Cytogenetic Analysis of Sporadic First-Trimester Miscarriage Specimens Using Karyotyping and QF-PCR: A Retrospective Romanian Cohort Study.

It is well known that first-trimester miscarriages are associated with chromosome abnormalities, with numerical chromosome abnormalities being the ones most commonly detected. Conventional karyotyping is still considered the gold standard in the analysis of products of conception, despite the extended use of molecular genetic techniques. However, conventional karyotyping is a laborious and time-consuming method, with a limited resolution of 5-10 Mb and hampered by maternal cell contamination and culture failure. The aim of our study was to assess the type and frequency of chromosomal abnormalities detected by conventional karyotyping in specimens of sporadic first-trimester miscarriages in a Romanian cohort, using QF-PCR to exclude maternal cell contamination. Long-term cultures were established and standard protocols were applied for cell harvesting, slide preparation, and GTG banding. All samples with 46,XX karyotype were tested for maternal cell contamination by QF-PCR, comparing multiple microsatellite markers in maternal blood with cell culture and tissue samples. Out of the initial 311 specimens collected from patients with sporadic first-trimester miscarriages, a total of 230 samples were successfully analyzed after the exclusion of 81 specimens based on unsuitable sampling, culture failure, or QF-PCR-proven maternal cell contamination. Chromosome abnormalities were detected in 135 cases (58.7%), with the most common type being single autosomal trisomy (71/135-52.6%), followed by monosomy (monosomy X being the only one detected, 24/135-17.8%), and polyploidy (23/135-17.0%). The subgroup analysis based on maternal age showed a statistically significant higher rate of single trisomy for women aged 35 years or older (40.3%) compared to the young maternal age group (26.1%) (p = 0.029). In conclusion, the combination of conventional karyotyping and QF-PCR can lead to an increased chromosome abnormality detection rate in first-trimester miscarriages. Our study provides reliable information for the genetic counseling of patients with first-trimester miscarriages, and further large-scale studies using different genetic techniques are required.

Five Variable Number of Tandem Repeats Loci (D17S5, APOB, TPO Intron 10, IL-1α Intron 6, and CIAS1) in Thais and Application in the Prenatal Diagnostic Laboratory.

Background: Prenatal diagnosis of genetic disease requires DNA analysis of fetal tissue of a responsible gene. Accurate diagnosis is useful for the appropriate management of pregnancy. However, maternal contamination of fetal specimens poses a high preanalytical risk of prenatal misdiagnosis. We have examined five variable number of tandem repeat (VNTR) polymorphisms for use in monitoring potential maternal contamination. Materials and Methods: A study was conducted to examine the heterozygosities of five VNTR loci including, D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 in 200 unrelated Thai subjects and applied to the monitoring of maternal contamination in 22 families at risk of having fetuses with severe thalassemia. Results: The heterozygosities of D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 VNTRs were 59.5, 19.5, 66.0, 35.5, and 42.0%, respectively. Therefore, the TPO intron 10 and D17S5 loci were chosen for prenatal diagnosis of thalassemia in 22 families. Analyses of these VNTRs demonstrated an increase of informative data from 59.1% provided by the routine D1S80 VNTR analysis to 90.9%. Conclusions: The VNTR diagnostic procedure described above is simple, cost-effective, rapid, and does not require the use of sophisticated instruments; it should prove useful in the prenatal diagnosis of thalassemia.

Ultrasound-guided Manual Vacuum Aspiration is an optimal method for obtaining products of conception from early pregnancy loss for cytogenetic testing.

OBJECTIVE: The culture failure rate of conventional karyotyping in products of conception evacuated from early pregnancy loss by traditional electrical vacuum aspiration and/or dilation & curettage remains high. We aim to determine whether obtaining products of conception from early pregnancy loss via another evacuation approach, ultrasound-guided manual vacuum aspiration, could decrease the culture failure rate of karyotyping. METHODS: For patients with early pregnancy loss, ultrasound-guided manual vacuum aspiration (Case group) and traditional electrical vacuum aspiration and/or dilation & curettage (Control group) were applied as surgical methods for pregnancy loss management respectively. The evacuated products of conception were subjected to cytogenetic karyotyping analysis. The primary outcome was the culture failure rate of karyotyping. Secondary outcomes included the chromosomal abnormality spectrum, maternal cell contamination, and complications from the manual vacuum aspiration procedure. RESULTS: For the case group, 132 products of conception were genetically analyzed by conventional karyotyping. The culture failure rate was significantly lower than that of the control cohort (2.3% vs 7.4%, p = 0.027). 65.2% of cases were abnormal within the detection scope of karyotyping. The euploid female to male ratio was 0.82 (18:22), suggesting a lower likelihood of maternal cell contamination. The efficacy in achieving complete evacuation was 99.6%. There were no significant complications from the procedure. CONCLUSION: Ultrasound-guided manual vacuum aspiration approach significantly decreased the culture failure rate of karyotyping and decreased maternal cell contamination tendency, enabling a high and accurate cytogenetic diagnosis. It is especially crucial when the cytogenetic analysis is required in the early pregnancy loss clinics.

