Chorionic Biopsy Research Articles

CLINICAL, ULTRASOUND AND CYTOGENETIC CHARACTERISTICS OF FETUSES WITH INCREASED NUCHAL TRANSLUCENCY THICKNESS IN THE FIRST TRIMESTER OF PREGNANCY

Background: Improving programs for early prenatal detection of congenital malformations remains a relevant scientific and practical problem. The prevalence of congenital anomalies in Ukraine is 23.7:1000 among live-born babies, and there is no significant decrease in it. The aim of this study was to compare clinical, ultrasound and cytogenetic data in swollen fetuses with increased nuchal translucency (NT) thicknesses from the group of pregnant women in the first trimester at high genetic risk to optimize the algorithm of prenatal diagnostics.
 Materials and Methods: Clinical examinations, ultrasound diagnostics, invasive methods of prenatal diagnostics (chorion biopsy, amniocentesis), genetic testing techniques, such as karyotyping and FISH, genetic counseling and statistical analysis were carried out. The results of complex examinations of 127 fetuses with an increased NT thickness from the group of pregnant women were analyzed. Fetuses were divided into two groups with an NT thickness of 2.5–3.5 mm (group 1) (38 cases) and with an NT above 3.5 mm (group 2)(89 cases).
 Results: Among pregnancies with fetuses with an increased NT thickness, there were 65.4% cases of adverse outcomes with chromosomal pathology (69.9%), congenital malformations of non-chromosomal etiology (25.3%) and pregnancy loss (4.8%). The frequency of chromosomal abnormalities in fetuses of group 1 was 55.3% and 41.6% in group 2. Congenital malformations of various systems and organs in fetuses, the most frequent of which were cardiac defects, were diagnosed. The ratio of congenital heart defects in the fetuses of groups 1 and 2 was 23.7% and 43.8%, respectively (p=0.03; OR=0.40).
 Conclusion: There is no significant difference between the frequency of chromosomal abnormalities in the fetuses of group 1 compared to group 2, which indicates a high informative value of an increased NT thickness, including the thickness of 2.5–3.5 mm in fetuses in the first trimester as a marker of chromosomal pathology. A significantly higher incidence of congenital malformations of non-chromosomal etiology was found in fetuses with venous duct pathology and NT thickness over 3.5 mm compared to fetuses with the same pathology and NT thickness of 2.5–3.5 mm. Proposed changes to the management algorithm for pregnant women with swollen fetuses include mandatory congenital heart defects screening in the first trimester.

