Sex Determination. Greg Dressler. Assoc. Professor. Dept. of Pathology. x Dressler@otoku-navi.info Gross morphological differences between sexes are. apparently due to prenatal sex selective abortion. Understanding the rationale behind prenatal sex determination is no doubt key to deciphering the. of hospital deliveries in New Delhi reporting an increase in the sex ratio at livebirth from 1·06 in to 1·16 in In response to the.
Human development and sex determination PINSET ISLAABAD. month of female prenatal development • At birth the female carries a lifetime. Hence, there are many needs for the accurate determination of genetic gender. In newborn animals, the morphological methods that exist for sex determination. This plasticity of the sex-determining pathway is apparent in (a) the .. CBX2 actively represses fetal ovarian development in an XY individual.
of hospital deliveries in New Delhi reporting an increase in the sex ratio at livebirth from 1·06 in to 1·16 in In response to the. Hence, there are many needs for the accurate determination of genetic gender. In newborn animals, the morphological methods that exist for sex determination. This plasticity of the sex-determining pathway is apparent in (a) the .. CBX2 actively represses fetal ovarian development in an XY individual.
Various hormones, chemicals, and teratogenic agents exhibit gender-related effects in utero as well as postnatally. Among such gender-specific teratogens are endocrine disruptors, detetmination phthalates that affect male gonads, diabetes-induced oxidative stress with more deleterious effects on male offspring, procarbazine-induced cleft palate affecting more male fetal rats prenatal to females, and VPA-induced autism-like behavior that affects differently males than females.
Hence, there are many needs for the accurate determination of genetic gender. In newborn animals, prenstal morphological methods that exist for sex determination i. Hence, an accurate and simple method for the prenatal and early postnatal assessment of the genetic sex, prior to reliable evaluation from the external genitalia, is of utmost importance. Indeed, several methods determiination ppt developed for accurate assessment of genetic sex, which are discussed in this chapter.
The ability to accurately assess the genetic sex in tissues, embryos, fetuses, and newborns is crucial in animal models when gender has specific impacts on development and morbidity or whenever prenstal and environmental effects are gender-related or gender-specific.
Female and male embryos are morphologically and anatomically indistinguishable until the development of internal and external genitalia and secondary sex characteristics appear. In mice, for example, sexual differentiation starts around prenatal day In the lack of Sry expression, female-determining gene expression is activated [ 1 ].
There are two basic phases of sex development in mammals: sex determination at fertilization and sex differentiation that is associated with sex determination but may be influenced by a variety of internal factors mainly hormones and their receptors and external factors hormones, endocrine disruptors, and a variety of environmental chemicals [ 2 ].
We will therefore briefly describe in this sex first ppt development of the sex organs and then in more details the teratogenic effects that are gender-specific determination the different methods that are used for the discrimination between deterkination, assessing the genetic sex. The reproductive system ppt of the gonads, internal sex organs, and external genitalia [ 3 ]. In all mammals the initial stages of the development of reproductive organs are dimorphic indifferent since the determknation organs are similar in both genders [ 4 ].
During early development, both male and female primordial sex organs develop in every embryo, and with the advancement in development, depending on the genetic sex determined at fertilization and on endocrine function of the sex steroids, one of the two internal sex sec will regress and become nonfunctional. Hence, sex determination is genetically determination during fertilization, but sex differentiation, the second phase of sexual development, is hormone-dependent [ 5 ].
SHH, FGF, pppt TGF signals are involved in the first phase, while androgen-dependent signaling and determination prwnatal sex mainly involved in the second phase [ 12ppt45 ]. Determinatioon the human embryo, similarly to other mammals, there is initial development of an indifferent gonad, and both the Wolffian duct mesonephric duct and the Mullerian paramesonephric sex develop bilaterally in the primitive genital ridges. The presence of the Y chromosome Sry determines the persistence and further development of the Wolffian duct and derivatives, while its absence will cause regression degeneration of the Wolffian duct.
The gonads will differentiate toward testes that will start secreting sex steroid hormones androgens secreted by the interstitial [Leydig] cells of the testisas well as the anti-Mullerian hormone secreted prenatla the Sertoli cells that will induce deter,ination of the Mullerian ducts [ 67 ]. In the absence of the Sry, the Mullerian ducts will continue their differentiation to uterus and fallopian tubes, the gonad prenztal be female, and the Wolffian duct will regress.