Maternal cell contamination in embryonic chromosome analysis of missed abortions.

AIM: The fetal sample used for embryonic chromosome analysis is often contaminated with maternal cells, making it difficult to evaluate the fetal chromosomes. We examined on the rate of maternal cell contamination and its relationship with maternal information in the embryonic chromosome analysis of missed abortions using the Giemsabanding method. METHODS: Chromosome analysis was performed in 200 cases of delayed miscarriages in first trimester between July 1, 2000 and May 31, 2019. Chorionic villi were collected and were analyzed using the Giemsa banding method. Among the 20 cells for which chromosomal examination was performed, cells wherein 46,XX chromosomes were found together with normal male karyotype or abnormal chromosomes were defined as maternal cell contamination. RESULTS: Of the 200 cases analyzed, 136 had abnormal chromosomes. The normal female karyotype (n = 52) was four times more prevalent than the normal male karyotype (n = 12). Maternal cell contamination was seen in 15.4% of the abnormal chromosome cases and 8.3% of the normal male karyotype cases. There was no significant difference in the gestational age between the contaminated and noncontaminated groups at the time of miscarriage diagnosis. However, miscarriage before fetal heartbeat confirmation was significantly associated with higher maternal cell contamination. CONCLUSION: We found maternal cell contamination in 15% of all the cases. Moreover, in many cases of the normal female karyotype, it was suspected that only maternal chromosomes were cultured. When performing embryonic chromosome analysis in recurrent miscarriages, we should pay attention to maternal cell contamination and interpret the results accordingly.

The Influence of Maternal Cell Contamination on Fetal Aneuploidy Detection Using Chip-Based Digital PCR Testing.

Prenatal samples obtained by amniocentesis or chorionic villus sampling are at risk of maternal cell contamination (MCC). In traditional prenatal analysis, MCC is recommended to be assayed by special tests, such as the short tandem repeat analysis and, if detected at a high level, may result in failed analysis report. The objective of this study was to test the ability of chip-based digital PCR to detect fetal aneuploidies in the presence of MCC. To determine the level of accuracy of MCC detection, an aneuploid male sample was subjected to serial dilution with an euploid female sample. DNA was extracted from prenatal samples and analyzed with QuantStudio 3D Digital PCR. Digital PCR analysis allowed the detection of trisomy 21, trisomy 18, and X monosomy accurately in samples with 90%, 85%, and 92% of MCC, respectively. Moreover, our results indicated that digital PCR was able to accurately confirm the presence of Y chromosome at up to 95% contamination. The amniotic fluid and chorionic villus sampling (CVS) received in our clinical laboratory was subjected to further analysis of MCC based on the aneuploidy assessment algorithm, resulting in the identification of 10 contaminated samples and four cases of true fetal mosaicism. We conclude that chip-based digital PCR analysis enables the detection of fetal aneuploidy with high levels of accuracy, even in cases of significant MCC. Importantly, the algorithm eliminates the need for maternal DNA and additional MCC tests, which reduces costs and simplifies the diagnostic procedure. The method is easy to set up and suitable for routine clinical practice.

Reducing misdiagnosis caused by maternal cell contamination in genetic testing for early pregnancy loss.

The analysis of products of conception (POC) is clinically important to establish the cause of early pregnancy loss. Data from such analyses can lead to specific interventions in subsequent natural or assisted conceptions. The techniques available to examine the chromosomal composition of POC have limitations and can give misleading results when maternal cell contamination (MCC) is overlooked. The aim of this study was to develop a protocol for MCC assessment and to formulate POC material handling, testing, and reporting recommendations. Using array comparative genomic hybridization, we tested 86 POC samples, of which 47 sample pairs (DNA extracted from the POC sample and maternal DNA) were assessed for the presence of MCC. MCC was evaluated using an approach we developed, which exploited the genotyping of 14 STR, AMEL, and SRY loci. POC samples showing the clear presence of villi (63.9%) did not contain any signs of the maternal genome and can therefore be reliably tested using conventional methods. The proportion of 46,XX karyotype in the unselected sample batch was 0.39, which fell to 0.23 in visually good samples and was 0.27 in samples having no signs of contamination upon MCC testing. MCC assessment can rescue visually poor samples from being discarded or wrongly genotyped. We demonstrate here that classification based on visual POC material evaluation and MCC testing leads to predictable and reliable POC genetic testing outcomes. Our formulated recommendations covering POC material collection, transportation, primary and secondary processing, as well as the array of pertinent considerations discussed here, can be implemented by laboratories to improve their POC genetic testing practices. We anticipate our protocol for MCC assessment and recommendations will help reduce the misconception regarding the etiology of miscarried fetuses and foster informed decision-making by clinicians and patients dealing with early pregnancy loss.

