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Back to Journal »International Journal of General Medicine» Volume 14
Fetal nucleated red blood cells are more useful than cell-free fetal DNA for early determination of the gender of invasive and non-invasive sources using the new four-gene multiplex PCR
Author: Alhur NF, Al Qahtani NH, AlSuhaibani ES, Alsulmi E, Almandil NB, AbdulAzeez S, Borgio JF
Published on December 13, 2021 2021 Volume: 14 pages 9697-9705
DOI https://doi.org/10.2147/IJGM.S345345
Single anonymous peer review
Editor approved for publication: Dr. Scott Fraser
Norah F Alhur, 1 Nourah H Al Qahtani, 2 Entissar S AlSuhaibani, 1 Eman Alsulmi, 2 Noor B Almandil, 3 Sayed AbdulAzeez, 4 J Francis Borgio 4 1 Department of Zoology, Faculty of Science, King Saud University, Riyadh, Saudi Arabia; 2 Imam A Department of Obstetrics and Gynecology, Abdul Rahman Ben Faisal University School of Medicine, Dammam, Saudi Arabia; 3 Imam Abdul Rahman Ben Faisal University Department of Clinical Pharmacy, Research and Medical Consulting Institute ( IRMC), Dammam, Saudi Arabia, 31441; 4 Department of Genetic Research, Institute of Research and Medical Consulting (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, Dammam, Saudi Arabia Tel +966 13 3330864 Email [Email protected]; [Email protected] Background: DNA from invasive, non-invasive, and 9th week embryos can be used as a resource for determining the sex of a fetus using highly sensitive and specific multiplex PCR. Method: A total of 402 DNA samples were used to test the newly developed multiplex PCR, including male-specific (3 genes: SRY, DAZ2 and TSPY1) Y biomarkers and internal control ACTB. The study isolated cffDNA (cell-free fetal DNA; n = 73) from mother’s plasma, serum and urine, fetal DNA from 9th week embryos and cord blood, and CD71+ve nucleated red blood cells (fNRBC; n = 73) fetal DNA was isolated). The paternal and maternal DNA from oral cells (n = 20) and blood (n = 232) were used for male and female identification. Results: The study observed that SRY alone cannot be a suitable Y biomarker. The confirmation of any two Y biomarkers is mandatory for male fetus identification. Direct sequencing of multiple and single amplicons eluted from the gel confirmed the specific sequence. The strength of two of the three Y biomarkers or a single Y biomarker> 1,000,000 is considered positive for men. Multiplex PCR is suitable for determining gender from all fetal DNA sources including highly degraded cffDNA, and 0.5 ng DNA can be used to detect gender. Real-time qPCR based on a single label, and then combined with melting curve analysis, showed a significant melting curve peak of the label. Conclusion: Multiplex PCR achieves 100% accuracy on fetal DNA from fNRBC and is used for early (<13 weeks) gender determination. The developed new simple multiplex PCR and individual qPCR can be used in all types of laboratories to determine the sex of human fetuses using fNRBC fetal DNA. Early identification of gender can support preparation for possible X-link analysis, reduce mother's anxiety, strengthen mother-to-child connection, and effective decision-making. The non-invasive source of fetal DNA from fNRBC is the first choice for gender identification to reduce the risk of invasive surgery in the first trimester (8-13 weeks). Keywords: cffDNA, male PCR, invasive, non-invasive, Y biomarker, fetal gender, fNRBC
Determining the sex of the fetus in the first trimester is very important for identifying pregnancies at risk of X-linked genetic diseases. To date, more than 100 X-linked genetic diseases have been discovered in humans, including muscular dystrophy, fragile X syndrome, and hemophilia. 1 The fetal sex determination test is designed to help manage or present X-linked genetic diseases. Early fetal sex determination is accurately achieved through invasive procedures, whether it is chorionic villus sampling in the first trimester or early amniocentesis in the second trimester. 2 However, these procedures are associated with the risk of miscarriage and are expensive, which makes these programs less acceptable to some people. 3 Therefore, many patients use 2D ultrasound to know the sex of the fetus, and its accuracy is 100% from the 20th week of pregnancy. 4 However, the high accuracy of ultrasound can only be achieved in the late second trimester. The accuracy of ultrasound in the first trimester is affected by gestational age and depends on the operator, which makes the accuracy at 11 weeks of gestation approximately 70%. 5 Therefore, it is vital that the search for cheaper, non-invasive, and highly accurate diagnostic methods can be completed early in the first three months. Table 1 Sample distribution of Y-Biomarker quantity