PRENATAL DIAGNOSIS OF CONGENITAL AND HEREDITARY PATHOLOGY IN WOMEN WHO HAD COVID-19 DURING PREGNANCY

The recent investigation of genetic and demographic processes in Ukraine has revealed an exacerbation of the demographic crisis due to a high level of reproductive losses in women that is manifested by an increased frequency of lost pregnancies and the birth of offspring being at a high risk of disability and mortality. Corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome due to infection with the SARS-CoV-2 coronavirus spread rapidly around the world is potentially associated with complicated pregnancy. Unforeseen consequences of the COVID-19 pandemic pose a threat to the health of pregnant women. The effects of COVID-19 can be expected to become evident over years. Therefore, epidemiological information on the identification of population groups that may be at risk of severe complicated reactions is critically important. It is also essential to evaluate the situation at the different levels of population in order to outline the general directions of state policy in terms of effective countermeasures against the COVID-19 pandemic.
 Aim: to investigate the frequency and structure of congenital and hereditary pathology diagnosed by non-invasive and invasive methods of prenatal diagnosis in women who contracted COVID-19 during pregnancy.
 Material and methods. Ultrasound diagnosis of pregnant women who contracted COVID-19 during pregnancy was carried out using the Voluson E8 device and chorion biopsy in pregnant women. Collection of chorionic villi in the 1st trimester of pregnancy was carried out by aspiration method through cervical access under constant ultrasound control using a TROFOCAN catheter. An analysis of the frequency and structure of congenital and hereditary pathology was conducted based on the findings of non-invasive and invasive methods of prenatal diagnosis.
 Results. The study had demonstrated that the frequency of diagnosis of congenital foetal malformations is 6.3%. In the structure of congenital pathology, 2.5% of women had developmental anomalies of the cardiovascular system, 2.5% had congenital anomalies of the bone system, and 1.3% had multiple congenital malformations. Invasive prenatal diagnosis performed in 3.7% of women detected chromosomal pathology in 3.7% of women, and in particular, trisomy 21 of chromosomes was diagnosed in 2.5% of women, Turner syndrome was found in 1.3% of women. The frequency of chromosomal pathology in women who contracted COVID-19 during pregnancy is 3.7%. In 51 (63.7%) women who contracted COVID-19 during pregnancy, ultrasound examination of pregnant women has revealed certain features and pathological changes, especially in the structure of the placenta and changes in uteroplacental blood flow: 26.3% of women were diagnosed on premature aging of the placenta; 13.7% of women had the impairment of uteroplacental blood flow, and 23.7% of women were diagnosed as having concomitant pathology.
 Conclusion. The frequency of congenital malformations diagnosed by non-invasive prenatal diagnosis in women who contracted COVID-19 during pregnancy was 6.3%. The frequency of chromosomal pathology in the women was 3.7%. In 63.7% of women who contracted COVID-19 during pregnancy, certain features and pathological changes were detected during ultrasound examination, especially in the structure of the placenta and changes in uteroplacental blood flow.

Resultados perinatales tras prueba diagnóstica invasiva en el embarazo

IntroductionInvasive prenatal diagnostic techniques allow us to conduct genetic tests. The development of non-invasive techniques has reduced their use. The foetal loss rate following an invasive procedure is considered to be around 1%. The published data is heterogeneous however, although everything indicates that the risk has been overestimated, we need to conduct further studies. Material and methodsIn our single-centre retrospective study we analysed the procedures carried out using invasive prenatal diagnostic techniques between 2011 and 2019. A total of 832 invasive techniques were performed. Perinatal results are compared with a control group of pregnant women (n=1734). ResultsThe early foetal loss rate for the different techniques were 1.1% for amniocentesis, 1.6% for transvaginal chorionic biopsy and 5% for abdominal chorionic biopsy, with a total rate of 1.1%, without statistically significant differences between them (P=.57). We found differences in foetal outcome, in terms of variable early foetal loss, related to the attempts made (when three attempts were made, the risk increased). When comparing the perinatal outcomes after delivery of the group that underwent techniques with the control group, a higher rate of caesarean sections was found in the study group (28.9% vs 20.5%), in addition to lower mean gestational age at delivery (38.33 vs. 38.95 weeks). DiscussionWhen the invasive technique is performed at the right time and with no more than two attempts, we consider that the risk of foetal loss is not affected, and is equal to that of the general population.

Использование трофобластов цервикального канала для неинвазивной пренатальной детекции частых анеуплоидий

The article presents an experience of isolation of trophoblast cells from cervical samples of pregnant women and demonstrates a possibility of determining aneuploid cells in cervical samples using FISH analysis. Objective. To study methods of effective isolation and reliable detection of trophoblasts for genetic pathology analysis. Patients and methods. The participants of the study were three pregnant women, who according to the findings of the combined first trimester screening test were referred to the group with a high risk for fetal aneuploidy. The terms of gestation varied from 12 to 14 wks. Immunocytochemical detection of trophoblast cells was performed with FITC-labelled HLA-G antibodies. FISH was performed with the use of locus-specific DNA probes on chromosomes 13/21 and a DNA probe on chromosome 18 centromere. The chromosome set of a fetus was verified by the results of standard cytogenetic analysis of semi-direct chromosome preparations from cytotrophoblast cells obtained by chorionic villi biopsy. Results. HLA-G-positive cells were found in all examined cytological specimens. FISH analysis with the use of DNA probes on chronomomes, whose trisomies are compatible with live birth, detected aneuploid cells in all three cases. Conclusion. The use of cervical trophoblasts for noninvasive collection of information about the genetic status of the developing fetus at present needs further study of effective and reliable methods of their isolation, detection and analysis. Key words: aneuploidy, throphoblast cells, NIPT, prenatal diagnosis, HLA-G