Assessment of Maternal Cell Contamination in Prenatal Samples by Quantitative Fluorescent PCR (QF-PCR).

When biopsying a fetal tissue like chorionic villi or amniotic fluid, there is a chance of getting some maternal material that could contaminate the fetal specimen and might lead to a misdiagnosis. Thus, all prenatal samples should be subjected to testing for maternal cell contamination. This is done using quantitative fluorescent PCR (QF-PCR) of short tandem repeat (STR) markers.

Next generation sequencing as a new detection strategy for maternal cell contamination in clinical prenatal samples.

OBJECTIVES: The maternal cell contamination in chorionic villus or amniotic fluid presents a serious preanalytical risk for prenatal misdiagnosis. The following study presents and validates a novel process for identifying MCC by detecting short tandem repeat markers on Ion Proton system. Initially, MCC testing was performed during the detection of chromosomal abnormalities so as to improve the detection efficiency and accuracy of this method. MATERIAL AND METHODS: More than 70 STR loci were selected to establish the detection progress. Capillary electrophoresis was used to compare the next generation sequencing detection results, as well as to identify the optimal STR on Ion Proton system. Evaluation criteria for maternal cell contamination were set, and the automated data analysis was performed. The detection sensitivity was validated via 4 groups with mixed samples and different proportions. RESULTS: Consequently, twenty-three clinical samples were tested to evaluate the detection accuracy. In addition, 14 reli-able STR loci, which were stably detected in more than 25 samples, were found. The detection sensitivity in maternal cell contamination was no less than 20%, while its accuracy reached 100% in clinical samples. CONCLUSIONS: Finally, we established and validated a novel detection procedure for maternal cell contamination in clinical prenatal samples using next generation sequencing. This procedure allowed us to simultaneously perform prenatal test-ing and MCC testing. Unlike the traditional capillary electrophoresis, this method is rapid, highly sensitive, and suitable for wide range of clinical applications.

Prevalence of maternal cell contamination in amniotic fluid samples.

PURPOSE: The purpose of this study is to evaluate the incidence of maternal cell contamination (MCC) in the first few milliliters of amniotic fluid withdrawn during amniocentesis. METHODS: A prospective observational study was performed. The initial 2-3 ml of amniotic fluid withdrawn during amniocentesis was divided into direct analysis (uncultured) and cultured samples. A matching maternal buccal swab was obtained for MCC testing. MCC was determined by short-tandem repeat analysis. The primary outcome was measurement of clinically significant contamination (MCC >5%). Secondary outcomes included the determination of risk factors associated with MCC >5%. Outcomes were assessed by fisher's exact, independent t-test, binary logistic regression, and ANOVA. RESULTS: Direct analysis measured clinically significant contamination (MCC > 5%) in 26% of specimens, while any amount of MCC was present in 68% of specimens. Cultured specimens had MCC > 5% in 2%, and any amount of MCC in 24%. Only blood-tinged fluid was associated with an increased risk for MCC > 5%. Larger volumes of the discard sample were not associated with increased incidence of MCC greater than 5%. CONCLUSION: A significant amount of MCC is present with direct analysis of the initial few milliliters of amniotic fluid withdrawn and is not influenced by the volume of the discard sample. Our results suggest that the first few milliliters of amniotic fluid be removed and discarded when direct analysis is utilized for prenatal genetic testing.

Prenatal diagnosis of cystic fibrosis: 10-years experience.

PURPOSE: We present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. PATIENTS AND METHODS: Based on family history, 40 Tunisian couples were selected for prenatal diagnosis. Fetal DNA was isolated from amniotic fluid collected by transabdominal amniocentesis or from chronic villi by transcervical chorionic villus sampling. The genetic analysis for cystic fibrosis mutations was performed by denaturant gradient gel electrophoresis and denaturing high-pressure liquid phase chromatography. We performed microsatellites analysis by capillary electrophoresis in order to verify the absence of maternal cell contamination. RESULTS: Thirteen fetuses were affected, 21 were heterozygous carriers and 15 were healthy with two normal alleles of CFTR gene. Ten couples opted for therapeutic abortion. The microsatellites genotyping showed the absence of contamination of the fetal DNA by maternal DNA in 93.75%. CONCLUSION: Our diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis.

Separation of miscarriage tissue from maternal decidua for chromosome analysis.

