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Background In human fetuses with Down syndrome, placental pathology, structural anomalies and growth restriction are present. There is currently a significant lack of information regarding the early life span in mouse models of Down syndrome. Objective The objective of this study was to examine embryonic day 18.5 and placental phenotype in the 3 most common mouse models of Down syndrome (Ts65Dn, Dp(16)1/Yey, Ts1Cje). Based on prenatal and placental phenotyping in 3 mouse models of Down syndrome, we hypothesized that one or more of them would have a similar phenotype to human fetuses with trisomy 21, which would make it the most suitable for in utero treatment studies. Study Design Here, C57BL6J/6 female mice were mated to Dp(16)1/Yey and Ts1Cje male mice and Ts65Dn female mice to C57BL/B6Eic3Sn.BLiAF1/J male mice. At embryonic day 18.5, dams were euthanized. Embryos and placentas were examined blindly for weight and size. Embryos were characterized as euploid or trisomic, male or female by polymerase chain reaction. A subset of embryos (34 euploid and 34 trisomic) were examined for malformations. Results The Ts65Dn mouse model showed the largest differences in fetal growth, brain development, and placental development when comparing euploid and trisomic embryos. For the Dp(16)1/Yey mouse model, genotype did not impact fetal growth, but there were differences in brain and placental development. For the Ts1Cje mouse model, no significant association was found between genotype and fetal growth, brain development, or placental development. Euploid mouse embryos had no congenital anomalies; however, 1 mouse embryo died. Hepatic necrosis was seen in 6 of 12 Dp(16)1/Yey (50%) and 1 of 12 Ts1Cje (8%) mouse embryos; hepatic congestion or inflammation was observed in 3 of 10 Ts65Dn mouse embryos (30%). Renal pelvis dilation was seen in 5 of 12 Dp(16)1/Yey (42%), 5 of 10 Ts65Dn (50%), and 3 of 12 Ts1Cje (25%) mouse embryos. In addition, 1 Ts65Dn mouse embryo and 1 Dp(16)1/Yey mouse embryo had an aortic outflow abnormality. Furthermore, 2 Ts1Cje mouse embryos had ventricular septal defects. Ts65Dn mouse placentas had increased spongiotrophoblast necrosis. Conclusion Fetal and placental growth showed varying trends across strains. Congenital anomalies were primarily seen in trisomic embryos. The presence of liver abnormalities in all 3 mouse models of Down syndrome (10 of 34 cases) is a novel finding. Renal pelvis dilation was also common (13 of 34 cases). Future research will examine human autopsy material to determine if these findings are relevant to infants with Down syndrome. Differences in placental histology were also observed among strains.

Women’s health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation—a fast, scarless healing process in healthy individuals—will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, “Menstruation: Science and Society” with an aim to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration—and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids—to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent “menstrual equity” and “period poverty” movements spreading across high-income countries.

Women and children bear a substantial part of the burden of opioid overuse in the United States. Opioid use during pregnancy can lead to neonatal opioid withdrawal syndrome, and both the mothers and babies may be at higher risk of opioid use and its consequences later in the life course, setting up intergenerational cycles of opioid overuse. As part of the HEAL (Helping to End Addiction Long-term) Initiative of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the Environmental influences on Child Health Outcomes program are together launching observational and intervention research programs to interrupt these cycles, beginning with opportunities in pregnancy and the newborn period. The Eunice Kennedy Shriver National Institute of Child Health and Human Development has also launched programs to find alternatives to opioids for painful conditions in women of reproductive age, including a range of gynecologic conditions. These coordinated efforts promise to help turn the tide against the opioid crisis by providing the necessary evidence to improve care for women and children affected by these substances.

Maternal obesity is associated with adverse neurodevelopmental outcomes in children, including autism spectrum disorders, developmental delay, and attention-deficit hyperactivity disorder. The underlying mechanisms remain unclear. We previously identified second-trimester amniotic fluid and term cord blood gene expression patterns suggesting dysregulated brain development in fetuses of obese compared with lean women.We sought to investigate the biological significance of these findings in a mouse model of maternal diet-induced obesity. We evaluated sex-specific differences in fetal growth, brain gene expression signatures, and associated pathways.Female C57BL/6J mice were fed a 60% high-fat diet or 10% fat control diet for 12-14 weeks prior to mating. During pregnancy, obese dams continued on the high-fat diet or transitioned to the control diet. Lean dams stayed on the control diet. On embryonic day 17.5, embryos were weighed and fetal brains were snap frozen. RNA was extracted from male and female forebrains (10 per diet group per sex) and hybridized to whole-genome expression arrays. Significantly differentially expressed genes were identified using a Welch's t test with the Benjamini-Hochberg correction. Functional analyses were performed using ingenuity pathways analysis and gene set enrichment analysis.Embryos of dams on the high-fat diet were significantly smaller than controls, with males more severely affected than females (P = .01). Maternal obesity and maternal obesity with dietary change in pregnancy resulted in significantly more dysregulated genes in male vs female fetal brains (386 vs 66, P.001). Maternal obesity with and without dietary change in pregnancy was associated with unique brain gene expression signatures for each sex, with an overlap of only 1 gene. Changing obese dams to a control diet in pregnancy resulted in more differentially expressed genes in the fetal brain than maternal obesity alone. Functional analyses identified common dysregulated pathways in both sexes, but maternal obesity and maternal dietary change affected different aspects of brain development in males compared with females.Maternal obesity is associated with sex-specific differences in fetal size and fetal brain gene expression signatures. Male fetal growth and brain gene expression may be more sensitive to environmental influences during pregnancy. Maternal diet during pregnancy has a significant impact on the embryonic brain transcriptome. It is important to consider both fetal sex and maternal diet when evaluating the effects of maternal obesity on fetal neurodevelopment.