Table 1 Sample distribution of Y-Biomarker quantity
Fetal DNA is more reliable and can be extracted and used early in pregnancy. 6 Fetal DNA can be obtained from maternal peripheral blood through non-invasive methods, such as cell-free fetal DNA (cffDNA) or fetal DNA circulating freely in maternal blood, 7 or through invasive procedures such as chronic chorionic villus and amniocentesis sampling. 8 Fetal nucleated red blood cells (fNRBC) are cells that circulate in the peripheral blood of pregnant mothers. FNRBC contains the entire genome of the fetus and can be used as a source of sex determination. 9 The availability and concentration of fetal DNA are important to obtain significant results. In a non-invasive method, low concentrations of DNA can be obtained. 10 This raises the need for specific, sensitive and optimal techniques for fetal sex determination. The placental extracellular vesicles of pregnant mothers are a relatively new field that is developing rapidly and is observed as a new communication method from the fetus to the pregnant mother that can affect the maternal cell population. 11 Exosomal DNA (exoDNA) or vesicle fetal DNA is also an emerging option for determining fetal sex. 12-15
Some studies have focused on Y chromosome-specific biomarkers and genes. Different Y chromosome sequences have been used for sex determination. Mainly, the single-copy sex determining region Y (SRY) has been well reported. 16 However, SRY is a single copy, so it is not sensitive enough to determine the sex of the fetus. The combination of multiple copies of sequences in the Y chromosome, such as azoospermia 2 gene deletion (DAZ2) and testis-specific protein Y-linked 1 gene (TSPY1), also known as (DYS14), will be more sensitive and specific for sex determination. The multiplex single-tube PCR with three Y biomarkers described here is sensitive and specific, and can use traditional multiplex PCR methods to detect male fetal DNA from any sample source.
The Institutional Review Board of Imam Abdul Rahman Ben Faisal University approved the study. IRB approval number: IRB-2017-13-137, dated June 7, 2017, and extended on December 14, 2020.
Primers of 541, 769, 269, and 329–356 bp were designed for ACTB, SRY, DAZ2, and TSPY1, respectively (the primer sequence is in the patent publication of the US Patent Office; the sequence is available on request). Manually strengthen the primers to obtain high precision, specificity and narrow annealing temperature difference, and use primer BLAST to confirm its applicability. The primers were synthesized by Integrated DNA Technologies, Inc. (Leuven, Belgium).
All samples (n=402) used in the study were collected at the King Fahd Hospital of Imam Abdul Rahman Bin Faisal University. The detailed sources of cffDNA and fetal DNA are shown in Figure 1. A total of 402 DNA samples used in the study: fetuses aborted in the 9th week of pregnancy (n=4) and fetuses in the uterus ≤13 weeks (n=24) [cffDNA: 8 weeks=2, 9 weeks=2, 10 weeks =2, 11 weeks = 2, 12 weeks = 3, 13 weeks = 1; Fetal DNA from fNRBC: 8 weeks = 2, 9 weeks = 2, 10 weeks = 2, 11 weeks = 2, 12 weeks = 3, 13 Week = 1]. Fetus in utero> 13 weeks (n = 122) [cffDNA = 61; fetal DNA from fNRBCs = 61]. In addition, adult gDNA from oral cells (n = 20; 10♀, 10♂) and blood samples (n = 232; 100♀, 132♂) were used to validate the single-tube novel multiplex PCR. Maternal blood (3 to 5 ml) and cord blood (3 to 5 ml) samples are collected from women’s lithium heparin or EDTA vacuum blood collection tubes during delivery. After collecting for 8-10 hours, the samples were processed to extract cffDNA. Serum (n=23) samples are collected in tubes without clot activator or gel, and urine (1 to 2 ounces) (n=12) samples are collected in sterile containers. Urine samples were centrifuged twice at 3000 rpm for 10 minutes at 20-24°C to remove cells, and then stored at -20°C for cffDNA extraction. Tissues (n=4) that were aborted from the 9th week of pregnancy (carefully separated from maternal tissues to avoid contamination) samples and blood samples were collected from the fetus. Abortion samples were collected in RNAprotect Cell Reagent (Qiagen, Hilden, Germany). Except for 4 aborted fetuses, the rest of the samples were intrauterine fetuses. All samples were collected in accordance with the standard protocol after signing the informed consent form. Figure 1 The flow chart of sample collection and the source of DNA used to identify the sex of the fetus. Abbreviations: PBMC, peripheral blood mononuclear cells; gDNA, genomic deoxyribonucleic acid; cff DNA, cell-free fetal deoxyribonucleic acid; PCR, polymerase chain reaction; fNRBC, fetal nucleated red blood cells.
