analysis was performed based on the amplification of chromosome-specific DNA sequences with short tandem repeats polymorphic in length between subjects. By means of fluorescent primers, the amplified segments could be visualized and quantified as peak areas on automated DNA scanners. Normal heterozygous subjects were expected to show two peak areas (peaks ratio 1:1) for each chromosome analyzed, while trisomies were visualized either as an extra peak (triallelic subjects) or as a 2:1 ratio peak between the two areas^[37]. Quantitative fluorescent-PCR amplifications were performed for the determination of any foreign DNA residues originating from the cells that could migrate from the babies to the brains of the mothers during the gestational period. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Quality and quantity of DNA samples were evaluated by Implen Nanophotometer^TM (Implen GmbH, Munich, Germany). Quantitative fluorescent-PCR amplifications were performed using the Aneufast^TM (Molgentix SL, Barcelona) trisomy detection kit, which includes fluorescently labeled primers for 27 predefined short tandem repeat marker sites on chromosomes 13, 18, 21, X and Y, and primer pairs for AMXY and SRY regions (AMXY, X22, HPRT, SRY, DXYS218, D135631, D135634, D135258, D135305, D18S391, D18S390, D18S635, D18S386, D21S1411, D211435 and D21S1446). The kit also contained dNTPs and Hot Start Taq DNA polymerase in an optimized reaction buffer. DNA (2 μL, 10–50 ng) and PCR-grade water (3 μL) were added to each of the reaction tubes containing 10 μL of the master mix. After initial denaturation at 95°C for 15 minutes, amplification was achieved by 28 cycles of 95°C for 40 seconds, 58°C for 80 seconds and 72°C for 40 seconds, and final extension was 30 minutes at 60°C. Quantitative fluorescent-PCR products (1.5 μL from each mix) were added to 40 μL Hi-DiTM Formamide (Applied Biosystems, Foster City, CA, USA) containing 0.3 μL of GeneScanTM- 500 LIZTM (Applied Biosystems) size standard. After denaturation at 95°C for 3 minutes, the mixture was cooled to 4°C and then capillary electrophoresis was carried out on an ABI 3130 Genetic Analyzer using POP7 polymer. Results were analyzed using GeneMapper 4.0 software (Applied Biosystems). By means of quantitative fluorescent-PCR together with short tandem repeat markers, amplifications were achieved from trace amounts of foreign DNA and distinguished from the amplification of maternal DNA samples.

Funding: The study was supported by Research Funds of University of Çukurova, Turkey.
Author contributions: Osman Demirhan, Necmi Çekin, Deniz Ta?temir, Erdal Tunç, Demet Meral and Bülent Demirbek designed this study. Deniz Ta?temir, Erdal Tunç and Ali ?rfan Güzel performed the research. Osman Demirhan, Deniz Ta?temir, Ali ?rfan Güzel, and Demet Meral analyzed the experimental data. Osman Demirhan, Necmi Çekin, Erdal Tunç, and Bülent Demirbek were responsible for writing the paper. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: This study was approved by the Ethics Committee, Faculty of Medicine, University of Çukurova , Turkey.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.

(1)   Fetal cells are known to enter maternal blood circulation during pregnancy.

(2)   Whether fetal cells in the maternal blood circulation can cross the blood-brain barrier to enter the material brain remains poorly understood.

(3)   We tested this possibility using 13 women who had one male infant in their lifetime.

(4)   We used quantitative fluorescent-PCR amplification peaks of 27 short tandem repeat markers on five chromosomes (chromosome 13, 18, 21, X and Y).

(5)   There were no additional (residual) peaks except the main peaks for all DNA samples from the two different brain regions (hippocampal dentate gyrus and the subventricular zone) of each woman.

1  妊娠期间，胚胎细胞可进入母体血液循环，但该细胞是否可穿越血脑屏障进入母体大脑尚不知晓。

2  因此，我们检测了13名产过1名男婴的女性。研究应用了5个染色体13，18，21号和X，Y染色体的27个STR基因座荧光复合扩增峰。分析显示，每名女性的海马齿状回和脑室下区几乎所有DNA样本无残峰。

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