The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.

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Currently, prenatal detection of Down syndrome and other most common aneuploidies relies on invasive procedures such as amniocentesis and villocentesis, and on non-invasive screening tests such as second trimester maternal serum screening (Triple test), and first trimester screening (ULTRA-screen). However, it well known that invasive techniques carry a small risk of fetal loss, while both Triple test and ULTRA-screen are not diagnostic, may miss from 15 - 40 % of cases of Down syndrome, in addition to having a 5 - 8 % rate of false-positives. We now report clear evidence that the number of fetal nucleated red blood cells (FNRBCs) in the maternal circulation is remarkably higher in pregnant women carrying aneuploid fetuses, especially in cases of Down syndrome. These results are in agreement with the findings of other investigators using different methods, and suggest that the number of FNRBCs present in the maternal blood sample could be used as additional marker, in concert with existing screening tests, to improve non-invasive detection of Down syndrome, and other most common aneuploidies.

To further investigate the relationship between oophorectomy (OF) and mineral bone loss, 15 women who underwent total hysterectomy and bilateral oophorectomy were studied for 12 months after surgery. Mineralometric and metabolic data were obtained before and after 3, 6, and 12 months. The women lost bone mineral content (measured by single photon absorptiometry) at the same rate they lost cortical and trabecular bone, suggesting that bone loss after OF is a generalized phenomenon. Our data also show that an increase in bone resorption takes place only in the first period after OF; the persistency of a negative bone balance up to 12 months, accompanied by a reduction of osteocalcin serum levels, may be dependent on a reduced bone formation, probably due to osteoblastic insufficiency.

Fetal nucleated red blood cells (FNRBCs) circulate in the maternal blood throughout pregnancy. Even if the frequency of fetal cells in the maternal circulation remains to be ascertained, complications of pregnancy such as fetal cells aneuploidies, preeclampsia, abnormal Doppler of the uterine artery without symptoms of preeclampsia, fetal growth restriction and polyhydramnios are associated with an increased feto-maternal trafficking. Based on these observations, previous studies have suggested that determination of the fetal nucleated red blood cell count (FNRBCC) might be a useful non-invasive screening test, either alone or in combination with existing maternal tests, for the non-invasive assessment of aneuploidies, in particular Down syndrome (DS). In this paper we have evaluated the distribution of FNRBCC in a set of 18 normal pregnancies and 18 pregnancies with a trisomy 21-affected fetus, matching for gestational age, maternal age, and, when possible, fetal gender, in order to quantify the difference in the number of fetal cells between the two populations. Maternal blood was collected from each pregnant woman two to three weeks after amniocentesis after knowing the cytogenetic results. Correlation of FNRBCC with the gestational week and clinical status (affected vs. non affected) by multiple regression analysis provided significant results (p < 0.001). Adjusted values of FNRBCC were 48 +/- 10.2 in controls and 301 +/- 17.01 in DS cases, corresponding to a 6.27 fold increase. These retrospective results prompt a prospective evaluation of the use of FNRBCC for screening purposes.