Fetal Fraction Research Articles

Trophoblast retrieval from the cervical canal to predict abnormal pregnancy early in gestation: a pilot study

BackgroundThe current detection of fetal chromosomal abnormalities by non-invasive prenatal testing (NIPT) mainly relies on the cell free DNA(cfDNA) in the maternal blood. However, a gestational age of less than 12 weeks or a high maternal BMI affects cfDNA fetal fraction and further the detection by NIPT negatively. In this study, we aim to retrieve the trophoblast cells from the maternal cervix to develop a new sampling method for NIPT enabling an earlier use of NIPT.MethodsWe enrolled three patients who wanted to undergo induced abortion at Beijing Hospital between January 2022 and March 2022. Peripheral blood, cervix specimen, and the abortion tissue were collected and processed for each patient. Allele frequencies of the mutated gene loci of the maternal blood and the cervix sample were compared and the Sex Determining Region Y (SRY) gene was tested.ResultsThe allele frequencies of the mutated gene loci showed no significant difference between the maternal blood and the cervix sample. But we successfully detected signal of the SRY gene in the cervix sample of the only patient carrying a male fetus.ConclusionsThe detection of the SRY gene in a cervix sample indicated a successful retrieval of trophoblast cells from the cervix canal. Further study needs to be conducted to verify our finding before its application to the clinical settings.

PBPK-based dose finding for sildenafil in pregnant women for antenatal treatment of congenital diaphragmatic hernia.

Sildenafil is a potent vasodilator and phosphodiesterase type five inhibitor, commercially known as Revatio® and approved for the treatment of pulmonary arterial hypertension. Maternal administration of sildenafil during pregnancy is being evaluated for antenatal treatment of several conditions, including the prevention of pulmonary hypertension in fetuses with congenital diaphragmatic hernia. However, determination of a safe and effective maternal dose to achieve adequate fetal exposure to sildenafil remains challenging, as pregnancy almost always is an exclusion criterion in clinical studies. Physiologically-based pharmacokinetic (PBPK) modelling offers an attractive approach for dose finding in this specific population. The aim of this study is to exploit physiologically-based pharmacokinetic modelling to predict the required maternal dose to achieve therapeutic fetal exposure for the treatment congenital diaphragmatic hernia. A full-PBPK model was developed for sildenafil and N-desmethyl-sildenafil using the Simcyp simulator V21 platform, and verified in adult reference individuals, as well as in pregnant women, taking into account maternal and fetal physiology, along with factors known to determine hepatic disposition of sildenafil. Clinical pharmacokinetic data in mother and fetus were previously obtained in the RIDSTRESS study and were used for model verification purposes. Subsequent simulations were performed relying either on measured values for fetal fraction unbound (fu = 0.108) or on values predicted by the simulator (fu = 0.044). Adequate doses were predicted according to the efficacy target of 15ng/mL (or 38ng/mL) and safety target of 166ng/mL (or 409ng/mL), assuming measured (or predicted) fu values, respectively. Considering simulated median profiles for average steady state sildenafil concentrations, dosing regimens of 130mg/day or 150mg/day (administered as t.i.d.), were within the therapeutic window, assuming either measured or predicted fu values, respectively. For safety reasons, dosing should be initiated at 130mg/day, under therapeutic drug monitoring. Additional experimental measurements should be performed to confirm accurate fetal (and maternal) values for fu. Additional characterization of pharmacodynamics in this specific population is required and may lead to further optimization of the dosing regimen.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

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Combined fetal fraction to analyze the Z-score accuracy of noninvasive prenatal testing for fetal trisomies 13, 18, and 21