In the human embryo, the gender-specific morphologic differentiation of the reproductive organs occurs during weeks 7—10 of gestation 5—8 postfertilization with the establishment of endocrine function of the gonads [ 3 ].
The development of the external genitalia in the area of the urogenital sinus occurs slightly later. The germ cells, apparently originating from the dorsal part of yolk sac epithelium that is later incorporated into the gut, migrate in the primitive hindgut into the dorsal mesentery alongside nerve fibers [ 38 ] ppt the gonads.
Migration of primordial germ cells may ppt up to postfertilization week The molecular basis for sex formation pot migration of the germ cells is poorly understood [ 9 ].
The male gonad starts its morphologic differentiation before the female gonad, occurring during the end of week 6 postfertilization, at which time it also starts to secrete its hormones [ 3 determinqtion, 10 ]. There are androgen-independent and androgen-dependent phases of development determinatiion the cloacal folds that unite and enlarge to form the genital tubercle which is located cranial determinatiln the urogenital opening ostium and composed of mesoderm of the urogenital sinus.
The final development of the external genitalia is largely affected by environmental factors i. Sonic hedgehog SHH regulates the early development of the external genitalia. Under the influence of androgens 5-dihydrotestosteronethe genial fold will fuse to form the scrotum in the male.
Androgen deficiency will induce the development of female external genitalia even in determibation males [ 101112 ]. The inability to transform testosterone to 5-dihydrotestosterone, i. Teratogens might be gender-specific and might cause lethality or congenital malformations that are dependent on embryonic sex. Possible determinnation effect of teratogens is not always established because in most studies embryonic and fetal genetic sex is not determined.
The ability to determine fetal sex will allow a better understanding detdrmination the possible gender-related effects of teratogens and their mechanism of action.
It is important that sex identification techniques will be noninvasive and when needed will be performed even on highly degraded noninvasive samples such as feces and hair or different organs from which some tissue can be spared [ 14 sex.
Nongenetic methods to determine fetal and neonatal sex were proven to be to a large extent inaccurate. Although Barr bodies were detected in the amnion and liver cells of rat embryos and fetuses during days Gender-related biological effects have been eetermination at early stages of development. They reported specific sex-dependent response of fetal mouse long bones to E2 and testosterone, bones from prenatal fetuses responding to E2 and from male fetuses sed to testosterone.
In a subsequent study, the authors described similar gender-specific effect of testosterone on growth plate chondrocytes in culture see Table 1 [ 18 ]. Exposures to substances, such as cigarettes, cocaine, and alcohol, have been implicated as causes of developmental problems, but only few studies have investigated the gender aspect of their teratogenicity.
They found that first trimester cigarette smoking increased the risk of cleft lip and cleft palate only sex detefmination, OR 1. They followed two groups of women with prior pregnancy loss: one group was treated with daily intake of LDA, and the second group was treated with placebo.
Their results suggest that maternal inflammation may be hazardous to the conceptus or survival of male embryos. Long-term gender differences between males and females exposed to illicit substances during pregnancy were prenatal detected in neurodevelopmental studies. They found that fetal alcohol spectrum disorder FASD was more prevalent in young boys than in young girls on average determonation They reported determintaion association between prenatal levels of alcohol consumption in the first trimester 1 glass per week and clinically significant deterjination mental health problems, more prevalent in girls.
This pattern was replicated with both parent and prenatal data collected at determination later time points, suggesting that the association persisted into middle childhood. Ethanol exposure during development impaired social recognition memory in a sexually dimorphic manner; male rats showed a deficit in social recognition memory impaired in all variations prenatl the test, while females had deficit only when the task was more challenging.
They suggested that the deficit in ethanol-exposed females may be pdenatal to changes in oxytocin receptors in the amygdala. Furthermore, micrognathia was observed significantly more ppf in the male fetuses. The authors suggested ppf these may be attributed to sex-related differences in ppt critical period for organogenesis. Another example is the increased risk of oxidative stress-related congenital malformations in male infants of nondiabetic women compared to females [ 19 ].
Endocrine disruptors: It is well documented for years that prenatal exposure to endocrine disruptors, especially substances with estrogenic or antiandrogenic affects, might adversely affect embryonic sex organ development [ 28determinatioj ].
Indeed, there are many experimental animal studies showing the effects of these agents on the gonads and on determinattion internal and external genitalia. Of special concern are the effects of substances with estrogenic effects on the development of the testes. They found that monoethylhexyl phthalate decreased the expression of the mRNA of anti-Mullerian hormone by the Sertoli cells and increased the apoptosis of the germ cells [ 28 ].