Chromosomal disorders in the fetus of pregnant woman from Aktau

The article presents the data of cytogenetic study on the fetal karyotype of 310 pregnantwomen from the city Aktau in 2013-2015. Chorionic biopsy and placental biopsy were carried out for248 pregnant women with amniocentesis in 18 cases and cordocentesis in 44 cases. Clinical indicators imposing invasive procedures to pregnant women at risk were maternal age factor, fetal ultrasoundmarkers, serum blood markers determined in pregnant women, the presence of children with multiplecongenital malformations, chromosomal pathologies, etc. Distribution frequencies of fetal chromosomalabnormalities have been determined based on clinical and laboratory studies. The highest frequency ofchromosomal abnormalities of the fetus was indicated in pregnant women with three salient indicators:the factor of the age of the pregnant woman, parameters of biochemical screening, and ultrasound markers. Chromosomal abnormalities of the fetus were detected in 94 (30.3%) pregnant women including 92cases (97.9%) represented by numerical chromosome disorders and 2 cases (2.1%) of structural disorders. Disorders of the autosome system were observed 9.4 times more often comparing to abnormalitiesin the sex chromosome system. Of the numerical chromosome abnormalities, a high specific weight isoccupied by trisomy of the 21-st chromosome which has reached 65.1%. A comparative analysis of thefrequencies of chromosomal abnormalities of the fetus of two port cities has demonstrated a 1.7-foldincrease in the city of Aktau (Kazakhstan) compared with the city of Murmansk (Russia), and the averagefrequencies of fetal karyotype anomalies have made up 19.6% and 11.6%, respectively. Higher level offrequencies for fetal karyotype anomalies in Aktau is possibly associated with unfavorable environmentalconditions in this city caused by the allocation of the oil and gas industry, the repository for tailing toxicand radioactive wastes, and abandoned uranium mines.Key words: karyotype, fetus, prenatal diagnosis, fetal chromosome disorders, ecological state ofurban agglomerations.

DEGUM, ÖGUM, SGUM and FMF Germany Recommendations for the Implementation of First-Trimester Screening, Detailed Ultrasound, Cell-Free DNA Screening and Diagnostic Procedures

First-trimester screening between 11 + 0 and 13 + 6 weeks with qualified prenatal counseling, detailed ultrasound, biochemical markers and maternal factors has become the basis for decisions about further examinations. It detects numerous structural and genetic anomalies. The inclusion of uterine artery Doppler and PlGF screens for preeclampsia and fetal growth restriction. Low-dose aspirin significantly reduces the prevalence of severe preterm eclampsia. Cut-off values define groups of high, intermediate and low probability. Prenatal counseling uses detection and false-positive rates to work out the individual need profile and the corresponding decision: no further diagnosis/screening - cell-free DNA screening - diagnostic procedure and genetic analysis. In pre-test counseling it must be recognized that the prevalence of trisomy 21, 18 or 13 is low in younger women, as in submicroscopic anomalies in every maternal age. Even with high specificities, the positive predictive values of screening tests for rare anomalies are low. In the general population trisomies and sex chromosome aneuploidies account for approximately 70 % of anomalies recognizable by conventional genetic analysis. Screen positive results of cfDNA tests have to be proven by diagnostic procedure and genetic diagnosis. In cases of inconclusive results a higher rate of genetic anomalies is detected. Procedure-related fetal loss rates after chorionic biopsy and amniocentesis performed by experts are lower than 1 to 2 in 1000. Counseling should include the possible detection of submicroscopic anomalies by comparative genomic hybridization (array-CGH). At present, existing studies about screening for microdeletions and duplications do not provide reliable data to calculate sensitivities, false-positive rates and positive predictive values.