OBJECTIVE: To demonstrate a technique for separation of miscarriage tissue from maternal decidua to reduce maternal cell contamination for chromosome analysis. DESIGN: Retrospective. SETTING: University-based infertility center. PATIENT(S): Not applicable. INTERVENTION(S): Retrospective collection of de-identified images and video from manual vacuum aspiration (MVA) performed after first-trimester pregnancy losses. This project was exempt from institutional review board approval as no patient-identifying data were used. MAIN OUTCOME MEASURE(S): Reduction of maternal cell contamination and improvement of the accuracy of chromosome analysis. RESULT(S): Video demonstration of separation of miscarriage tissue from maternal decidua after MVA. CONCLUSION(S): Chromosome analysis of a miscarriage is performed to assess the etiology of miscarriage and recurrent pregnancy loss. However, maternal cell contamination can limit the accuracy. This video demonstrates a technique for separating miscarriage tissue from maternal decidua after MVA to reduce maternal cell contamination prior to sending tissue for analysis. The same technique has been used in our clinic with first-trimester dilation and curettage using mechanical suction.

Absence of maternal cell contamination in mesenchymal stromal cell cultures derived from equine umbilical cord tissue.

This study aimed to determine whether maternal cell contamination exists in cells derived from equine umbilical cord tissue, a perspective material for cell-based therapies in veterinary medicine. Potential maternal cell contamination was analyzed at DNA level via a set of 16 microsatellite markers in cells originating from the cord tissue of 22 foals. In these cells no maternal cell contamination was detected at a sensitivity level of 0.01%. Our results suggest that equine umbilical cord tissue-derived cells are entirely of fetal origin.

A 26-Year Experience in Chorionic Villus Sampling Prenatal Genetic Diagnosis.

This report describes the trends of chorionic villus sampling (CVS) referred for prenatal genetic diagnosis in the past two and a half decades in a Portuguese Center. Our cohort of 491 CVS was mostly performed by the transcervical method at the 12th gestational week. Data collected within the framework of this study relate to the following: sampling method, referral reason versus abnormality and incidence of procedure-related pregnancy loss, that declined to about 0.5% over the last 15 years. The year 2000 represented a change in referral reasons for chorionic tissue collection, shifting from almost exclusively for cytogenetic testing to an increasing number of molecular tests for monogenic disorders. Herein, success rates as well as cytogenetic and/or molecular DNA results are presented. These latter include not only tests for several monogenic disorders, but also aneuploidy and maternal cell contamination screening. This retrospective analysis reiterates that CVS is a safe and reliable first trimester technique for prenatal diagnosis in high genetic risk pregnancies.

Reliability of 46,XX results on miscarriage specimens: a review of 1,222 first-trimester miscarriage specimens.

OBJECTIVE: To examine the rate of maternal contamination in miscarriage specimens. DESIGN: Retrospective review of 1,222 miscarriage specimens submitted for chromosome testing with detection of maternal cell contamination (MCC). SETTING: Referral centers requesting genetic testing of miscarriage specimens at a single reference laboratory. PATIENT(S): Women with pregnancy loss who desire complete chromosome analysis of the pregnancy tissue. INTERVENTION(S): Analysis of miscarriage specimens using single-nucleotide polymorphism (SNP) microarray technology with bioinformatics program to detect maternal cell contamination. MAIN OUTCOME MEASURE(S): Chromosome content of miscarriages and incidence of 46,XX results due to MCC. RESULT(S): Of the 1,222 samples analyzed, 592 had numeric chromosomal abnormalities, and 630 were normal 46,XX or 46,XY (456 and 187, respectively). In 269 of the 46,XX specimens, MCC with no embryonic component was found. With the exclusion of maternal 46,XX results, the chromosomal abnormality rate increased from 48% to 62%, and the ratio for XX to XY results dropped from 2.6 to 1.0. CONCLUSION(S): Over half of the normal 46,XX results in miscarriage specimens were due to MCC. The use of SNPs in MCC testing allows for precise identification of chromosomal abnormalities in miscarriage as well as MCC, improving the accuracy of products of conception testing.

Six-years' Experience of Pseudomosaicism and Maternal Cell Contamination in Cultured Amniocytes

PURPOSE : To present our experiences in pseudomosaicism or maternal celi contamination in genetic mid-trimester amniocentesis confirmed through percuraneous umbilical blood sampling. METHODS : From 1992 to 1997, repeated cytogenetic evaluation with fetal cord blood was carried out in 14 cases showing mosaic patterns. RESULTS : We confirmed pseudomosaicsm in 12 cases (85.7%) by repeated cytohenetic evaluation, and also maternal cell contamination in 2 cases. CONCLUSIONS : Repeated cytohenetic evaluation via percutaneous umbilical blood sampling was a rapid and useful method fof the confirmation of mosaicism resulted from genetic mid-trimester amnicentesis.