Background Cell-free RNA in amniotic fluid supernatant reflects developmental changes in gene expression in the living fetus, which includes genes that are specific to the central nervous system. Although it has been previously shown that central nervous system–specific transcripts are present in amniotic fluid supernatant, it is not known whether changes in the amniotic fluid supernatant transcriptome reflect the specific pathophysiologic condition of fetal central nervous system disorders. In myelomeningocele, there is open communication between the central nervous system and amniotic fluid. Objectives The purpose of this study was to identify molecular pathophysiologic changes and novel disease mechanisms that are specific to myelomeningocele by the analysis of amniotic fluid supernatant cell-free RNA in fetuses with open myelomeningocele. Study Design Amniotic fluid supernatant was collected from 10 pregnant women at the time of the open myelomeningocele repair in the second trimester (24.5±1.0 weeks); 10 archived amniotic fluid supernatant from sex and gestational age–matched euploid fetuses without myelomeningocele were used as controls (20.9±0.9 weeks). Differentially regulated gene expression patterns were analyzed with the use of human genome expression arrays. Results Fetuses with myelomeningocele had 284 differentially regulated genes (176 up- and 108 down-regulated) in amniotic fluid supernatant. Known genes that were associated with myelomeningocele ( PRICKLE2 , GLI3 , RAB2 3, HES1 , FOLR1 ) and novel dysregulated genes were identified in association with neurodevelopment and neuronal regeneration (up-regulated, GAP4 3 and ZEB1 ) or axonal growth and guidance (down-regulated, ACAP1 ). Pathway analysis demonstrated a significant contribution of inflammation to disease and a broad influence of Wnt signaling pathways ( Wnt1 , Wnt5A , ITPR1 ). Conclusion Transcriptomic analyses of living fetuses with myelomeningocele with the use of amniotic fluid supernatant cell-free RNA demonstrated differential regulation of specific genes and molecular pathways relevant to this central nervous system disorder, which resulted in a new understanding of pathophysiologic changes. The data also suggested the importance of pathways that involve secondary disease, such as inflammation, in myelomeningocele. These newly identified pathways may lead to hypotheses that can test novel therapeutic targets as adjuncts to fetal surgical repair.

Objective The purpose of this study was to evaluate whether there is a nuchal translucency (NT) measurement, independent of gestational age, above which immediate diagnostic testing should be offered without waiting for first trimester serum markers. Study design Thirty-six thousand one hundred twenty patients had successful measurement of simple NT at 10 3/7 to 13 6/7 weeks and had first trimester serum screening. No risks were reported until second trimester serum screening was completed. Results Thirty-two patients (0.09%) had NT ≥4.0 mm; the lowest combined first trimester trisomy 21 risk assessment in euploid cases was 1 in 8 and among aneuploidy cases was 7 in 8. One hundred twenty-eight patients (0.3%) had simple NT ≥3.0 mm: the lowest combined first trimester trisomy 21 risk assessment of any patient in this group was 1 in 1479 and the lowest risk assessment among aneuploid cases was 1 in 2. Ten patients (8%) had first trimester trisomy 21 risk assessments lowered to less that 1:200 and none of these 10 cases had an abnormal outcome. Conclusion During first trimester Down syndrome screening, whenever an NT measurement of 3.0 mm or greater is obtained there is minimal benefit in waiting for serum screening results, and no benefit for NT of 4.0 mm or greater. Differentiation between cystic hygroma and enlarged simple NT (≥3.0 mm) is now a moot point as both are sufficiently high risk situations to warrant immediate CVS.

The aim of this study was to examine fetal gene expression in maternal plasma after fetoscopic intervention for twin-twin transfusion syndrome or congenital diaphragmatic hernia.Twelve women with pregnancies that were complicated by twin-twin transfusion syndrome and 10 women carrying fetuses with congenital diaphragmatic hernia were sampled before and sequentially after treatment. Levels of glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, and gamma globin messenger RNA were measured by real-time reverse transcriptase polymerase chain reaction amplification.At all time points, glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels were higher in the congenital diaphragmatic hernia cases than in the twin-twin transfusion syndrome cases (P.05), but during the immediate postoperative observation period, there were no significant changes in glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, or gamma globin messenger RNA levels in individual patients or patients who were grouped by procedure.Fetoscopic intervention of complicated pregnancies does not affect circulating fetal messenger RNA levels, which is in contrast to earlier observations that circulating fetal DNA levels increase after laser ablation for twin-twin transfusion syndrome. Plasma glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels could be a potential novel biomarker for fetal trauma.