ObjectiveThis study aims to evaluate the correlation combined fetal fraction and Z-score for fetal trisomies 13, 18, and 21 of NIPT by the semiconductor sequencing platform and further analyze the differences of different sequencing depths.MethodsA cohort of 61,581 pregnancies were recruited for NIPT. Invasive prenatal diagnostic confirmation is recommended in all high-risk NIPT cases. Logistic regression and rank correlation analysis were applied to analyze the relationship between different parameters. ROC curve analysis was adopted to analyze the cutoff values of Z-score and fetal fraction.ResultsA total of 278 common trisomy pregnancies were verified in 377 NIPT-positive results. The fitted logistic regression models revealed that Z-scores of NIPT-positive results were significantly associated with PPVs (p < 0.05). The ROC curve analysis showed that the optimal cutoff value of Z-scores for T21, T18, and T13 was 7.597, 4.944, and 9.135 for NIPT and 9.489, 8.004, and 12.4 for NIPT-plus. If combing fetal fraction as another evaluation factor, the PPV of trisomy 21 gradually improved. We analyzed the correlation between the fetal fraction and the PPV, which revealed that the fetal fraction was significantly correlated with PPV. By analyzing the PPV of different groups divided by the associated criteria obtained from ROC curve, the PPV of high Z-score and high fetal fraction is higher in groups of Z-score > the optimal cutoff value.ConclusionThe results of this study show that the fetal fraction is significantly correlated with the PPV. Combining fetal fraction with Z-score is significantly better than in groups of Z-score-associated criteria; clinicians can give more accurate and efficient prenatal genetic counseling.

Screen-positive rate in cell-free DNA screening for microdeletion 22q11.2.

To examine the impact of the fetal fraction (FF) on the screen-positive rate in screening for microdeletion 22q11.2. This study is based on samples that were analyzed using the Harmony® Prenatal Test (Roche Inc). The study cohort comprised samples from women with singleton pregnancies who were at least 16years old and at least at 11weeks' gestation. Logistic regression analysis was used to determine significant covariates that carry an impact on the screen-positive rate. The study population consisted of 52,019 pregnancies, including 309 pregnancies with a high-risk result for microdeletion 22q11.2. Thus, the overall screen-positive rate was 0.59%. In the low-risk group, the FF was 10.1%, and in the high-risk group, it was 7.3%. Regression analysis indicated a strong correlation between the FF and the screen-positive rate. In the cases with an FF of <11.0%, the screen-positive rate was 0.92%, while it was 0.13% in the group with a higher FF. The screen-positive rate depends on the FF. In order to keep the rate low, we recommend restricting the analysis to samples with a FF of 11% and more.

Niet-invasieve prenatale test bij meerlingzwangerschappen: een retrospectieve analyse van de distributie van de foetale fractie

Noninvasive prenatal testing in multiple pregnancies: retrospective analysis of the fetal fraction distribution This article describes the distribution of the fetal fraction (FF) in noninvasive prenatal testing (NIPT) in multiple pregnancies. A retrospective study of 514 NIPT experiments was performed in 484 women with an (originally) multiple pregnancy from January 1st, 2017 to December 31st, 2019 at the Center for medical genetics in Ghent (Belgium). The FF distribution in vanishing twin, twin and triplet pregnancies was significantly different with a mean of 14%, 18.1% and 23.7% respectively. Test failure due to a low FF was higher in dichorionic-diamniotic (DCDA) twins (9.5%) compared to monochorionic-diamniotic (MCDA) twins (0%). The mean ratio FF based on the Y-chromosome (FFY)/total fetal fraction in male-male pregnancies was 0.96, in male-female pregnancies 0.43 and in female-female pregnancies 0.0. In vanishing twin pregnancies, the duration of persistence of cell-free fetal DNA (cffDNA) from the demised cotwin was up to 7 weeks. Test failure due to a low FF seems to appear more frequently in DCDA twins compared to MCDA twins, which could be explained by the higher FF threshold in DCDA twins (8%) in comparison to MCDA twins (4%). In mixed pregnancies, on average each fetus seems to contribute equally to the FF, although this does not seem to apply to all mixed pregnancies, giving rise to the possibility of an unequal contribution and a higher risk of a false negative result.

White blood cell count affects fetal fraction and test failure rates in noninvasive prenatal screening.