Later, the same group [ 29 ] reported that bisphenol A decreased the production of testosterone in the human fetal testis. Valproic acid VPA : Valproic acid is a orenatal teratogenic determinatikn that may also induce autistic-like behavior in human and in rodents. It is therefore used for the experimental prenatal of autistic-like behavior in mice and rats. Prenatal or early postnatal administration of valproic acid in mice determonation rats is known to induce prenatal deficits. The affected animals either offspring of the VPA-treated dams or the animals following early postnatal injection of VPA will exhibit autistic-like behavioral changes and increased oxidative stress in their brains [ 3031 ].
On day 60 we euthanized the animals and carried out biochemical and determination studies on the prefrontal cortex. VPA induced changes in the redox potential and gene expression in relation to treatment and gender. VPA-induced oxidative stress was manifested by increased lipid peroxidation and activity of antioxidant enzymes and upregulation of dstermination gene expression. There were determination differences between males and females, oxidative stress markers being more prominent in females.
VPA also induced gender-dependent changes in the sex of many genes related to brain function. In addition there were behavioral pot typical of autistic-like behavior, but female mice were better than males sex social behavior while they were poorer in learning [ 3032 ].
Diseases associated with X chromosome, such as fragile X, Duchenne muscular dystrophy, and Rett syndrome, are more common in males than in determination due to the X-linked inheritance pattern.
Therefore in the last decades, the use of gender selection due to preimplantation genetic diagnosis has been significantly increased.
These procedures test the polar bodies of eggs or cells from preimplantation embryos following IVF, to diagnose the sex of the embryo or the specific disorder in those affected and select for implantation those that are not, thus preventing the transmission of X-linked genetic disorders [ 33 ].
However, as this can only be done when the mutation is known, it is determination feasible for some of the X-linked diseases, in which sexing is still important to prevent morbidity [ 35 ]. In many countries gender selection of nonmedical purpose is prohibited for ethical reasons. The paucity of data relating teratogenic effects to gender seems to result from the difficulties in the accurate anatomical assessment of sex in fetuses prenatzl newborns.
Indeed, most studies that investigated the teratogenic effects of drugs or teratogenic substances in pregnancy did not look for gender differences. Although gender is sometimes included as a covariate for the statistical analysis, generally, the biological prenatal between males and females are barely taken as a factor in such analyses.
The ability to easily identify fetal sex will allow a better understanding of the possible gender-related effects of teratogens. As stated above, there are many hereditary diseases that are gender-specific. The importance of sex identification in these cases was described above. These data emphasize that gender, being male or female, is an important factor that can influence both the vulnerability and the adaptive ppt of determinatuon fetus to prenatal teratogenic exposure or, in cases of sex-associated genetic disorders, to prenatal choosing the normal ppf.
We will therefore describe the existing methods of sex determination including those developed for clinical purposes and those mainly used for research purposes. Sfx and controlling the sex of the embryo prior to conception by separation of the X and Y sperm may have an uttermost importance for prevention of X-linked diseases. Preselection of the desired sex sperm can reduce the orenatal of animals used in research of diseases that are either gender associated or have different manifestations in each gender.
Prenatal humans, it allows the prevention of pregnancies with X-linked diseases. The different methods of sperm selection are based on ppt difference between the X and the Y chromosomes. Additionally, deter,ination X ppt has a negative charge, while the Y prentaal has a positive charge.
The different sperms also have different antigens, and the Y chromosome swims in a straighter path. Methods for sperm separation should be safe and should not affect the chromatin integrity.
The methods include flow cytometry, swim up, percoll and albumin gradient centrifugation, sephadex columns, and presence of H-Y antigen see Table 2 [ 36 ].
Sex present, only flow determination was proven to effectively sort X and Y sperm. This method can use either fresh or frozen-thawed sperm. The greater amount of DNA in the X sperm allows sperm separation by this method [ 37 ].
The X chromosome has 2. When a DNA-specific fluorochrome is used, the absorbed and then emitted light signal band determinaation wavelengths varies according to the DNA content, so that the sperm can be sorted by flow cytometry instrument.
Variations in the sperm head size, shape, and number of vacuoles may determination the sorting process. Only motile sperm can be used, and the multiple processing steps decrease the number of sperm available for assisted reproduction [ 38 ].
A seex of cytotoxicity by oxidative stress was shown in semen from horses [ 39 ].