Objective The purpose of this study was to determine whether cell-free fetal DNA is detectable in the cerebrospinal fluid of women during pregnancy and after delivery. Study design Cerebrospinal fluid was collected from 39 women who underwent an indicated spinal anesthesia procedure. Twenty-six samples were from women who carried at least 1 male fetus, and 13 samples were from women with only a female fetus. DNA was analyzed with the use of real-time polymerase chain reaction for DYS-1 (which represented male fetal DNA) and β-globin (which represented maternal and fetal DNA). Results β-Globin DNA was detected in all cerebrospinal samples. DYS-1 gene sequences were detected in 4 cerebrospinal fluid samples from women who had male fetuses (2 samples were from women who underwent cesarean delivery of singleton pregnancies, 1 sample was from a triplet pregnancy, and 1 sample was from a woman after delivery). No male DNA was detected in the cerebrospinal fluid of women who carried female fetuses. Conclusion Male fetal cells and/or cell-free fetal DNA is detectable in the cerebrospinal fluid of some pregnant women or some women after delivery.

Increased levels of cell-free fetal DNA (f-DNA) in the maternal circulation are a potential noninvasive marker for fetal Down syndrome. Our objectives were to (1) determine whether f-DNA could be quantified by using archived serum and amniotic fluid, (2) examine whether serum f-DNA levels are elevated in Down syndrome pregnancies in a case-control series matched for gestational age and duration of sample storage, and (3) determine whether f-DNA levels are elevated in the amniotic fluid of Down syndrome fetuses.Eleven serum and six amniotic fluid samples previously collected and stored at -20 degrees C from gravid women carrying a 47,XY,+21 fetus were each paired with five matched control samples of identical specimen type from gravid women carrying a presumed euploid male fetus. f-DNA concentration was quantified blindly by real-time polymerase chain reaction amplification for a Y-chromosome sequence. Matched rank-sum analysis and analysis of variance were used for analysis.The mean observed rank of 5.0 in the Down syndrome group was significantly higher than expected (P/=.005). Adjusted mean serum f-DNA concentrations were 41.2 genomic equivalents (GE) per milliliter for the Down syndrome cases and 24.2 GE/mL for the euploid controls (P =.002). Differences among amniotic fluid samples were not statistically significant. There was a suggestion of a sample storage effect on f-DNA concentration on the order of -0.66 GE/mL per month (P =.071).Down syndrome pregnancies exhibit 1.7-fold higher levels of maternal serum cell-free f-DNA compared with matched controls. No such association is observed in amniotic fluid. Archived serum appears to be a useful source of clinical material for retrospective analyses but may require controlling for the duration of sample storage.

Objective: Umbilical cord blood is a source of hematopoietic stem cells for transplantation. Although the first clinical applications have been encouraging, concern has been raised about contamination of umbilical blood by maternal cells, which might constitute a theoretical risk of graft-versus-host disease. The aim of this study was to assess the frequency of maternal deoxyribonucleic acid (DNA) contamination in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA markers. Study Design: Fifty-seven mother/child pairs were tested for the presence of maternal DNA sequences in cord plasma. After delivery, cord blood samples were collected via gravity. Maternal specific alleles were detected by using polymerase chain reaction amplification of 9 highly polymorphic short tandem repeat markers (D21S11, D21S1411, D21S1412, D18S386, D18S535, MBP-A, MBP-B, D13S631, and D13S634). Results: All 57 mother-child pairs were informative for the identification of uniquely maternal alleles in at least 2 of 9 different short tandem repeat markers used per case. Uniquely maternal DNA sequences were found in 43 of 57 (75%) cord plasma samples. Conclusion: The results of our study demonstrate that maternal DNA is present in the majority of umbilical cord blood plasma samples. The technique described herein might have application in the screening of umbilical cord blood samples for the presence of contaminating maternal genetic material. (Am J Obstet Gynecol 2002;186:117-20.)