To investigate the effects of white blood cell (WBC) count on fetal fraction (FF), which is an essential quality control for obtaining reliable results, and on the rate of screen failures in noninvasive prenatal screening (NIPS). Noninvasive prenatal screening, serum lipid and liver enzyme level measurements, and WBC count were performed for 4,281 pregnancies with male fetuses. After adjusting for confounders, including the maternal characteristics and alanine aminotransferase (ALT) levels, the effect of WBC count on FF and test failure rate was measured by linear and logistic regression analyses. Fetal fraction was negatively associated with BMI, ALT, IVF conceptions, and WBC count and positively correlated with gestational age in the multivariate linear regression model. Moreover, WBC count was the most important factor affecting FF after BMI according to the standardization coefficient analysis. In the 4,281 pregnancy samples with male fetuses, FF decreased with WBC count from 11.45% at ≤8 to 9.02% at >12, and FF markedly decreased to 7.40% in pregnancies with a higher WBC count (>12) and higher BMI (≥25 kg/m2). Meanwhile, the test failure rates were significantly higher in the WBC count > 12 group (4.29%) than in the WBC count ≤ 8 group (0.89%). Notably, when the BMI of pregnancies with a WBC count of >12 was >25, the rate reached 7.53%. Subsequently, multivariate logistic regression analysis further confirmed that an increased BMI and WBC count were independently and significantly associated with the test failure rates. An increased WBC count was associated with lower FF and higher test failure rates, suggesting that these important factors should be carefully considered during genetic counseling in pregnant women who decide to undergo blood collection or resampling.

Evaluation of The Relationship between Cell-Free DNA Fetal Fraction of The Circulatory System and Fetal and Maternal Pregnancy Prognosis: A Prospective Study.

Non-invasive prenatal testing (NIPT), sometimes called noninvasive prenatal screening (NIPS), is a non-invasive prenatal genetic test using cell-free DNA in maternal blood. This method is used to diagnose fetal aneuploidy disorders such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13), which causes disability disorders or significant postpartum defects. The aim of this study was to investigate the relationship between high and low fetal fraction (FF) and prognosis of maternal pregnancy. In this prospective study, after obtaining informed consent, 10 ml of blood was collected from 450 mothers with singleton pregnancies with gestational age above 11 weeks (11-16) at the request of NIPT for cell-free DNA BCT test. After obtaining the test results, maternal and embryonic results were evaluated based on the amount of non-cellular DNA FF. Data analysis was performed by using SPSS software version 21 and independent t test, chi-square statistical tests. Based on test results, 20.5% of women were nulli par. The mean FF index in the studied women was 8.3% with a standard deviation of 4.6. The minimum and maximum values were 0 and 27, respectively. The frequency of normal, low and high FFs was 73.2, 17.3 and 9.5%, respectively. High FF has fewer risks to the mother and fetus than low FF. The use of FF level (high or low) can help us determining the prognosis of pregnancy and using it to better manage the pregnancy.

Predicting fetal growth retardation: mathematical modeling

Introduction. Annually, more than 13 million neonates are born with fetal growth retardation (FGR) worldwide. FGR increases prenatal mortality and morbidity. Due to no effective treatments for FGR are available, its prevention and prognosis are of extreme relevance.Aim: development of prognostic clinical and anamnestic mathematical model for assessing a risk of developing FGR during pregnancy.Materials and Methods. A prospective, controlled, open, continuous study was performed. The main group (1) included 75 patients who had FGR during pregnancy; the control group (2) consisted of 414 women with favorable pregnancy outcome. All subjects underwent examination, including collecting medical history, a complex of prenatal diagnostics in the first trimester of pregnancy – ultrasound, Doppler uterine arteries, serum level of pregnancy-associated plasma protein-A (PAPP-A), free beta-subunit of human chorionic gonadotropin (β-hCG), placental growth factor (PlGF), and non-invasive prenatal test (NIPT).Results. To determine the relative contribution of each individual trait to the formation of FGR risk and develop a prognostic index, a discriminant analysis was carried out, on the basis of which a prognostic F-index was developed. The formula for calculating the F-index includes the age of pregnant woman, obstetric history data, method of conception, recorded nicotine addiction in pregnancy, detected uterine fibroids, body mass index, biochemical parameters (PAPP-A, β-hCG, PlGF), nuchal translucency of the fetus, the pulsation index of the uterine artery, the level of the fetal fraction and fetal gender (determined during NIPT). The parameters of sensitivity and specificity of the FGR prognosis were 90.1 and 82.18 % respectively, the method effectiveness was 83.97 %.Conclusion. The method developed for predicting FGR can be used in clinical practice to form risk groups for FGR development and choose tactics for pregnancy management.