Preimplantation genetic diagnosis of X-linked Charcot-Marie-Tooth disease by indirect linkage analysis

ObjectiveTo present methodical approach of preimplantation genetic diagnosis (PGD) as an option for an unaffected pregnancy in reproductive-age couples who have a genetic risk of the X-linked dominant peripheral neuropathy Charcot-Marie-Tooth type 1 disease. Patients and methodsWe performed PGD of X-linked Charcot-Marie-Tooth type 1 disease using haplotyping/indirect linkage analysis, when during analysis we reach to exclude embryos that carry a high-risk haplotype linked to the causal mutation p.Leu9Phe in the GJB1 gene. ResultsWithin the PGD cycle, we examined 4 blastomeres biopsied from cleavage-stage embryos and recommended 3 embryos for transfer. Two embryos were implanted into the uterus; however, it resulted in a singleton pregnancy with a male descendant. Three years later, the couple returned again with spontaneous gravidity. A chorionic biopsy examination of this gravidity ascertained the female sex and a pericentric inversion of chromosome 5 in 70% of the cultivated foetal cells. ConclusionUsing indirect linkage analysis, PGD may help to identify genetic X-linked defects within embryos during screening, thereby circumventing the potential problems with abortion.

Diagnóstico genético preimplantacional de la enfermedad de Charcot-Marie-Tooth ligada al cromosoma X mediante análisis de ligamiento indirecto

ObjectiveTo present methodical approach of preimplantation genetic diagnosis (PGD) as an option for an unaffected pregnancy in reproductive-age couples who have a genetic risk of the X-linked dominant peripheral neuropathy Charcot-Marie-Tooth type 1 disease. Patients and methodsWe performed PGD of X-linked Charcot-Marie-Tooth type 1 disease using haplotyping/indirect linkage analysis, when during analysis we reach to exclude embryos that carry a high-risk haplotype linked to the causal mutation p.Leu9Phe in the GJB1 gene. ResultsWithin the PGD cycle, we examined 4 blastomeres biopsied from cleavage-stage embryos and recommended 3 embryos for transfer. Two embryos were implanted into the uterus; however, it resulted in a singleton pregnancy with a male descendant. Three years later, the couple returned again with spontaneous gravidity. A chorionic biopsy examination of this gravidity ascertained the female sex and a pericentric inversion of chromosome 5 in 70% of the cultivated foetal cells. ConclusionUsing indirect linkage analysis, PGD may help to identify genetic X-linked defects within embryos during screening, thereby circumventing the potential problems with abortion.

Missed abortion: anembrionia and normal karyotype of chorion

The most probable cause of an anembryonic gestation is an abnormal karyotype. The analysis of 59 cases of missed abortion with anembrionia prior to 12 weeks of gestation after spontaneous conception and the assisted reproductive technologies (ART) was carried out. Patients were divided into two groups: those pregnant after natural conception and those with pregnancies after the ART programs. Upon embryoscopy and direct chorion biopsy, samples were sent for karyotyping. High frequency of normal karyotypes of chorion was found: 28.6% and 44.7%, respectively.