OBJECTIVE: This study examines the role of fluorescence in situ hybridization on uncultured amniocytes for prenatal diagnosis in a population at high risk for aneuploidies. STUDY DESIGN: All patients undergoing amniocentesis for fetal structural abnormality on ultrasonographic examination (performed from 13 to 39 weeks), abnormal maternal serum aneuploidy screening results, or advanced maternal age with substantial parental anxiety were offered both fluorescence in situ hybridization on uncultured cells and conventional metaphase karyotyping on dividing cells. RESULTS: From 1992 to 1995, 315 patients were studied. Mean time to obtain results was 2.8 days for fluorescence in situ hybridization and 8.3 days for karyotype. Fluorescence in situ hybridization was informative in 254 samples (80.6%), and within this group 21 aneuploidies were correctly identified. Among informative specimens there was 100% sensitivity and specificity, with 100% positive and negative predictive values. Of the 315 samples, 61 (19.4%) were uninformative or unreportable. Of 25 total cases of karyotype-proved aneuploidy, 4 were reported as uninformative by fluorescence in situ hybridization, for a total detection rate of 84%. Overall, amniocenteses performed after 24 weeks were significantly more likely to be uninformative than those performed in the second trimester (45% vs 16%, p = 0.01), peaking at a 56% uninformative rate after 33 weeks. Logistic regression analysis showed an 8% increase in the uninformative rate per week of gestational age (odds ratio 1.08, 95% confidence interval 1.04 to 1.14). CONCLUSIONS: Fluorescence in situ hybridization on uncultured amniocytes is a rapid, clinically useful tool for prenatal diagnosis, with informative specimens being highly accurate. The combination of a structural fetal anomaly and an abnormal fluorescence in situ hybridization result should allow for definitive management decisions. The significant increase in uninformative specimens at later gestational ages limits its usefulness in the third trimester. (Am J Obstet Gynecol 1997;176:769-76.)

OBJECTIVE: The aim of this study was to determine the accuracy of noninvasive fetal RhD genotyping by fetal cell isolation from maternal blood. STUDY DESIGN: Candidate fetal cells from 18 pregnant women (one twin gestation) were flow-sorted. Polymerase chain reaction amplification of a 261 bp fragment of the RhD gene was performed on sorted fetal cells. The presence of amplified product was considered predictive of the RhD-positive genotype in the fetus. RESULTS: Sixteen of the 19 fetal RhD genotypes were correctly predicted in fetal cells isolated from maternal blood (10 were Rh positive, 6 were Rh negative). In 3 cases no amplification products were detected in RhD-positive fetuses. The association between presence of the fragment and RhD-positive genotype was significant ( p = 0.003, Fisher's exact test). CONCLUSIONS: Noninvasive prenatal diagnosis of the fetal RhD genotype is feasible. Absence of amplification products in the reaction requires confirmation that fetal material is present. Improvements in fetal cell purity and yield should increase diagnostic accuracy, although the current protocol has a positive predictive value of 100% and a negative predictive value of 67%. (AM J OBSTET GYNECOL 1996;174:818-22.)

OBJECTIVE: At birth the fetus emerges from a sterile environment into a nonsterile one. This process is associated with activation of the fetal immune system which protects the fetal against infection in the newborn period. We conducted this study to determine whether activation of the monocyte-neutrophil system occurs in fetuses before premature birth. STUDY DESIGN: Forty patients in premature labor with intact membranes underwent cordocentesis for research purposes. Fetal blood was analyzed with the use of flow cytometry to measure the cell surface markers CD11c, CD13, CD15, and CD67, which are associated with monocyte and neutrophil activation, and CD14 and CD63, which were used as controls. RESULTS: Twenty-eight percent ( 11 40 ) of the infants were delivered prematurely within 72 hours of entering the study while the remainder were delivered at term. Our data clearly indicate that premature infants delivered within 72 hours had a higher percentage of CD11c, CD13, CD15, and CD67 than those delivered at term. In contrast, there were no significant differences in the percentages of CD14 and CD63. CONCLUSION: Activation of the monocyte-neutrophil system exists in fetuses destined for premature delivery. These findings indicate that premature parturition is associated with in utero immune system activation.

Objective: We studied transferrin receptor (CD71) expression in peripheral blood mononuclear cells from healthy pregnant women, to determine if a relationship existed between gestational age and circulating CD71 + mononuclear cells. Study Design: Cell suspensions were prepared from venous blood from 139 pregnant women (7 to 26 weeks of gestation), incubated with monoclonal anti-CD71 antibody, and analyzed by flow cytometry. Results: When only the first sample from each woman was analyzed, extensive biologic variation between women was shown. An apparent biphasic increase in the percentage of CD71 + cells with advancing gestation was suggested. A subgroup of 13 women studied on multiple occasions demonstrated linear increases in CD71 + cells as pregnancy progressed. Conclusions: Pregnant women, when compared with each other, may have differences in the baseline number of circulating CD71 + cells. The increases seen in individuals studied repeatedly are likely to reflect maternal hematopoiesis and current fetomaternal transfusion.