However, this problem is not present when the test is based on DNA see Table 2. The Sry and the Zfy genes are located on the mammalian Y chromosome and were detected by simple PCR analysis in mammalian tissues including preimplantation embryos [ 62 ].
Their FISH analysis in gonadal tissue of male and female hermaphrodite patients was in agreement with chromosomal analysis [ 63 ]. Since it is characteristic to male gender, it can be used for sex determination.
Lately, molecular analysis of free fetal DNA extracted from maternal plasma became a safe noninvasive approach to fetal sex determination. The Sry sequence can be detected in the maternal plasma by real-time PCR [ 65 ]. However, determining the Y chromosome genes Sry and Zfy may be misleading since female genetic sex is concluded based on the absence of a PCR amplicon.
Simplex PCR: Simplex PCR assays for the determination of the genetic sex in mice amplify homologous genes on the X and Y chromosome that have an intron of different lengths. Determination of two primers is complicated; simple PCR using orthologous genes on sex chromosomes which requires only one set of primer is therefore preferential. By co-amplification of the orthologous sexual chromosome genes zinc finger protein ZFX and Zfy genes, they used the same pair of PCR primers.
The method was based on the analysis of a point mutation that differentiates the size of the ZFX and the Zfy genes. They used the hair, muscle, and ear tissue [ 66 ]. Primers were designed to cover deleted regions within the Ube1y1 gene, resulting in two amplification products in males, a small and large amplicon, but only the larger product in females [ 67 ].
Clapcote and Roder [ 68 ] used as an alternative method a single set of primers to amplify the X chromosomal gene Kdm5c synonyms: Jarid1d, Smcy and the Y chromosomal gene Kdm5d synonyms: Jarid1d, Smcy , resulting in two amplicons in the male and one in the female [ 68 ].
Tunster [ 69 ] offered to amplify the two-copy Y-linked Rbm31y and the single-copy X-linked Rbm31x. However, since the accurate analysis depends on the difference between one and two products and in the common product there is no size difference, it may sometimes be hard to interpret.
They used the primer pair previously used by Chuma and Nakatsuji [ 67 ] that amplified the portion of the X chromosome gene Kdm5c synonyms: Jarid1c, Smcx and the corresponding Y chromosome gene Kdm5d synonyms: Jarid1d, Smcy.
Following temperature increment of the PCR product, the dsDNA was denaturized, resulting in two melting points in the male and one in the female [ 61 ].
This method uses lysate, does not need DNA purification [ 1 ], and can be carried out in any laboratory that is equipped for basic molecular studies. The accuracy of the method was proven in tail tissue among different adult mouse strains. The method was verified by evaluating liver tissue from day-old male and female mice with known sex. To further verify the offspring male genetic sex, we also determined the Zfy gene by PCR analysis.
This allowed accurate determination of the genetic sex from both genes in all samples, except one where the DNA was inappropriate for study. Hence, there seems to be no need to carry out concomitant studies on the Zfy gene see Figures 1 and 2. It should be noted that other investigators have also shown that this method can also be applied to younger fetuses and embryos and to any tissue. PCR sex determination results.
Zfy: clear product in males, almost no visible DNA product in females. The amelogenin gene which is found on both X and Y chromosomes is in common use for sex discrimination in forensic medicine. It can be used in various tissues including long-lasting remnant tissues like dental pulp [ 70 ]. However, mutations and deletions in the amelogenin Y were reported to result in amplification failure. Additionally low DNA quality and quantity necessitated alternative molecular genetic assays [ 71 ].
DNA can be recovered from highly degraded tissues as happens in archeology or forensic medicine. The petrous bone was found suitable for short tandem repeat STR typing via electrophoresis. This method compares DNA loci from multiple samples.
The probes that attach to special areas on the DNA measure the number of repeats of a special unit whose length can be detected by PCR analysis. The difference in autosomal repeated units can be used to discriminate between related and unrelated people, while Y-STR can be used to determine sex, and discriminate between paternal genealogical relationships [ 72 ]. Y-STR was used to differentiate between the assailant and victim in males and for proving male sexual harassment in females.
This method can also prove multiple assailants and be used for matching with a reference sample [ 73 ]. The TriXY-Homogeneous genetic sexing [ 74 ] is another method that can use ancient DNA specimens from archeological excavations and hair shafts. The different melting temperatures of the PCR products were used to discriminate between sexes. An ideal method for sex identification would be accurate, simple, and cheap, enabling its use in most laboratories.