Prenatal Diagnosis of Hemoglobinopathies: A Case Study on Tunisia

Hemoglobinopathies are the most common genetic disease in Tunisia with a total carrier prevalence of 4.48%, reaching 12.5% at certain affected regions. The β-thalassemia trait frequency is 2.21% and the sickle cell trait is 1.89%. A prenatal diagnosis (PND) unit has been progressively installed since 1986 at the biochemistry and molecular biology department of children’s hospital of Tunis. The present study tries to explore the prenatal diagnosis of the Hemoglobinopathies in Tunisia for the period of 1994-2012 and tries to share its experiences and the progress achieved in overcoming this challenge. 340 out of 461 fetuses examined for this study were at risk and couples that have agreed for pre-diagnosis have benefited a lot in averting the major health risks like beta-thalassemia (41%), sickle cell anemia (40.3%), S/beta-thal (14.7%) and the remaining fetuses were at risk for a compound heterozygote hemoglobinopathies (S/O, O/beta-thal, S/C). 25.8% of the couple covered in this study have voluntarily asked for PND several times, as they were worried about giving birth to a child with Hemoglobinopathy. Fetal DNA was collected from chorionic villis biopsy of 53 cases, amniotic fluid samples are considered in 397 cases and CVS was followed in 7 cases for lack of results. Out of 461 tested foetuses, 26.2% of them were affected, 50.5% were carriers of the disease and 19.3% were completely healthy. PND fail to detect the problem in 3.9% of the cases. Except 13% of the affected fetuses, the entire number of cases detected with defected fetuses had been aborted. Although abortion is legal in Tunisia, 13% of pregnant women with affected fetus have refuted to abortion due to religious reasons. The total number of fetus aborted remained 1.53%. Although PND was successful in our lab, it was insufficient to cover the entire Tunisian territory. The researchers hence recommend to the health authorities to establish widespread PND services in places that are worst affected with these cases in Tunisia to prevent hemoglobinopathies

β-Thalassemia: Early prenatal diagnosis

A case of beta-thalassemia major diagnosed by fetal DNA analysis on a chorion biopsy during the 11th, week of gestation is described. A 30 year-old Gravida 2, Parite 1 patient whose firstborn died of beta thalassemia major (Cd8/A) when 2 years old, was admitted to our clinic on her 10th week of pregnancy. Chorionic villus sampling was performed and resulted as beta thalassemia major (Cd8/Cd8). β-Talasemi: Dogum oncesi erken tani Bu vakada 11 hafta gebelik sirasinda koryon villus biopsisiyle beta talasemi major tespit ettik. Hasta 30 yasinda ve bu ikinci gebeligiydi. Ilk bebegi 2 yasinda beta talasemi major nedeniyle olmus. Biz bu vakayi 10 hafta gebelikte degerlendirdik. Beta talasemi major tespit ettik ve gebeligi sonlandirdik. J. Exp. Clin. Med., 2012; 29:229-230

Aborting a malformed fetus: a debatable issue in saudi arabia.

Congenital anomalies contribute a significant proportion of infant morbidity and mortality, as well as fetal mortality. They are generally grouped into three major categories: structural/metabolic, congenital infections, and other conditions. The most prevalent conditions include congenital heart defects, orofacial clefts, Down syndrome, and neural tube defects. Several prenatal diagnostic procedures have been introduced, both cytogenetic (such as chorion biopsy, amniocentesis and funiculocentesis) and biophysical (ultrasound 2-D, 3-D and 4-D, ultrasonography with Doppler, etc.). Insufficient data are currently available from Saudi Arabia on the epidemiology of the lethal congenital abnormalities which should be a priority due to high rate of consanguineous marriages among first cousins and their association with congenital anomalies. In terms of consanguinity and birth defects, a significant positive association has been consistently demonstrated between consanguinity and morbidity, and congenital defects with a complex etiology appear to be both more prevalent in consanguineous families and have a greater likelihood of recurrence. A debate regarding aborting a malformed fetus still exists among the senior Islamic scholars in many of the Islamic countries. The progressive interpretations of Islam have resulted in laws allowing for early abortion on request in two countries; six others permit abortion on health grounds and three more also allow abortion in cases of rape or fetal impairment. In Saudi Arabia, efforts to legalize abortion in certain circumstances have been recently discussed among Senior Religious Scholars and specialized physicians to permit abortions in certain circumstances. In this mini-review we discuss the current debate regarding aborting a malformed fetus in Saudi Arabia with a focus on the Islamic perspective.