► To examine the performance of noninvasive prenatal testing for sex chromosome aneuploidy (SCA) and fetal sex using genome-wide massively parallel sequencing in a CLIA-licensed, CAP-accredited clinical laboratory. ► SCA testing became clinically available in July 2012. All US-based NIPT laboratories now offer testing for 45,X (monosomy X/Turner syndrome), 47,XXX (triple X syndrome), 47,XXY (Klinefelter syndrome), and 47,XYY (Jacobs syndrome). — Some labs automatically test for SCAs, while for others it is optional. — If NIPT does not identify a SCA, the fetal sex is reported as XX or XY. ► Limited studies have shown high accuracy with NIPT for sex chromosomes and SCAs; however, its accuracy is slightly lower than for autosomal aneuploidies [1-3]. ► Maternal mosaicism as an explanation for discordant results is likely to be more common for sex chromosomes than for autosomes [4]. ► Reports of discordance between fetal sex by NIPT and fetal sex by clinical determination (karyotype or ultrasound) present counseling and management dilemmas for which no medical guidelines currently exist. Study Objective and Background ► Data are included from verifi® prenatal test samples with the SCA test option. ► Clinical follow-up information was collected and documented according to standard laboratory practice and quality procedures. ► SCA “Detected” results were categorized in one of three ways based on follow-up information: — ‘Concordant’ if the NIPT SCA result matched the fetal/neonatal karyotype result. — ‘Discordant’ if the NIPT SCA result did not match the fetal/neonatal karyotype result. — ‘No Discordance Reported’ if no follow-up information was reported back to the laboratory. ► Fetal sex chromosome (XX or XY) results were categorized in one of four ways based on follow-up information: — ‘Concordant’ if the NIPT sex result matched the fetal/neonatal karyotype result. — ‘Discordant (karyotype)’ if the NIPT sex result did not match the fetal/neonatal karyotype result. — ‘Discordant (ultrasound)’ if the NIPT sex result did not match ultrasound or post-mortem examination determination of fetal sex. — ‘No Discordance Reported’ if no follow-up information was reported back to the laboratory. Results ► 18,161 samples were tested for SCAs during the study period. — No SCAs were detected in 17,957 cases (98.88%) (Figure 1). – In these cases, XX or XY was reported. – XY to XX ratio of 1.06, which is on par with 1.05 as reported in 2012 U.S. birth statistics [5]. – XX and XY performance is well within, or better than, previous performance parameters in a blinded clinical research trial [1]. — SCAs were detected in 204 cases (1.12%) (Figure 2). – A false positive rate of 0.26% is estimated based on available clinical follow-up information.                                               

Objective This study was undertaken to determine whether laser thermocoagulation for twin-twin transfusion syndrome (TTTS) causes increased cell-free fetal DNA levels in maternal plasma, potentially as a result of placental injury. Study design We enrolled 34 patients with twin pregnancies complicated by severe TTTS who underwent fetoscopic selective laser ablation of placental vascular anastomoses. Blood samples were drawn before and sequentially after the procedure. Fetal DNA in maternal plasma was quantified by polymerase chain reaction amplification of a Y-chromosome sequence. Results Compared with baseline, median elevations of fetal DNA levels were 0.8% at 30 minutes ( P =.32), 15.8% at 60 minutes ( P =.1), 179.5% at 24 hours ( P =.003), and 172.9% at 48 hours ( P =.003). Factors associated with increased fetal DNA levels at 24 hours after procedure included longer operation time, higher number of vessels ablated, and subsequent in utero fetal death ( P =.01, .04, and .04, respectively). Conclusions Persistent elevation of fetal DNA levels in maternal plasma after laser ablation suggests that circulating fetal DNA could derive from placental injury. Plasma fetal DNA analysis may be an additional prognostic marker for fetal outcome after laser therapy.

Objective Measurement of cell-free fetal (cff) DNA in maternal plasma may have clinical application in prenatal screening for fetal Down syndrome and preeclampsia. Little is known regarding the tissue of origin of these fetal-derived sequences. We tested the hypothesis that if the placenta is the major contributor of cff DNA, then an increased placental volume should be associated with higher maternal plasma cff DNA levels. Study design We enrolled 143 pregnant women who underwent first-trimester placental volume measurement using 3-dimensional ultrasonography. Cff DNA in maternal plasma on the day of the scan was quantified by real-time polymerase chain reaction (PCR) amplification of a Y-chromosome sequence. The association between measured placental volume and maternal plasma cff DNA levels was analyzed along with relevant clinical variables. Results The median (25th, 75th percentiles) maternal plasma cff DNA level was 16.9 genome equivalents (GE)/mL (10.8, 28.7). Raw values were adjusted for gestational age and maternal body mass index. The median (25th, 75th percentiles) placental volume was 53.2 mL (43.0, 64.7), and median placental quotient (ratio of placental volume to fetal crown-rump length) was 1 mm 2 (0.8, 1.1). Based on multivariate linear regression analyses, neither of the above placental measurements showed a significant association with maternal plasma cff DNA levels ( P =.43 and .43, respectively). A modest association was found between plasma cff DNA levels and gravidity ( P =.03). Conclusion Our data did not show a significant association between either the placental volume or placental quotient, and maternal plasma cff DNA levels. We speculate that it is the extent of placental apoptosis that primarily affects the amount of cff DNA released into the maternal circulation.