Non-invasive prenatal testing for everybody or contingent screening?

To compare the advantages and disadvantages of two main implementation strategies for non-invasive prenatal testing (NIPT) for the detection of trisomies 21, 13 and 18 in public healthcare settings. These concern NIPT as a first-tier screening test for all pregnant women, or NIPT contingent on an increased risk first-trimester combined test (FCT) result. Comparison based on (un)published data and literature. Despite the lower prevalence of trisomies in the low-risk population, data show that the positive predictive value (PPV) and detection-miscarriage ratio of first-tier NIPT are comparable to NIPT used as contingency test. Advantages of NIPT as a first-tier test compared to FCT with contingent NIPT are that there is no time-window for testing, sensitivity is higher, fewer women need follow-up testing (PPV of NIPT is higher than PPV of FCT), less anxiety for pregnant women and partners. When given the choice, pregnant women prefer first-tier NIPT. Although contingency testing still seems to be the cheaper option, the differences in costs are becoming increasingly smaller and might soon disappear. We argue that NIPT should preferably be offered as a first-tier test for the detection of fetal aneuploidies for all pregnant women, provided that women are supported to make informed decisions. This article is protected by copyright. All rights reserved.

Low fetal fraction on cell free DNA screening is associated with preterm birth and preeclampsia

Low fetal fraction on cell free DNA screening is associated with preterm birth and preeclampsia

Non-invasive prenatal diagnosis (NIPD): current and emerging technologies

Prenatal testing is important for the early detection and diagnosis of rare genetic conditions with life-changing implications for the patient and their family. Gaining access to the fetal genotype can be achieved using gold-standard invasive sampling methods, such as amniocentesis and chorionic villus sampling, but these carry a small risk of miscarriage. Non-invasive prenatal diagnosis (NIPD) for select rare monogenic conditions has been in clinical service in England since 2012 and has revolutionised the field of prenatal diagnostics by reducing the number of women undergoing invasive sampling procedures. Fetal-derived genomic material is present in a highly fragmented form amongst the maternal cell-free DNA (cfDNA) in circulation, with sequence coverage across the entire fetal genome. Cell-free fetal DNA (cffDNA) is the foundation for NIPD, and several technologies have been clinically implemented for the detection of paternally inherited and de novo pathogenic variants. Conversely, a low abundance of cffDNA within a high background of maternal cfDNA makes assigning maternally inherited variants to the fetal fraction a significantly more challenging task. Research is ongoing to expand available tests for maternal inheritance to include a broader range of monogenic conditions, as well as to uncover novel diagnostic avenues. This review covers the scope of technologies currently clinically available for NIPD of monogenic conditions and those still in the research pipeline towards implementation in the future.

High fetal fraction on cell-free fetal DNA screening: is it associated with adverse perinatal outcomes?

High fetal fraction on cell-free fetal DNA screening: is it associated with adverse perinatal outcomes?

Variability in Fetal Fraction Estimation: Comparing Fetal Fractions Reported by Noninvasive Prenatal Testing Providers Globally.

