Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis

ABSTRACT: BACKGROUND: Cell-free fetal DNA (cffDNA) can be detected in maternal blood during pregnancy, opening the possibility of early non-invasive prenatal diagnosis for a variety of genetic conditions. Since 1997, many studies have examined the accuracy of prenatal fetal sex determination using cffDNA, particularly for pregnancies at risk of an X-linked condition. Here we report a review and meta-analysis of the published literature to evaluate the use of cffDNA for prenatal determination (diagnosis) of fetal sex. We applied a sensitive search of multiple bibliographic databases including PubMed (MEDLINE), EMBASE, the Cochrane library and Web of Science. RESULTS: Ninety studies, incorporating 9,965 pregnancies and 10,587 fetal sex results met our inclusion criteria. Overall mean sensitivity was 96.6% (95% credible interval 95.2% to 97.7%) and mean specificity was 98.9% (95% CI = 98.1% to 99.4%). These results vary very little with trimester or week of testing, indicating that the performance of the test is reliably high. CONCLUSIONS: Based on this review and meta-analysis we conclude that fetal sex can be determined with a high level of accuracy by analyzing cffDNA. Using cffDNA in prenatal diagnosis to replace or complement existing invasive methods can remove or reduce the risk of miscarriage. Future work should concentrate on the economic and ethical considerations of implementing an early non-invasive test for fetal sex.     

Technical note: A blind test of mandibular ramus flexure as a morphologic indicator of sexual dimorphism in the human skeleton

Loth and Henneberg (1996, Am. J. Phys. Anthropol. 99:473–487) identified a single morphological feature of the mandible, the presence or absence of a distinct flexure or angulation of the posterior margin of the mandibular ramus at the level of the occlusal plane, which appears to be an extraordinarily accurate predictor of sex. Using only this feature, Loth and Henneberg were able to predict sex with 94% accuracy in a large sample of mandibles. In this article, we report the results of a blind test of mandibular ramus flexure as a predictor of sex. In our blind test, only 62.5% of the mandibles were correctly sexed, and virtually identical results were obtained when the same sample of mandibles was examined by a second observer. Overall, our results demonstrate that: 1) the association between ramus flexure and sex is weak; 2) the predictive accuracy of Loth and Henneberg's method is better than chance for only one sex, males; and 3) the method is based on a trait that cannot be reliably or consistently identified. Am J Phys Anthropol 107:363–366, 1998 © 1998 Wiley-Liss, Inc.     

Test of Phenice's technique for determining sex from the os pubis

Pubic bone morphology was examined to test the accuracy of Phenice's visual method for determining sex from the os pubis. Twelve participants scored 50 pubic bones from individuals of known sex aged 52-92 years. The sample is of modern males and females, all presumed whites. An accuracy of approximately 83% in determining sex was recorded, compared to 95% reported by Phenice. This accuracy difference may reflect different age distributions of the two samples. Through replication of test results on two series of 25 specimens, the technique was found to be reliable. Previous experiences in human osteological analysis was shown to have no effect on accuracy in this test, confirming Phenice's assertion that the technique does not require extensive experience to yield accurate results. Results suggest that there is a moderate negative correlation between accuracy in determining the sex of an individual and that individual's age.     

Brief communication: a study of the predictive accuracy of mandibular ramus flexure as a singular morphologic indicator of sex in an archaeological sample

Loth and Henneberg ([1996] Am J Phys Anthropol 99:473-485) assert that they have discovered a single morphologic indicator of sexual dimorphism in the human mandible that rivals the predictive accuracy of the complete pelvis at 94.2% for all samples (99% for healthy samples). To test the accuracy of their method, mandibles (n = 150) from the Tepe Hissar collection were assessed for the presence or absence of mandibular ramus flexure. These results were then compared to a separate sex assessment based on morphologic indicators from the corresponding skull and innominates (where possible) to yield an overall accuracy of only 78.2%. As a means of independent assessment, the mandibular results were also compared with Krogman's ([1940] Racial Types from Tepe Hessar, Iran, from late fifth to early second millennium, BC. Amsterdam: Koninkliijke Nederlandsche Akademie van Wetenschappen) assessment of sex based on craniofacial measurements and morphologic indicators from the skull. This comparison produced an even lower accuracy of 67.2%. Such results question the predictive potential of mandibular ramus flexure as a single indicator of sexual dimorphism and suggest caution when applying this method, especially in the case of fragmentary forensic and fossil remains.     