Figure 1 Flow chart of sample collection and DNA source used to identify the sex of a fetus.
Abbreviations: PBMC, peripheral blood mononuclear cells; gDNA, genomic deoxyribonucleic acid; cff DNA, cell-free fetal deoxyribonucleic acid; PCR, polymerase chain reaction; fNRBC, fetal nucleated red blood cells.
Use histopaque -1077 to perform density gradient centrifugation on maternal and cord blood samples. Centrifuge at 1430 rpm for 45 minutes. Collect the plasma in a 1.5 mL tube, recover the cell layer between the plasma and tissue hyaluronic acid, and then wash with PBS. For anti-CD71 positive selection, cells were incubated with 1:10 CD71 MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4-8°C for 30 minutes. Use mini-MACS system (Miltenyi Biotec) to achieve magnetic selection. The dual gradient separation method is also suitable for the separation of fNRBCs. 17
According to the manufacturer's instructions, DNA was extracted from fetal blood using QIAamp DNA Blood Mini Kit (Qiagen, Germany). According to the manufacturer's protocol, the Puregene cell and tissue kit (Qiagen, Germany) was used to extract DNA from fetal tissue. The QiAamp MinElute ccfDNA mini Kit (Qiagen, Germany) was used to extract cffDNA from maternal plasma, serum and urine. Use nanodrop (Nanodrop 8000, Thermo Scientific, DE, USA) to estimate the concentration of extracted DNA, and ensure that the quantity>0.5 ng/µL and the quality A260/A280 ratio is 1.8±0.2. A Qubit 2.0 Fluorometer (Qubit™ dsDNA HS Detection Kit; Life Technologies, CA, USA) was used to quantify approximately 5% of the samples.
Separate PCR of ACTB (541 bp), SRY (769 bp), DAZ2 (269 bp), and TSPY1 (329–356 bp) amplicons was completed before designing multiple formulations. The total volume of the PCR amplification reaction is set to 25 µL, which contains: Absolute master mix 12.5 µL (MOLEQULE-ON, Auckland, New Zealand), primer 1 µL (10 nM) SRYaF, primer 1 µL (10 nM) SRYaR, primer 1 µL (10 nM) ActinF, primer 1 µL (10 nM) ActinR, primer 1 µL (10 nM) DYS14F, primer 1 µL (10 nM) DYS14R, primer 1 µL (10 nM) DAZ2aF, primer 1 µL (10 nM) DAZ2aR, template DNA 25 ng and Dis H2O to 25 µL. Multiplex PCR thermal cycling: Step 1: 95 °C for 10 minutes, Step 2: 95 °C for 1 minute, Step 3: 68.3 °C for 1 minute, Step 4: 72 °C for 1 minute, Step 5: Go to step 2, 35 cycles, step 6: 72 °C for 5 minutes, step 7: store at 4 °C. Observe the amplicons using 2% agarose electrophoresis at 100 volts for one hour.