Chorion biopsy in mongrel dogs

With the great development of the gestational studies in all of the species, we noticed the necessity of adaptations of these techniques for prenatal diagnosis in dogs. Based on this, we studied the feasibility of chorion biopsy guided by ultrasound. Our results demonstrated accuracy on the sex determination being 2 males and 12 females, as well as it would be possible to identify chromosome alteration due to the quality of samplings. Sex determination was accomplished with the identification of Y gene chromosomes in PCR technique. After the collection, fragments were prepared for light microscopy studies and revealed fetal chorion tissue, blood colloid and erythrocyte. In the whole material we found hemosiderin impregnations due to the hemolysis and to the residue of blood of the placental marginal hematomes. The submitted female dogs to this technique demonstrated normal puppy births without death.

Use of HbA estimation by CE-HPLC for prenatal diagnosis of β-thalassemia; experience from a tertiary care centre in north India: a brief report

Prenatal diagnosis (PND) of β-thalassemia is offered by DNA based mutation analysis at 10–12 weeks gestation using chorionic villous biopsy specimens. Most centers offering PND search for five common mutations prevalent in India, which cover more than 90% of patients. However in those families where the mutation cannot be identified in the parents, PND becomes difficult by mutation detection. The technique thus followed is estimation of globin chain synthesis ratio in second trimester cord blood samples. However, this technique is cumbersome and not available in most of the centres. We evaluated 112 cord blood samples (74 were informative) for analyzing hemoglobin A (HbA) levels by cation-exchange high performance liquid chromatography (CE-HPLC) using the BioRad β-thal short program between 18 and 22 weeks of gestation. A normal range for the population was calculated by running 14 cord blood samples of non-thalassemic abortuses of the same gestation. A 6 month post-natal follow-up was possible in 18 cases. All but one of these corroborated their PND.

Gezielte genetische Diagnostik nach Ersttrimester-Ultraschallscreening bei letaler fetaler Skelettdysplasie

Einleitung: Skelettdysplasien sind eine wichtige Differenzialdiagnose bei chromosomal unauffälligen Feten mit Wachstumsretardierung. Skelettdysplasien beinhalten eine Reihe von Störungen mit unterschiedlichen Folgen für das fetale Überleben und differierende Wiederholungsrisiken. In dem vorliegenden Fall möchten wir zeigen, wie die Kombination aus Ersttrimesterultraschall und gezielter DNA-Diagnostik auch ohne Indexpatient eine exakte Diagnose liefern kann, welche für die Beratung betroffener Familien essenziell ist. Methodik: Eine gesunde Nullipara stellte sich in der 12 + 4 SSW bei ausgeprägt verbreiterter Nackentransparenz (Nackenfalte > 10 mm) vor. Die eingehende fetale Sonografie zeigte verkürzte Rippen, eine negative A-Welle im Fluss des Ductus venosus und eine Tetramikromelie, welche eine letale Form einer Skelettdysplasie vermuten ließ. In Betracht gezogen wurden die thanatophore Dysplasie und die 3 Subtypen der Achondrogenesie (ACG). Daraus folgerte die Begrenzung möglicher Genlokalisationen auf 3 Gene. Ergebnis: Eine homozygote Frame-Shift-Mutation im Sulfattransportergen SLC26A2 wurde in der DNA der Chorionzottenbiopsie identifiziert. Daraus resultierte die Diagnose der autosomal rezessiven ACG Typ Ib (ACG1B; OMIM 600972). Die Gewissheit der letalen Prognose und das Wiederholungsrisiko von 25 % für ACG 1B ermöglichte es, der Familie eine aufgeklärte Entscheidung bezüglich der laufenden Schwangerschaft und ihrer weiteren Familienplanung zu treffen. Zusammenfassung: Durch die Kombination von Ultraschall und Genetik ist auch schon im ersten Trimester selbst bei fehlendem Indexpatienten eine exakte Diagnosestellung einiger letaler Skelettdysplasien möglich. Dies ist sowohl für die aktuelle sowie für zukünftige Schwangerschaften von weitreichender Bedeutung.