Sex diagnosis of subadult specimens from Medieval Polish archaeological sites: metric analysis of deciduous dentition.

The subject of this work is the characterisation of the metric features of deciduous dentition in a Medieval population of central Poland with the use of the jackknife technique leave one out (LOO)-supporting multivariate methods, which are important for deriving discrimination equations that would result in sex determination of children's skeletal remains. The sex of the individuals was assessed through analysis of sex-specific DNA sequences (AMELY/AMELX, SRY and alpha satellite sequences). Discriminant analysis concerned only teeth of those individuals whose sex was confirmed by the primary structure of three DNA sequences. The deciduous tooth diameters of males were found to be significantly larger than those of females in four respects: MD diameter of the maxillary second molar, MD and BL diameters of the mandibular first molar and BL diameter of the mandibular second molar. A two-group discriminant analysis considered all those measurements as independent variables. A multiple regression procedure produced a linear equation predicting the sex of children's skeletons with a significant probability amounting to approximately 78%. The accuracy of the sex assessment of an individual, using dental measurements, was established at 69% in deciduous male and 88% in deciduous female teeth.     

Effect of cell cycle synchronization on the accuracy of murine and bovine embryo sex determination

Different cell cycle synchronization methods were used to increase the mitotic index and accuracy of sex determination in murine and bovine embryos. For sexing purposes, colchicine treatment for 2, 4, 6 and 8 h and the FdU-thymidine-colchicine combination were tested in murine embryos. The best results were obtained with colchicine treatment for 8 h (96.88% accuracy) and with FdU-thymidine-colchicine (97.22% accuracy). Mitotic indexes differed significantly between the 2 treatments (21.71% for colchicine and 32.95% for FdU-thymidine-colchicine). For sex identification of murine and bovine demi-embryos, both treatments were demonstrated to be equally effective (nearly 90%). The mitotic index for the FdU-treated murine demi-embryos (19.04%) was higher than the one obtained for the 8-h colchicine treatment (15.62%).     

Cattle fetal sex determination by polymerase chain reaction using DNA isolated from maternal plasma

The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35-48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle.     

Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.     

Computer-aided rodent carcinogenicity prediction

The potential of the computer program PASS (Prediction Activity Spectra for Substances) to predict rodent carcinogenicity for chemical compounds was studied. PASS predicts carcinogenicity of chemical compounds on the basis of their structural formula and of structure-activity relationship analysis of known carcinogens and non-carcinogens. The data on structures and experimental results of 2-year carcinogenicity assays for 412 chemicals from the NTP (National Toxicological Program) and 1190 chemicals from the CPDB (Carcinogenic Potency Database) were used in our study. The predictions take into consideration information about species and sex of animals. For evaluation of the predictive accuracy we used two procedures: leave-one-out cross-validation (LOO CV) and leave-20%-out cross-validation. In the last case we randomly divided the studied data set 20 times into two subsets. The data from the first subset, containing 80% of the compounds, were added to the PASS training set (which includes about 46,000 compounds with about 1500 biological activity types collected during the last 20 years to predict biological activity spectra), the second subset with 20% of the compounds was used as an evaluation set. The mean accuracy of prediction calculated by LOO CV is about 73% for NTP compounds in the 'equivocal' category of carcinogenic activity and 80% for NTP compounds in the 'evidence' category of carcinogenicity. The mean accuracy of prediction for the CPDB database is 89.9% calculated by LOO CV and 63.4% calculated by leave-20%-out cross-validation. Influence of incorporation of species and sex data on the accuracy of carcinogenicity prediction was also investigated. It was shown that the accuracy was increased only for data on male animals.     