Fetal fraction (FF) measurement is considered important for reliable noninvasive prenatal testing (NIPT). Using minimal FF threshold as a quality parameter is under debate. We evaluated the variability in reported FFs of individual samples between providers and laboratories and within a single laboratory. Genomic quality assessment and European Molecular Genetics Quality Network provide joint proficiency testing for NIPT. We compared reported FFs across all laboratories and stratified according to test methodologies. A single sample was sequenced repeatedly and FF estimated by 2 bioinformatics methods: Veriseq2 and SeqFF. Finally, we compared FFs by Veriseq and SeqFF in 87 351 NIPT samples. For each proficiency test sample we observed a large variability in reported FF, SDs and CVs ranging from 1.7% to 3.6% and 17.0% to 35.8%, respectively. FF measurements reported by single nucleotide polymorphism-based methods had smaller SDs (0.5% to 2.4%) compared to whole genome sequencing-based methods (1.8% to 2.9%). In the internal quality assessment, SDs were similar between SeqFF (SD = 1.0%) and Veriseq v2 (SD = 0.9%), but mean FF by Veriseq v2 was higher compared to SeqFF (9.0% vs 6.4%, P < 0.001). In patient samples, reported FFs were on average 1.12%-points higher in Veriseq than in SeqFF (P < 0.001). Current methods do not allow for a reliable and consistent FF estimation. Our data show estimated FF should be regarded as a laboratory-specific range, rather than a precise number. Applying strict universal minimum thresholds might result in unnecessary test failures and should be used with caution.

The effects of heparin, aspirin, and maternal clinical factors on the rate of nonreportable cell-free DNA results: a retrospective cohort study

Technological advances in the analysis of cell-free DNA in maternal serum have allowed expanded prenatal screening possibilities for fetal aneuploidies. The sensitivity and positive predictive value of the assay are partly dependent on the amount of cell-free DNA present in maternal circulation. Thus, it is important to know what fetal and maternal factors influence the level of cell-free DNA in maternal circulation. Maternal heparin use has been associated with an increase in nonreportable cell-free DNA results because of a low fetal fraction in some, but not all, previous studies. In addition, there are likely additional factors that affect cell-free DNA that remain uncharacterized. This study aimed to determine whether heparins, low-dose aspirin, and maternal clinical factors affect the rate of nonreportable cell-free DNA testing results. A retrospective cohort study was conducted using pregnant people receiving cell-free fetal DNA testing from January 1, 2014, to June 30, 2018. Data were collected on patient demographics, medical comorbidities, medication use, and cell-free DNA test results. Univariate and multivariate analyses were performed to determine which factors were independently associated with the rate of nonreportable results. From an original sample of 1117 pregnant people, 743 met the inclusion criteria. Maternal weight (odds ratio, 1.02), heparin use (odds ratio, 12.06), aspirin use (odds ratio, 4.70), chronic hypertension (odds ratio, 5.26), pregestational diabetes mellitus (odds ratio, 2.46), and autoimmune disease (odds ratio, 3.59) were significantly associated with an increased rate of nonreportable results in the univariate analysis. Moreover, the association was present for maternal weight (odds ratio, 1.02), heparin use (odds ratio, 21.87),and aspirin use (odds ratio, 2.85) in the multivariate analysis. The previously seen association between maternal heparin use and an increase in nonreportable cell-free DNA results was confirmed. Furthermore, there seems to be an increase in nonreportable results in pregnant people taking low-dose aspirin. Providers should consider the effect of these medications when counseling patients on prenatal genetic screening options.

Inconsistency between non-invasive prenatal testing (NIPT) and conventional prenatal diagnosis due to confined placental and fetal mosaicism: Two case reports.

We aimed to identify the causes of inconsistent results between non-invasive prenatal testing (NIPT) and invasive testing methods for trisomy 21. In the first case, NIPT was performed at 11 weeks of pregnancy, and the result showed a high risk of trisomy 21 [fetal fraction (FF) = 6.98%, 21 chromosome Z-score = 3.6]. The patient underwent quantitative fluorescent (QF)-PCR and karyotyping at 14 + 0 weeks of pregnancy through CVS showing mosaicism of 47, XX, + 21[11] and 46, XX [39] in karyotyping. The patient underwent amniocentesis at 15 + 6 weeks, showing a normal pattern in QF-PCR and 46, XX karyotyping in long term culture. The second case underwent NIPT at 16 + 5 weeks of pregnancy (FF = 7.52%, 21 chromosome Z-score = 2.503). She underwent an invasive test at 19 weeks through amniotic fluid sampling. As a result, trisomy 21 was detected by QF-PCR, and mosaicism of XX, +21[22]/46, XX [4] was identified by karyotyping. Despite significant advances in fetal chromosome analysis using NIPT, invasive testing is still needed as placenta-derived DNA does not reflect 100% fetal genetic information. Placental mosaicism can be detected by NIPT, but more research is needed to increase its sensitivity. Therefore, if the NIPT result is positive, an invasive test can confirm the result, and continuous monitoring is required even if the NIPT result is negative.