The same primers are used separately in real-time PCR (qPCR) amplification to verify the specificity of these primers in qPCR. The PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA) containing 5 to 10 ng DNA was used to analyze samples from different maternal sources and male and female samples as positive and negative controls by qPCR assay, and repeat the procedure. The reaction was performed on the 7500 Fast Real-Time PCR system (Applied Biosystems, USA) using 7500 software v2.0.6 (Applied Biosystems, Life Technologies). Amplification method: 50°C/2 minutes; 95°C/10 minutes; 45 amplification cycles, including denaturation steps at 95°C/15 seconds, annealing at 60°C/1 minute, and annealing at 72°C/1 minute extend. The melting curve stage is carried out as follows: 95°C/15 seconds, 60°C/1 minute to 95/1 minute, 0.5% increase.
Primers designed for ACTB (541 bp), SRY (769 bp), DAZ2 (269 bp) and TSPY1 (329–356 bp) genes were tested to determine the specific application of each gene in males, without non-specificity Amplify women at a specific temperature (Supplementary File 1). All four pairs of primers were amplified individually to confirm that the amplicons in females (n=110; buccal cells=10; blood=100) and males (n=142; buccal cells=10; blood=132) did not contain non-specific amplicons. Heterosexual amplicon) Sample with gDNA (Supplementary File 1). Perform temperature gradient multiplex PCR to confirm the annealing temperature (68.3°C) for specific amplicons and no non-specific amplicons. The amplicons of concentration-dependent multiplex PCR confirmed stable specific products in different concentrations of DNA. Multiplex PCR amplification based on multiple samples, such as father DNA from father’s blood, mother DNA from mother’s blood, father DNA from father’s oral cells, mother DNA from mother’s oral cells, cffDNA from mother urine, and mothers Serum cffDNA, cffDNA from maternal plasma, cffDNA from umbilical cord blood, fetal DNA from fetal blood, fetal DNA from fetal tissue, and fetal DNA from fetal fNRBC confirm this possibility and specificity (Figure 2). Achieved 100% accuracy when identifying the gender in adult gDNA [♀n=110; oral cell=10; blood=100 and ♂ n=142; oral cell=10; blood=132. Figure 2 (A and B) Representatives of DNA multiple amplicons from various sources. (C) The intensity of a single Y-labeled amplicon. The red box indicates acceptable strength. The blue box indicates that the intensity is below the acceptable intensity. Abbreviations: L, 100 bp ladder; F, father DNA from father’s blood; M, maternal DNA from maternal blood; PDY3, father DNA from father’s oral cells; PDY8, maternal DNA from mother’s oral cells; U, from CffDNA from maternal urine; S, cffDNA from maternal serum; MP, cffDNA from maternal plasma; C, fetal DNA from fetal cells isolated from cord blood; UKN3 and UKN4, fetal DNA from fetal blood; UKN4 TISSUE, Fetal DNA from fetal tissue; M59, fetal DNA from fetal cells isolated from maternal blood; C59, fetal DNA from fetal cells isolated from cord blood; M5+ and C13H, fetal DNA from cord blood CD71+ve cells.
Figure 2 (A and B) Representatives of DNA multiple amplicons from various sources. (C) The intensity of a single Y-labeled amplicon. The red box indicates acceptable strength. The blue box indicates that the intensity is below the acceptable intensity.
Abbreviations: L, 100 bp ladder; F, paternal DNA from father’s blood; M, maternal DNA from maternal blood; PDY3, paternal DNA from paternal oral cells; PDY8, maternal DNA from maternal oral cells; U, from CffDNA from maternal urine; S, cffDNA from maternal serum; MP, cffDNA from maternal plasma; C, fetal DNA from fetal cells isolated from cord blood; UKN3 and UKN4, fetal DNA from fetal blood; UKN4 TISSUE, Fetal DNA from fetal tissue; M59, fetal DNA from fetal cells isolated from maternal blood; C59, fetal DNA from fetal cells isolated from cord blood; M5+ and C13H, fetal DNA from cord blood CD71+ve cells.
