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Objectives The aim of the study was the detection, quantification and correlation of cell-free fetal (cff) DNA in maternal urine and plasma in normal and complicated pregnancies during the third trimester. Methods One hundred and fifty-one urine and plasma samples obtained from 96 women carrying male fetuses, and 55 carrying female fetuses were collected and analyzed for cff-DNA using fluorescent PCR and quantitative real-time PCR. DNA was extracted from 1 mL maternal urine and analyzed with two different primer sets (SRY and DYS-14). The concentrations of cff and total DNA in maternal plasma were correlated with maternal and obstetric parameters using appropriate correlation analyses. Results Y-chromosome-specific sequences were detected in 31 of 96 (32.3%) urine samples collected from women pregnant with male fetuses using DYS-14 and in 6 of 96 (6.3%) urine samples using SRY as primers using real-time PCR. All 96 plasma samples obtained from women carrying male fetuses were positive for cff-DNA using real-time PCR. Cff-DNA exhibited a correlation with gestational age (R = 0.244; P = 0.018) and an inverse correlation with the latency between blood collection and birth (R = − 0.218; P = 0.036). Total DNA showed a correlation with placental weight (R = 0.182; P = 0.034) and pregnancy-associated complications (R = 0.280; P < 0.001). Conclusion Our data confirm that cff-DNA is cleared by the kidneys in detectable amounts, but due to its low concentration or problematic detection in maternal urine this source seems inappropriate for noninvasive prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.

Objectives The aim of this study was to investigate a possible relationship between fetal cell microchimerism and lichen sclerosus of the vulva. We searched for the presence of male cells and DNA in vulval tissue samples. Methods Paraffin-embedded skin biopsy samples from 15 women affected with vulval lichen sclerosus who gave birth to at least one son were analyzed for the presence of microchimeric male cells using fluorescence in situ hybridization (FISH) and fluorescent PCR. We included three lichen sclerosus samples originating from women without male offspring, six vulval specimens without pathological finding originating from autopsies and seven male gingival specimens as controls. Results Nucleated cells containing Y-chromosome specific sequences were neither detected at any site of the lesions nor in normal vulval specimens by using FISH. These results were confirmed by the use of PCR amplification demonstrating only DNA sequences specific for the X chromosome. No female microchimerism was detected in the male gingival samples. Conclusion Despite the limited number and size of the samples, we conclude that persistent male fetal cells are not involved in the pathogenesis of lichen sclerosus of the vulva, since we consistently could not detect Y-chromosome specific sequences by using two molecular techniques. Copyright © 2006 John Wiley & Sons, Ltd.

Objective The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. Methods CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbγ-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbγ and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. Results In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. Conclusion On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis. Copyright © 2003 John Wiley & Sons, Ltd.

Background: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC): class I, CD45 and cytokeratin expression. Methods: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. Results: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. Conclusions: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape. Cytometry 36:319–323, 1999. © 1999 Wiley-Liss, Inc.

Recently, maternal DNA was detected in umbilical cord blood using PCR amplification of minisatellite sequences. The presence of maternal DNA was demonstrated in 1% to 100% of umbilical cord blood samples. The objective of this study was to determine the frequency of cord blood contamination by maternal genetic material. We used fluorescent PCR amplification of highly polymorphic short tandem repeat (STR) markers to detect maternal DNA in umbilical cord plasma.

Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.