title: Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR.
abstract: OBJECTIVE
  Clinical indications for fetal sex determination include risk of X-linked disorders, a family history of conditions associated with ambiguous development of the external genitalia, and some fetal ultrasound findings. It is usually performed in the first trimester from fetal material obtained through CVS and is associated with an approximately 1% risk of miscarriage. Ultrasound fetal sex determination is often performed after 11 weeks of gestation. This study aims to validate a reliable method for non-invasive prenatal diagnosis of fetal gender using maternal plasma cell-free fetal DNA (cffDNA) for fetal sex assessment in the first trimester of pregnancy and test its clinical utility in the diagnosis of potentially affected pregnancies in carriers of X-linked disorders.
  
  
  METHODS
  In the validation study, blood samples from 100 pregnant women at 6-11 weeks of gestation were analysed. In the clinical study, 17 pregnancies at risk of having an affected fetus were tested. 7 ml of maternal blood in EDTA were obtained and cffDNA was extracted using a commercially available kit. DNA was enzymatically digested using a methylation sensitive endonuclease (AciI) to remove maternal unmethylated sequences of the RASSF1A gene. A multiplex PCR was performed for the simultaneous amplification of target sequences of SRY and DYS14 from chromosome Y, along with RASSF1A and ACTB sequences. Amplification of these loci indicates fetal gender, confirms the presence of cffDNA and allows assessment of digestion efficiency.
  
  
  RESULTS
  After establishing the appropriate experimental conditions, validation studies were successful in all 100 cases tested with no false negative or false positive results. Y chromosome-specific sequences were detected in 68 samples, and 32 cases were diagnosed as female based on the amplification of RASFF1A sequences only, in the absence of ACTB. In the clinical studies, fetal sex was correctly diagnosed in 16 pregnancies, and one case was reported as inconclusive.
  
  
  CONCLUSIONS
  Fetal sex assessment by detecting Y chromosome sequences in maternal blood can be routinely used from the 6th week of gestation. Reliable fetal sex determination from maternal blood in the 1st trimester of gestation can avoid conventional invasive methods of prenatal diagnosis.
authors.affiliation: Department of Medical Genetics, Athens University School of Medicine, Athens, Greece. akolial@med.uoa.gr
authors.forename: Aggeliki
authors.initials: A
authors.lastname: Kolialexi
authors.forename: Georgia
authors.initials: G
authors.lastname: Tounta
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Apostolou
authors.forename: Christina
authors.initials: C
authors.lastname: Vrettou
authors.forename: Nikos
authors.initials: N
authors.lastname: Papantoniou
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
authors.forename: Aris
authors.initials: A
authors.lastname: Antsaklis
authors.forename: Ariadni
authors.initials: A
authors.lastname: Mavrou
mesh_terms: D000199:Actins / Q000235:genetics; D000328:Adult; D001483:Base Sequence; D041322:Chromosomes, Human, Y / Q000737:chemistry / Q000235:genetics*; D042241:Early Diagnosis; D005260:Female; D040181:Genetic Diseases, X-Linked / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011296:Prenatal Diagnosis / Q000379:methods*; D055748:SOXB1 Transcription Factors / Q000235:genetics; D012732:Sex Determination Analysis / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics; D016216:Ultrasonography, Prenatal
chemical_list: D000199:Actins; C411762:RASSF1 protein, human; D055748:SOXB1 Transcription Factors; D025521:Tumor Suppressor Proteins