The purpose was to determine whether preeclampsia (PE) is caused by microfragments of syncytial trophoblast shed into the maternal circulation that stimulate an exaggerated inflammatory response.A nested case control study was performed within the Calcium for Preeclampsia Prevention trial cohort of healthy nulliparous women. Each preeclampsia case was matched to 1 normotensive control. One hundred twenty pairs were randomly chosen for analysis of serum cell-free fetal DNA (cffDNA), a marker of placental debris, and C-reactive protein (CRP), a marker of inflammation, in all 658 specimens obtained before labor.At 29 to 41 weeks of gestation, cffDNA concentrations were significantly higher after preeclampsia than before (219 vs 112 genome equivalents [GE]/mL, P.001). Before preeclampsia, cffDNA in cases exceeded controls at 17 to 28 weeks (36 vs 16 GE/mL, P.001), but at 29 to 41 weeks, only within 3 weeks before preeclampsia (176 vs 75 GE/mL, P.001). CRP serum concentrations were neither associated with cffDNA nor elevated before preeclampsia.Preeclampsia is accompanied by a 2-stage elevation of fetal DNA, but not by elevation of CRP. Elevated cffDNA at 17 to 28 weeks may be due to placental necrosis and apoptosis. Subsequent elevations may reflect impaired DNA elimination. The 2-stage elevation suggests the possibility of measurement of fetal DNA both to screen for preeclampsia and to indicate impending clinical disease.

Objective: Recent reports that an association exists between fetal cell microchimerism and autoimmune disease has increased interest in the postpartum persistence of fetal cells. The purpose of this study was to determine, by means of quantitative polymerase chain reaction amplification, whether a significant fetalmaternal hemorrhage occurs after elective termination of pregnancy. Study Design: Blood samples were obtained from 23 women who underwent termination of pregnancy immediately before venipuncture; these samples were subjected to analysis by quantitative polymerase chain reaction amplification with the use of Y-chromosome primers. There were 21 male and 2 female fetuses. Results were equilibrated to 16 mL and analyzed by a weighted linear regression analysis to evaluate the correlation between detected fetal nucleated cell equivalents and gestational weeks. Results: Among the 21 known male fetuses, the median number of detected fetal nucleated cell equivalents was 1552 (range, 50-37,618). The female fetuses had no fetal nucleated cell equivalents detected. A positive dependence of male fetal nucleated cell equivalents on gestational age was shown ( P Conclusion: Analysis by quantitative polymerase chain reaction amplification demonstrated a large fetal-maternal transfusion after elective abortion. Consideration of the biologic consequences of pregnancy and the potential for future development of fetal cell microchimerism must now extend to a larger population of women. (Am J Obstet Gynecol 2001;184:703-6.)

Objective: Our aim was to develop a new technique, which we have termed fetal cell recycling, that combines the 2 powerful methods of fluorescence in situ hybridization and polymerase chain reaction to maximize the genetic information available from a small number of fetal nucleated erythrocytes obtained noninvasively from the blood of pregnant women. Study Design: Blood samples were obtained from 4 Rh-negative women after elective termination of pregnancy at 7 to 17 weeks' gestation. Fetal nucleated erythrocytes were separated by flow sorting with antibody to the γ chain of fetal hemoglobin. Fluorescence in situ hybridization with chromosome-specific probes was used to diagnose fetal gender. After fluorescence in situ hybridization analysis the fetal nucleated erythrocytes were recycled by a micromanipulation technique and deoxyribonucleic acid diagnosis was performed with polymerase chain reaction amplification of the RhD gene. Results: Among the 4 case patients we detected a total of 101 fetal nucleated erythrocytes. All targeted cells were successfully retrieved with a micromanipulator. In each case we successfully performed both fluorescence in situ hybridization and polymerase chain reaction analysis. The predicted fetal gender and Rh status corresponded to the results obtained from fetal tissue. Conclusions: Fetal cell recycling combines the powers of highly sensitive molecular methods to maximize the genetic information available from a single fetal cell. This technique will permit noninvasive diagnosis of recessively inherited single-gene disorders. (Am J Obstet Gynecol 1999;181:1237-42.)

Objective: We evaluated monoclonal antibodies to 3 cell surface and 3 intracellular antigens for their relative usefulness as markers to identify fetal cells in maternal blood. Study Design: With indirect immunocytochemical labeling techniques, antigen expression was studied in 52 fetal blood samples as a function of gestational age, fetal karyotype, the presence of multiple anomalies detectable on ultrasonography, and anemia. Results: A decline in the expression of these antigens as gestational age advanced was demonstrated. Samples from karyotypically abnormal fetuses, fetuses with multiple anomalies, and anemic fetuses showed an antigenic distribution that was immature for gestational age. In normal fetuses ζ globin and ϵ globin expression decreased after 12 to 14 weeks, potentially limiting the utility of these proteins as fetal cell markers in the isolation of fetal cells from maternal blood. Conclusions: The results of this study demonstrate a fetal developmental hematologic profile that varies with gestational age and also with pathologic condition. Antibodies to the γ chain of fetal hemoglobin and the transferrin receptor (CD71) are the most useful fetal cell–identifying reagents. (Am J Obstet Gynecol 1999;180:1234-9.)