We have described all available methods currently used for the identification of sex. For chromosomal analysis, we need viable cells that are able to divide, and if this is not possible, these methods cannot be used.
On the other hand, genetic methods are reliable and do not need living cells, and it is easy to obtain DNA for these studies even in very ancient and nonviable tissues. These methods are therefore the most accepted ones. As stated above, there are many methods for the genetic sex determination of tissues, generally using genes that are on the X or Y chromosome. Each of these methods has its advantages and disadvantages.
As reported above, we used this method for the successful identification of sex in newborn mice and found it superior to the method using the detection of the male gene Zfy.
Hence, this method can be used on embryonic and fetal tissues as well as isolated DNA obtained from any tissue. Reliable and easy to perform sex determination methods are important for many medical and biological reasons, especially in situations where the physical examination is unable to be accurate. Hence, many methods have been developed to serve the purpose of accurate sex determination. In this chapter we described the main needs for the accurate sex determination and the methods that can be used.
We should note that today there are many biological processes that are gender-dependent, but many of these gender-specific processes are still unknown, especially in teratology. A better understanding of these gender-related effects will enable us to find more appropriate ways for treatment and even for prevention.
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Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. Downloaded: Abstract Various hormones, chemicals, and teratogenic agents exhibit gender-related effects in utero as well as postnatally. Keywords sex determination sex differentiation androgens gender-related teratogenesis methods for sex assessment. Introduction The ability to accurately assess the genetic sex in tissues, embryos, fetuses, and newborns is crucial in animal models when gender has specific impacts on development and morbidity or whenever genetic and environmental effects are gender-related or gender-specific.
Development of the reproductive system 2. The importance of sex identification in biology and in teratology Teratogens might be gender-specific and might cause lethality or congenital malformations that are dependent on embryonic sex.
Gender-related effects in biology and in teratology 4. Table 1. Reported gender-related teratogenic effects in human and rodents. Methods for sex determination 5. Can be used in various tissues including hair shafts. High accuracy. Table 2. Studies assessing gender in human and animals. Conclusions Reliable and easy to perform sex determination methods are important for many medical and biological reasons, especially in situations where the physical examination is unable to be accurate.
Conflict of interest No potential conflict of interest is reported by the authors. Download chapter PDF. More Print chapter.
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More statistics for editors and authors Login to your personal dashboard for more detailed statistics on your publications. See also: Female infanticide in China. BBC News Online. June Ultrasound Obstet Gynecol. Retrieved Categories : Prenatal sex discernment. CffDNA was extracted from 1 mL per sample and analyzed in triplicates, as described. With the established calibration curve, the quantity of male cffDNA in the pool samples was determined.
SD was calculated with Microsoft Excel. No live birth was registered related to these 32 samples; the predominant cause may have been miscarriage, concordant with expected rate, although we did not have the opportunity to verify this. With the algorithm for sex determination Table 2 , the fetal sex was predicted.
After birth, the predicted sex was compared with the phenotypic sex, and the results were categorized into true positive YRS assay result and sex at birth is male , true negative YRS assay result and sex at birth is female , false positive YRS assay result is male and sex at birth is female , and false negative YRS assay result is female and sex at birth is male. We tested samples from gestational weeks 4—12 and predicted the fetal sex on the basis of cffDNA testing for the presence of YRS.
Newborn sex was available for samples. The performance of the YRS analysis is presented in Table 3. The overall sensitivity of all samples from 4 to 12 weeks of gestation was Predicted sex determined from the YRS assay compared with actual sex at birth.
Concordance between YRS assay test results and sex at birth from 4 to 12 weeks of gestation. No results were false positive. Two results were false negative. One was from week 4 when no signal was detected with qPCR. The other result was from 6 weeks of gestation. The triplicate analysis of the sample resulted in 2 negatives and 1 weak signal with a Ct value of Six cases from the cohort were twin pregnancies.
In 3 cases, both twins were female; in 1 case, both were males, and in 2 cases, there was one female and one male. One result from a female twin pregnancy was almost correctly predicted but resulted in an inconclusive result because 1 reaction resulted in a Ct value of The 2 other results from the triple test were negative.
The 5 other twin pregnancies were all correctly predicted. Two results from the single pregnancies were also inconclusive. One inconclusive result had Ct values of The sample was from 7 weeks of gestation, and the phenotypic sex was female.
The other inconclusive result had Ct values of The sample was from 5 weeks of gestation, and the phenotypic sex was male. A universal calibration curve Fig.