The presence of two amplicons in the three Y tags should be considered male positive. Some amplicons were observed to be very weak, and their presence could not be confirmed. In order to confirm the presence of amplicons and avoid human intervention to determine the presence or absence of amplicons, Image Lab (Bio-Rad, USA) was used to calculate the amplicon intensity of a single Y biomarker (Figure 2). A single Y biomarker amplicon with an intensity greater than 1,000,000 is considered positive for men. CffDNA from urine (n = 2; >13 weeks gestation) was positive for two Y biomarkers and negative for the ACTB gene. Fetal DNA from NRBC (n = 1; <13 weeks of gestation) was also positive for two Y biomarkers and negative for ACTB.
Direct sequencing was used to confirm single PCR amplicons of specific gene products and gel-eluted multiple PCR amplicons (Figure 3). The gel-eluting amplicons of the SRY, ACTB, DAZ2, and TSPY1 genes from multiplex PCR were confirmed to have specific sequences and no background noise. For early sex determination of ≤ 13 weeks of pregnancy and> 13 weeks of pregnancy, multiplex PCR obtained 100% accuracy on fetal DNA from fNRBC. However, the accuracy of cffDNA from plasma (n=36), serum (n=23) and urine (n=12) in determining gender is 83.56%. In plasma, serum and urine, the study observed that the accuracy of plasma cffDNA was very low (Table 1). Figure 3 Electrophoresis of multiple PCR amplicons eluted from the gel. Electropherogram generated from the forward primers of multiplex PCR amplicons. The sequence highlighted in blue inside represents the complete sequence of the amplicon.
Figure 3 Electrophoresis of multiple PCR amplicons eluted from the gel. Electropherogram generated from the forward primers of multiplex PCR amplicons. The sequence highlighted in blue inside represents the complete sequence of the amplicon.
The qPCR results show that the primers have high specificity and can detect male fetal DNA in the test sample (Figure 4). The melting curve analysis of individual markers (Y-biomakers and housekeeper) showed that the derivative of fluorescence with respect to temperature showed that DAZ2, SRY, ACTB and TSPY1 genes were at 78.5 °C, 86 °C, 88.5 °C and 91 °C, respectively. Use any type of Y-biomakers and the housekeeping gene considered to perform combinatorial analysis and melting curve analysis for individual markers. Figure 4 qPCR melting curve analysis of 3 Y-markers (SRY, DAZ2 and TSPY1) and housekeeping (ACTB) genes.
Figure 4 qPCR melting curve analysis of 3 Y-markers (SRY, DAZ2 and TSPY1) and housekeeping (ACTB) genes.
The ability to determine the sex of a fetus early in pregnancy is a huge leap forward in prenatal diagnosis, especially in the prevention or management of X-linked genetic diseases. The easiest way to determine the sex of a fetus is massively parallel sequencing (MPS). 18 However, it would be expensive to test for MPS in all pregnancies at risk for X-linked genetic diseases. The single-tube multiplex PCR developed in the research is more cost-effective. The use of SRY, TSPY1 and DAZ has been well studied19-21 and has shown high sensitivity for fetal sex determination, mainly through real-time PCR. However, a good DNA concentration is required. One of the main advantages of this study is that it can detect low concentrations of DNA (< 5 ng/µL). This study includes DNA from all possible samples from the mother or fetus, including invasive and non-invasive samples. The study isolated cffDNA (cell-free fetal DNA) from mother’s plasma, serum and urine, fetal DNA from 9th week embryos and cord blood, and fetal DNA from nucleated red blood cells (CD71 positive). The male and female DNA derived from oral cells are used as the source of positive DNA for males and females, respectively. Two of the three Y biomarkers or a single Y biomarker with an intensity greater than 1,000,000 are considered positive for men. The sex of the fetus is confirmed by ultrasound after 20 weeks of pregnancy or at birth. Multiplex PCR is a suitable method to determine gender from all fetal DNA sources (including highly degraded cffDNA), but the accuracy is not very high.