Technical note: evaluating mandibular ramus flexure as a morphological indicator of sex

Described as a highly reliable method of sex identification, mandibular ramus flexure is a morphological trait expressed on the posterior border of the ramus at the occlusal plane (Loth and Henneberg [1996] Am. J. Phys. Anthropol. 99:473-485). In a blind test, 158 mandibles were examined for the presence of flexure as defined by Loth and Henneberg, resulting in 79.1% accuracy, which is well below the reported 91-99% accuracy. Twenty-five of these mandibles were assigned the ambiguous score of 0, an outcome of a +1 score for one side, and a -1 score for the other. Seventeen mandibles were examined twice to measure intraobserver error. Only 64.7% of the scores were duplicated in the second session, suggesting difficulty in consistent identification of flexure. Low overall accuracy, an invalid scoring system, and high intraobserver error indicate that mandibular ramus flexure is an unreliable technique for estimation of sex.     

上肢长骨的性别判别分析研究

本文对100例(男女各50)国人上肢骨进行了36项测量。统计结果显示所有测量项目均值男性都大于女性并具有显著性的差异。本研究表明可以采用单一指标对破损严重的肢骨进行性别鉴定。36个测量项目中有23项单一指标性别判别率达75％以上,其中9项在80％以上。本文采用Fisher判别分析法建立了56项单一肢骨性别判别函数,判别率为80％—87％。为进一步提高判别效果,采用逐步判别分析法建立了四项逐步性别判别函数,判别率达90％以上。     

Sex determination of adolescent skeletons using the distal humerus

Accurate determination of the sex of immature skeletal remains is difficult in the absence of DNA, due to the fact that most sexually dimorphic features of the human skeleton develop as secondary sex characteristics during adolescence. Methods of assessment of adult skeletons cannot reliably be applied to adolescent skeletons because of the transitional nature of the skeleton at puberty and the variability of the adolescent growth spurt. The purpose of this work was to evaluate the accuracy of Rogers's method of morphological sex determination using the distal humerus (Rogers: J Forensic Sci 44 ( 1999 ) 55–59) to assess the sex of adolescent skeletons. The sample consists of 7 documented adolescent skeletons from the Christ Church Spitalfields collection at the British Museum of Natural History and 35 from the Luis Lopes skeletal collection housed in the National History Museum (Museu Bocage) of the University of Lisbon, Portugal. Ages range from 11 to 20 years. The technique achieved an accuracy of 81% on the combined sample of 42. This method can be applied to adolescent skeletons once the trochlea begins fusing to the humeral diaphysis, which occurred by age 11 years in the test samples. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Fetal gender determination by first-trimester ultrasound in dairy cows under routine herd management in Northwest Spain

Ultrasonography (US) provides detailed visualization of the fetus in early pregnancy in cows, thus allowing for fetal sex determination. The objective of this prospective observational study was to determine the feasibility and accuracy of a single US examination to diagnose fetal sex in dairy cattle under routine reproductive management conditions. For this purpose, 953 Holstein cows at 7-16 weeks of gestation were examined. Gender assignment was performed in 822 cows, while the genitalia could not be clearly visualized in 131 (13.7%) of the fetuses. After calving, it was verified that 99.3% of the diagnoses were accurate. Fetal sex was correctly determined by US in 99.5% of male fetuses and 98.8% of female fetuses. Fetal sex determination was less accurate when conducted before d 55 of gestation. Likewise, it was verified that fetal sex, cow age and ultrasonographic diagnosis section did not have a significant influence (P>0.05) on diagnostic accuracy. With respect to the plane used for diagnosis, the sagittal view was poorly used for early pregnancy diagnosis, whereas the longitudinal and cross-sectional planes were used most frequently. These results demonstrate that US can be routinely applied under farm conditions to accurately determine the fetal sex in cattle between days 51 and 111 of gestation without apparent influence of cow age, US scanning plane or fetal sex. Conversely, days of gestation affected the accuracy and feasibility of US gender determination, showing poorer results when the diagnosis was made before day 55 of gestation.     

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.     