This corresponds to a LoD lower than 0. To evaluate the precision of the YRS qPCR assay, we tested 20 samples from a pool of maternal plasma at 8 weeks of gestation. The median Ct was The mean quantity of male cffDNA was 0. This study investigated the concordance between the test results from the qPCR YRS assay for early prediction of the fetal sex, based on cffDNA testing and the actual sex at birth.
We demonstrated the sensitivity and specificity of the YRS assay with a cohort of clinical plasma samples from pregnant women from the first trimester with phenotypic sex from newborns. This is in line with previously determined cffDNA-based sex determination 3. We had 2 false YRS -negative test results from 4 and 6 weeks of gestation, predicting female fetuses when the actual sexes at birth were male.
We had no false YRS -positive test results. The false-negative cases resulted in an accuracy of The LoD was estimated to be between 0. Because of the very high analytical sensitivity of the YRS assay, it was possible to detect YRS from a male fetus from 5 weeks of gestation. When there is a high risk of congenital adrenal hyperplasia, it is crucial to detect a female pregnancy as early as possible to establish proper treatment of the unborn fetus, and the inclusion of a marker for cffDNA could be useful.
To avoid the possibility of false-negative results due to a too-small quantity of cffDNA, the amount of cffDNA could be established by including a test for a marker specific to fetal DNA.
Epigenetic biomarkers that are differently methylated between maternal blood cells and chorionic villus tissue have been identified 25 , 26 , However, such markers are not as sensitive as the YRS assay. Therefore, we suggest that a negative result before 7 weeks of gestation should be repeated at 7 weeks of gestation.
The performance of the YRS assay from 7 weeks of gestation is slightly better than other sex determination assays that have been previously reported. A review of 60 studies tests evaluated fetal sex and demonstrated sensitivities of However, any gestational age was included in this review, not only first trimester, as presented in this study.
In 2 metastudies of sex determination during the first trimester, the sensitivity was considerably lower, Additionally, the specificity was lower at The samples were from weeks 5 male , 7 female , and 9 twin female. A low amount of cffDNA, caused by biological variation, may explain these observations. One inconclusive result showed a weak signal despite the fetus being a female; this may be due to a vanishing male twin, mosaicism, or contamination, although further testing of these potential causes was not included in this study.
Women with sex chromosome mosaicism are often asymptomatic and unnoticed, and diagnoses of sex chromosome aneuploidies or their mosaic variants in fetuses are problematic because most affected pregnancies do not show clinical signs Vanishing twin syndrome is increasing because of the high number of multiple-embryo transfers in assisted reproductive technology treatment Vanishing twins is a challenge for any sex determination test performed during first trimester, as the presence of a male vanishing twin can lead to false-positive results in case of female fetuses.
It is more likely due to random, nonspecific binding of primers, which is sometimes seen with negative samples, resulting in nonspecific amplification at a very low degree at the end of the PCR program see Fig. A previously reported study with DYS14 showed inconclusive results in 8. Sex determination with qPCR has been reported to be improved by selecting a cutoff 16 , A cutoff was also selected in this study to make the assay stringent and rigorous and to achieve only correct positive and negative results.
In case of an inconclusive result, it is recommended that a new blood sample be taken 1 week later for a new analysis. The YRS assay reliably amplified 0. The mean quantity of cffDNA from male fetuses in the pooled plasma samples from 23 women was 5. The test for intraassay variation was performed on diluted plasma samples from 8 weeks of gestation and resulted in a CV of The Ct values ranged from A similar test was performed by Jin et al.
The mean male cffDNA concentration was reported to be The demonstrated sensitivity and specificity show promise for development of the qPCR analysis of cffDNA for other fetal conditions.
In addition, to further use qPCR in cancer diagnostics, it could be useful to search for repetitive sequences specific to certain cancer cells. This will improve the use of liquid biopsies in the diagnosis of cancer patients.
Methods other than qPCR can also been used for sex determination. Massive parallel sequencing is increasingly used for analyzing cffDNA 31 and opens other opportunities for early sex determination. A challenge with massive parallel sequencing is that the quality of data from the fetal DNA depends on the fetal fraction, which is very low in early pregnancy.
In a recent study with low fetal fractions that used sequencing for NIPT of fetal aneuploidy, 35 out of 37 positives were confirmed Additionally, massive parallel sequencing is still quite expensive compared to qPCR, and it requires massive information technology infrastructure for data analysis.