OBJECTIVE: Although polymorphonuclear leukocytes are the inflammatory cells most frequently recovered from the amniotic cavity in cases of suspected intrauterine infection, the source of these cells has not been definitively determined. We took advantage of the gender difference between the mother and her male fetus, and we report four cases in which amniotic fluid polymorphonuclear leukocytes were identified as fetal by fluorescence in situ hybridization with probes specific for X and Y chromosomes. Fetal membranes were intact at the time amniotic fluid was obtained in all cases. STUDY DESIGN: Amniotic fluid was obtained from women with male fetuses in premature labor with clinical or laboratory evidence of infection. Cytospin preparations of amniotic fluid samples with polymorphonuclear leukocytes were prepared and sequentially stained with fluorescent reagents. To determine which cells were polymorphonuclear leukocytes, all replicate samples were stained with the fluorescent nuclear stain 4′-6-diamidino-2-phenyl-indole. This allowed definition of the characteristic multilobed polymorphonuclear leukocytes nuclear morphologic features. The sample was then probed with a rhodamine-labeled probe specific for the X chromosome and a fluorescein-labeled probe specific for the Y chromosome to assess whether the polymorphonuclear leukocytes were male or female. RESULTS: Ninety percent to 99% of polymorphonuclear leukocytes identified by normal multiple lobed nuclear morphologic study on 4′-6-diamidino-2-phenyl-indole staining had an X and Y chromosome and were therefore fetal cells. CONCLUSION: These data demonstrate a fetal response during intraamniotic infection. Further investigation of the roles for maternal and fetal polymorphonuclear leukocytes in chorioamnionitis may provide valuable information about the critical interaction of the two immune responses in this setting. (Am J Obstet Gynecol 1997;176:77-81.)

amniotic fluid supernatant reveals major alterations in nervous and cardiovascular system gene expression LISA HUI, Heather Wick, Kenneth Moise, Anthony Johnson, Michael Belfort, Kirby Johnson, Diana Bianchi Tufts Medical Center, Mother Infant Research Institute, Boston, MA, Tufts University, Computer Science, Medford, MA, Baylor College of Medicine, Obstetrics and Gynecology, Houston, TX OBJECTIVE: To understand the molecular mechanisms underlying the recipient’s response to twin-twin transfusion syndrome (TTTS) and develop fetal biomarkers by performing functional genomic analysis of amniotic fluid supernatant (AFS). STUDY DESIGN: We have previously shown that AFS contains RNA from multiple fetal organs, including brain. This was a prospective study analyzing cell-free RNA transcripts in recipient AFS from women undergoing clinically-indicated laser surgery. Normal AFS was obtained from singleton fetuses undergoing genetic amniocentesis. The independent t test with the Benjamini-Hochberg correction was used to evaluate up-regulated genes in TTTS vs normal controls, and Quintero Stage III vs stage II cases. Functional analyses of the significantly up-regulated genes were performed with Ingenuity software. RESULTS: AFS samples from 8 recipient twins (4 Stage II and 4 Stage III, EGA 17-21 w) and 4 euploid controls (EGA 17-22 w) were included. Compared with controls, 811 genes were significantly upregulated in TTTS cases. The most statistically significant biological process that differed between cases and controls was nervous system development and function. There were 19 up-regulated nervous system genes in TTTS cases including microtubule-associated protein tau, a biomarker of adult neurodegeneration. Other important upregulated transcripts in TTTS were vascular endothelial growth factor 1 and angiotensin II receptor type 2. The most statistically significant up-regulated biological process in Stage III vs Stage II fetuses was cardiovascular disease. Among the 58 cardiovascular disease genes up-regulated in Stage III recipients were some specifically associated with hypertension and left ventricular dysfunction (TRPC4) and congenital pulmonary stenosis (NTRK3). CONCLUSION: Our study provides novel molecular evidence of the impact of TTTS on the developing nervous system. We also identified the cardiovascular genes specifically associated with Stage III vs Stage II disease. These results further our understanding of the pathophysiology of TTTS and provide potential prognostic fetal biomarkers. 424 Gender equality in fetal reduction (FR) patient decision preferences: major cultural change in the USA Mara Rosner, Stephanie Andriole, Avishai Alkalay, Rachel Greenbaum, Juliana Gebb, Mark Evans Montefiore Medical Center/Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, NY, Comprehensive Genetics, Genetic Counseling, New York, NY, Albert Einstein College of Medicine/ Montefiore Medical Center, Obstetrics and Gynecology & Women’s Health, Bronx, NY, Comprehensive Genetics, Ultrasound, New York, NY, Comprehensive Genetics & Mt. Sinai School of Medicine, Obstetrics & Gynecology, New York, NY OBJECTIVE: 20 years ago, a disproportionate % of gender preference requests for prenatal diagnosis, terminations, and reductions were from patients of ethnic groups that valued boys over girls. We refused to participate. As patient interest expanded to all ethnic groups and gender preference seemed to equalize, our bioethicist had us re-evaluate and incorporate patient preference. STUDY DESIGN: We prioritize FR decisions by: 1. documented major (M) anomaly; 2. suspicious or minor (m) findings; 3. if neither, we consider gender preference. We retrospectively reviewed patient choices in our last 400 CVS/FR cases starting with triplets or twins. Patients were categorized based on type of reduction (3i2, 3i1, or 2i1), presence or absence of M or m findings, and whether all fetuses were the same gender (SG) or a gender choice was possible. Higher order and non-CVS pregnancies were excluded. RESULTS: 178/321 (56%) had either M/m or SG. Of 3i2 with option, 71/79 reduced to M/F of which about 1/3rd had no preference. 1 chose MM and 7 chose FF. Most 3i1 included MZ twins for whom we reduced MZ in 33/35 cases. Of 20 3i1 with option, 10 chose M and 10 chose F. Of 44 2i1 with option, 20 chose M & 24 chose F. CONCLUSION: There has been a major shift in culture from a generation ago. Male preponderance has ended in the USA. The vast majority of our patients who reduced to 2 preferred one of each or had no preference. When reducing to 1, a non-significant trend towards female preference was found. In addition to identifying abnormalities, CVS prior to FR expands patient autonomy. Poster Session III Doppler Assessment, Fetus, Prematurity www.AJOG.org