New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma

Background Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. Methods We have used a mini-sequencing based method, the SNaPshot®, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. Results The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. Conclusions The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot® technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

Births Resulting from Oocyte Cryopreservation Using a Slow Freezing Protocol with Propanediol and Sucrose

The human mature oocyte is particularly sensitive to cooling and low temperatures in addition to freeze-thaw damage. The efficiency of oocyte cryopreservation including the pregnancy outcome is still low. The aim of our study is to briefly introduce our preliminary clinical results achieved with oocyte cryopreservation (CP). Our work focused on the use of a slow cooling procedure using the cryoprotectants propanediol (1.5 M) and sucrose (0.3 M). Following a short incubation of 4–6 hours thawed oocytes were injected with a single sperm (ICSI) and fertilization was assessed 12–16 hours later. Laser assisted hatching (LAH) was performed on all transferred embryos and embryo transfer (ET) was carried out 48–72 hours after ICSI. One-hundred and ten eggs were thawed and a survival rate of 76% (84/110) was obtained. Of the 84 oocytes which survived, 64 subsequently fertilized (64/84; 76%) following ICSI and on the following day 55 of those had cleaved (55/64; 86%). Fifty-two embryos were transferred in 29 patients (1.8 embryo/patient), and 7 (7/29; 24%) resulted in clinical pregnancy (1 twin pregnancy). One of the pregnancies encountered first trimester abortion (1/7; 14%). Implantation rate of 15.4% per embryo transferred (8/52) and 7.3% per egg thawed (8/110) were obtained. In all cases, chorion biopsy was performed and chromosomal anomalities were not detected. Our results provide further evidence that the procedure can be applied safely and with good success in clinical assisted reproduction. However, more work is needed since the survival and implantation rate should be improved.

Protein expression profiling of extremely preterm placentas suggests distinct pathways of intrauterine inflammation

DISTINCT PATHWAYS OF INTRAUTERINE INFLAMMATION THOMAS MCELRATH, RAINA FICHOROVA, ELIZABETH ALLRED, JONATHAN HECHT, OLAF DAMMANN, ALAN LEVITON, Brigham and Women’s Hospital, Department of Obstetrics and Gynecology, Boston, Massachusetts, Children’s Hospital of Boston, Neuroepidemiology Division, Boston, Massachusetts, Beth Israel Deaconess Medical Center, Department of Pathology, Boston, Massachusetts, Hannover Medical School, Neurology, Hannover, Germany OBJECTIVE: Preeclampsia (PE), preterm premature membrane rupture (pPROM), and preterm labor (PL) are disorders that may arise from alternative pathways in inflammatory pathology. We hypothesize that placental protein expression profiles are not random and will cluster into patterns corresponding to different types of intrauterine inflammation. STUDY DESIGN: 30 singleton placentas (23-28 wks) were obtained immediately after delivery for the 3 clinical indications. A lysate was prepared from a systematic chorionic biopsy and protein expression levels evaluated on a multiplex array. Data was analyzed in 4 functional categories: Inflammatory (IL1b IL6 TNFa IL18), Chemokine (IL8), Endothelial (Pselectin Eselectin VCAM1 ICAM1 ICAM3 VEGF), Modulatory (IL10 TGFb IL1ra Tmodulin). A K-means cluster analysis, T-test and ANOVA were performed. The clusters represent the consensus of 5x10 iterations. Unless otherwise noted a p-value of !0.05 with Bonferroni correction is considered significant. RESULTS: Profile#1: Characterized by significantly lower expression of all 4 functional categories, except VEGF, whose expression was elevated (p!0.001). Only PE samples were contained in this pattern. Profile#2: Characterized by significantly increased expression of inflammatory/chemokine proteins and contained both PL and pPROM placentas. Profile#3: Characterized by significantly decreased expression of inflammatory/chemokine proteins and also contained both PL and pPROM placentas. CONCLUSION: This protein profiling of extremely preterm placentas distinguished three subprofiles and suggests that: PE is characterized by globally low levels of inflammatory, chemokine, endothelial and modulatory protein expression but with increased expression of placental VEGF. PL and pPROM share two expression profiles, one inflammatory and the other not. This observation suggests that common etiologies underlie both PL and pPROM despite different clinical presentations. These etiologies may involve alternative inflammatory pathways including potential differences in maternal/ fetal inflammatory origins.