Comparison of The Foetal Fraction (ff) of Cell-Free DNA in In Vitro Fertilisation Versus Natural Conception. Evaluation of ff with IVF Parameters

Comparison of The Foetal Fraction (ff) of Cell-Free DNA in In Vitro Fertilisation Versus Natural Conception. Evaluation of ff with IVF Parameters

Factors affecting low fetal fraction in fetal screening with cell-free DNA in pregnant women: a systematic review and meta-analysis

Cell-Free DNA (cfDNA) is a non-invasive perinatal test (NIPT) used to assess fetal anomalies. The ability to detect fetal chromosomal aneuploidies is directly related to a sample's fetal to total DNA fraction, known as the fetal fraction (FF). The minimum FF is considered 4%, and the test result below 4% is uncertain due to low fetal fraction (LFF). This study aimed to conduct a systematic review and a meta-analysis to determine the possible factors affecting LFF in cfDNA testing for fetal screening. PubMed, Web of Science, Google Scholar, Since Direct, Scopus, CINHAL, Cochrane Library, and Persian databases, including Scientific Information Database, Irandoc, and Magiran were searched for studies investigating factors affecting LFF in cfDNA testing from 2000 until the end of 2021. Gathered data were analyzed using Comprehensive Meta-Analysis (CMA) software version 3.3.070. The quality of the included studies was assessed using the Joanna Briggs Institute Critical Appraisal of Cohort Studies tool. Thirteen articles related to the topic were included, and seven related articles were reviewed for meta-analysis. The other six were reviewed qualitatively. Four factors were identified that might have a potential effect on the LFF, of which only gestational age had a significant association with LFF (Pooled mean difference= -1.111, SE = 0.515, 95% CI= -2.121, -0.101, (P-value < 0.05)). Maternal age (P-value = 0.573), maternal weight (P-value = 0.113), and Body Mass Index (P-value = 0.104) had no statically significant effect. The effect size was pooled by mean difference and 95% confidence interval. Lower gestational age is significantly associated with LFF. Thus, this factor can be considered when interpreting prenatal cfDNA screening tests.

High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed—a 12-plex and a 57-plex—and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

Maternal Copy Number Imbalances in Non-Invasive Prenatal Testing: Do They Matter?

Non-invasive prenatal testing (NIPT) has become a routine practice in screening for common aneuploidies of chromosomes 21, 18, and 13 and gonosomes X and Y in fetuses worldwide since 2015 and has even expanded to include smaller subchromosomal events. In fact, the fetal fraction represents only a small proportion of cell-free DNA on a predominant background of maternal DNA. Unlike fetal findings that have to be confirmed using invasive testing, it has been well documented that NIPT provides information on maternal mosaicism, occult malignancies, and hidden health conditions due to copy number variations (CNVs) with diagnostic resolution. Although large duplications or deletions associated with certain medical conditions or syndromes are usually well recognized and easy to interpret, very little is known about small, relatively common copy number variations on the order of a few hundred kilobases and their potential impact on human health. We analyzed data from 6422 NIPT patient samples with a CNV detection resolution of 200 kb for the maternal genome and identified 942 distinct CNVs; 328 occurred repeatedly. We defined them as multiple occurring variants (MOVs). We scrutinized the most common ones, compared them with frequencies in the gnomAD SVs v2.1, dbVar, and DGV population databases, and analyzed them with an emphasis on genomic content and potential association with specific phenotypes.