Objective: The detection of fetal aneuploidy and gene mutations by analysis of fetal cells in maternal blood has demonstrated the feasibility of noninvasive prenatal diagnosis. Fetal cells are rare in the maternal circulation; all current methods used for their isolation also yield maternal cells. We developed a method that permits a quantitative assessment of the relative numbers of fetal and maternal cells. Study Design: Samples from 40 pregnant women were flow sorted with different monoclonal antibodies. Deoxyribonucleic acid was subsequently purified from candidate fetal cells; polymerase chain reaction was performed with synthetic primers specific for sequences on chromosomes Y and 7. Results: The maximum number of fetal cells detected was 52 in 1080 maternal cells. Fetal cell purity ranged from 0.001 % to 4.8%. Fetal cells were detected with antibodies to CD71, CD36, and glycophorin A. Conclusion: Quantitative polymerase chain reaction enables the determination of the purity and yield of fetal cells remaining after isolation from maternal blood, facilitating rapid comparisons between different cell separation techniques.

SEQUENCE ANALYSIS FOR THE GENDER-INDEPENDENT ASSESSMENT OF FETAL CELLS IN MATERNAL BLOOD DONGHYUN CHA, KIARASH KHOSROTEHRANI, DIANA BIANCHI, KIRBY JOHNSON, Pochon University, Obstetrics and Gynecology, Seoul, South Korea, Tufts University, Pediatrics, Boston, Massachusetts, Tufts University, Pediatrics, Obstetrics & Gynecology, Boston, Massachusetts OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (fNRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fNRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. STUDY DESIGN: fNRBCs were isolated by density gradient separation, CD15/ 45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (4 cases), 18 (1 case), 13 (2 cases), and other genetic abnormalities (2 cases). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms. RESULTS: In all cases candidate fetal NRBCs were accurately identified based on morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSION: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications. Women and Infants’ Hospital, Maternal Fetal Medicine, Providence, Rhode Island, University of North Carolina, Department of Obstetrics and Gynecology, Chapel Hill, North Carolina OBJECTIVE: Vaginal bleeding in the first trimester may result in a disruption in the maternal-fetal interface with subsequent transfer of hormones into the maternal circulation. Elevated maternal serum concentrations of AFP have been observed in the first and second trimester in women with a history of first trimester vaginal bleeding. Our goal was to examine the effect of first trimester vaginal bleeding on first trimester serum levels of PAPP-A, free b-hCG, and nuchal translucency (NT) using a prospectively-collected large database. STUDY DESIGN: Women enrolled in the FASTER trial had a NT measurement and PAPP-A and free b-hCG levels drawn at 10 3/7-13 6/7 weeks. Patients with fetal anomalies, a diagnosis of diabetes prior to becoming pregnant or who conceived with invitro fertilization were excluded from this analysis. Patients were divided into three groups: (1) no bleeding, (2) light bleeding, or (3) heavy bleeding. The log transformed multiples of the medians (MoMs) of NT, PAPPA, and free b-hCG for the three groups were compared using analysis of variance for crude effects and analysis of covariance adjusting for possible confounding variables. For presentation, the results were back transformed to medians in MoMs. RESULTS: The study included 34,548 patients: 29,639 patients without bleeding, 4341 patients with light bleeding, and 547 patients with heavy bleeding. Adjusted medians of the MoMs of the markers are listed in the table. CONCLUSION: First trimester vaginal bleeding does not appear to affect PAPP-A or free b-hCG levels nor does bleeding impact NT measurement in this large study.