Studies on the Isolation and Identification of Fetal Nucleated Red Blood Cells in the Circulation of Pregnant Women before and after Chorion Villus Sampling

Objective: To investigate the feasibility of various molecular forms of hemoglobin as markers for fetal nucleated red blood cells (NRBCs). Methods: The presence of epsilon and gamma globin positive NRBCs was investigated in pure fetal blood and in blood from pregnant women before and after chorion biopsy. Maternal samples were enriched for NRBCs by various conventional methods, including limited enrichment by only positive CD71 selection or single density gradient. We searched for fetal cells on slides by automated scanning. Fetal cells were defined by (1) the presence of epsilon or gamma globin and (2) simultaneously by the presence of a Y chromosome signal. Results: 18 of 25 gamma globin positive cells identified in blood samples after chorion biopsy were chromosome Y signal positive, and 1 cell had two X chromosome signals. 263 of 339 epsilon globin positive cells identified in blood samples after chorion biopsy were hybridized with X and Y chromosome probes. None had two X signals, and 249 were Y positive. In blood samples before chorion biopsy, only 1 epsilon globin positive fetal NRBC and no epsilon globin positive maternal NRBCs were found. Conclusions: Epsilon globin may be specific for fetal NRBCs. Only 1 epsilon globin positive fetal cell was identified in 1 of 12 blood samples before chorion biopsy, representing a total of 182 ml of maternal blood. This suggests that most fetal cells found in maternal blood by fluorescence in situ hybridization methods may not be NRBCs.

Prenatal Diagnosis in Rett Syndrome

Background/Aim: Rett syndrome (RTT) is an X-linked neurodevelopmental dominant disorder that affects almost exclusively girls. The disease is mainly sporadic, caused by de novo mutations at MECP2 gene (Xq28), but a low percentage of familial cases have been reported. We present the results of RTT prenatal diagnosis in three families and discuss the usefulness of such analyses in diseases caused mainly by de novo mutations. Methods: For adult individuals, DNA was extracted from peripheral lymphocytes; for fetus analysis it was obtained from cultured amniotic fluid or from chorionic biopsy specimens. Mutation detection at MECP2 gene was first carried out in the patients by SSCP/HD analysis and subsequent sequencing. Family studies and prenatal diagnoses were done by direct analysis of previously characterized patients’ mutations using SSCP/HD or restriction analysis. Results: Heterozygous mutations identified in the 3 patients were: 1061del96bp, 473C→T, and 763C→T, respectively. Mutations were not present in the mothers’ DNAs obtained from peripheral lymphocytes. None of the 3 fetuses analyzed carried the mutation of the affected sister. Conclusions: Recurrence within RTT families can be due to asymptomatic nonpenetrant carrier mothers or to parental germinal mosaicism for the MECP2 mutation. Since germline mosaicism can neither be predicted nor detected, families with 1 affected patient whose RTT-causing mutation has been previously identified can benefit from prenatal diagnosis which contributes to a decrease in the recurrence risk in a new pregnancy comparable to that of the normal population.