Mandibular ramus flexure and gonial eversion as morphologic indicators of sex

Recently, two mandibular traits--ramus flexure and gonial eversion--have come under close scrutiny (Loth & Henneberg 1996, 2000). The present study investigates the reliability of these two traits when each is applied as a single and independent indicator of sex, including the question of repeatability. The investigation was designed to give insights into possible confounding factors such as age and remodeling after tooth loss. Two samples, one of forensic (N = 153) and one of archaeological provenance (N = 80), were examined. The forensic sample was evaluated by a single observer while the archaeological sample was independently scored by three different observers. The results document that age and localized tooth loss seriously reduce the accuracy of these traits. For ramus flexure, male accuracy was only 66%, while female accuracy was even lower (32%). Overall accuracy was 59%. It is believed that the original scoring system devised by Loth and Henneberg (1996) creates an inherent bias in favor of males. For gonial eversion, a similar picture emerged (75.4% for males, 45.2% for females and 69.3% overall accuracy). Furthermore, both indicators are prone to intra- as well as inter-observer bias. While both possess some merit as sex indicators, they show marked functional and adaptive responses and may not be suitable for all samples.     

太行山猕猴掌骨性别判别分析

为了了解成年太行山猕猴5根掌骨的性差大小。本文对太行山猕猴掌骨标本40例(雄:10, 雌:30)进行观察并选择掌骨的9项形态变量进行测量。数据用SPSS19.0软件进行多变量判别分析。结果表明: 5根掌骨长度等变量在性别之间有明显差异。用全模型法5根掌骨的性别正确判别率范围为94.1%~100.0%, 用逐步判别法掌骨性别正确判别率范围为93.8%~97.5%。有关掌骨的9个形态变量, 长度变量首先被挑选出来, 说明在性别判别中起重要作用。分别用左右侧掌骨的判别函数来判别性别时侧别差异不明显。结论: 用每根掌骨的形态变量建立判别函数可以有效地区分性别, 对非人灵长类掌骨标本的性别鉴定有一定的理论意义和应用价值。     

Sex determination of fragmentary crania by analysis of the cranial base

The cranial base can be used to determine the sex of fragmentary or deformed skulls. An initial study used nine measurements taken from 100 crania in the Terry Collection. The sample was divided equally by race and sex. Six regression models were formulated that predicted correctly the sex of the sample with 71-90% accuracy. In a separate test, a control sample of 20 skulls, also drawn from the Terry Collection but not involved with formulating the regression equations, was correctly classified with 70-85% accuracy.     

基于形态特征判定五种鹬类性别的可靠性

快速准确地鉴定两性同型鸟类个体性别在鸟类生态学研究中具有重要意义。本文选择2008年春季迁徙期在崇明东滩停歇的大滨鹬(Calidris tenuirostris)、红腹滨鹬(C.canutus)、红颈滨鹬(C.ruficollis)、尖尾滨鹬(C.acuminata)及翘嘴鹬(Xenus cinereus)5种两性同型的鹬类,用分子生物学方法进行性别鉴定,并基于个体的形态特征(体重、翅长、喙长、头喙长及跗跖长)采用判别分析方法对性别进行判定。结果表明,尖尾滨鹬雄性各形态特征均显著大于雌性,其他4种鹬类则相反。5种鹬类形态特征的性别差异指数在0.5%~25.3%之间,重叠度在29.4%~98.6%之间。5种鹬类判别分析判定性别的准确率在(0.69±0.06)~(0.96±0.01)之间,其中,尖尾滨鹬判别准确率(0.96)最高,翘嘴鹬判别准确率(0.69)最低。形态特征在两性间的差异程度影响性别的判别准确率。另外,两性性比对性别判别的准确率也有影响:性比偏雄性鸟类的雄性判别准确率高于雌性,而性比偏雌性鸟类的雌性判别准确率高于雄性。采用判别分析估测的性比与分子生物学鉴定结果相似,表明判别分析在判定种群的性比方面具有较高的可靠性。     

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.     

Determination of sex from the tibia

Determination of sex from the tibial shaft as well as the entire bone itself has not been generally investigated by osteologists . This paper is an attempt to fulfill this need. The purpose is to determine sex from both the complete tibia and the shaft at the nutrient foramen level. The sample was obtained from the Terry Collection and included 159 specimens of whites and blacks. The tibial length, and circumference, anteroposterior, and transverse measurements at the nutrient foramen level were analyzed by means of step-wise discriminant function analysis. It was found that the circumference alone could give, on the average, 80% accuracy in determining sex. The addition of other dimensions to the function increased the accuracy by about 4%. Comparisons of this study with those on the femur indicated that sexual dimorphism in the tibia was a result not only of the general growth and musculo-skeletal activity, but also of the genetic structure of the population, i.e., racial variation.