According to our research, a single non-invasive maternal cffDNA sample is not sufficient to determine the sex of the fetus most accurately. Among 73 people, only 61 had male DNA detected at different stages of pregnancy. This result is contrary to the scientific literature, which reports higher sensitivity in early pregnancy (87.5-99.1%) and increases as pregnancy progresses (97-100%). 20,22 This is due to the use of The sample size is small. Due to the low availability of blood samples, current studies only use <1 mL of plasma or serum to extract cffDNA. If the blood sample volume is larger, then we can obtain higher accuracy due to the availability of high cffDNA. However, due to the restrictions approved by the IRB, we cannot get more blood. In a study conducted by Kazachkova et al., the effects of Y chromosome biomarker gestation age and low cffDNA concentration in maternal samples were not detected in cffDNA. 19 In addition, the type of PCR used was proposed by Costa et al. and Levi et al. as an influence on the detection of Y chromosomes. 23,24
Fetal DNA from fNRBC is most suitable for early determination of fetal sex because it shows 100% accuracy in early pregnancy samples. The two studies used different techniques to enrich fNRBC: Byeon et al. used the dual enrichment process of RBC hyper-aggregation and WBC consumption, while Zhang et al. used a microfluidic chip. 25,26 Both studies reported the complete accuracy of the Y chromosome detected in enriched fNRBC. This study used a double enrichment process. 17
Multiplex PCR uses the fetal DNA from fNRBC to correctly identify the fetal gender of mothers carrying male fetuses (n=67) and female (n=6) fetuses. Gender is confirmed by ultrasound after 20 weeks of pregnancy or at birth. Multiplex PCR has 100% accuracy and can identify fetal gender from all adult samples.
Use multiplex PCR to determine the sex of the four fetal tissue samples. A fetal tissue reported to have a homozygous ASIC5Saudi mutation. 27 Since fetal tissue samples are collected from aborted 9-week fetuses, clinical confirmation of fetal gender cannot be achieved. Early prenatal diagnosis of gender and identification of disease-related mutations can provide effective decisions for mothers in high blood relatives to make effective decisions within the allowed time. 27,28 The eight pairs of primers designed in the study have high specificity, sensitivity and uniqueness. They are very suitable for simultaneous detection of male-specific Y biomarkers SRY, DAZ2 and TSPY1 genes and internal control ACTB genes for routine PCR and real-time qPCR . Confirm the specificity of the primers by sequencing. This reduces costs, improves accuracy due to 3Y biomarkers, minimizes the risk of invasive sampling, 16 simplifies the process due to a single tube, and can be used in low-income areas and laboratories with minimal facilities.
The gender of adult gDNA is correctly identified with 100% accuracy. Using the currently developed new multiplex PCR based on Y chromosome markers can be a reliable method for determining fetal gender using invasive and non-invasive sources of fetal DNA, with 100% accuracy on fetal DNA from fNRBC, This is due to incomplete external genitalia, early pregnancy through ultrasound examination. The shortest possible time is sufficient to determine the use of single-tube multiplex PCR with 3 Y biomarkers or separate real-time qPCR. The study fully and accurately confirmed the gender determination procedures for male and female pregnancy using fNRBC. Studies have confirmed that the non-invasive and invasive source of fetal DNA can be applied to early gender identification, which can help reduce mother’s depression and anxiety, strengthen the bond between mother and fetus, prepare for possible X-linkage analysis, and make effective decision-making. Reduce the risk of invasive surgery.
All data will be provided at the reasonable request of the corresponding author.
The research was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of Imam Abdul Rahman bin Faisal University. IRB approval number: IRB-2017-13-137, dated June 7, 2017, and extended on December 14, 2020.
The author thanks the Dean of the Imam Abdul Rahman Bin Faisal University (IRMC) in Dammam, Saudi Arabia for his continued support and encouragement. The author thanks Mr. Ranilo. Mr. M. Tumbaga, Mr. Horace T. Pacifico and Mrs. Jee Entusiasamo Aquino thank them for their help. This research was supported by Imam Abdul Rahman Bin Faisal University Director of the Institute of Science (to Dr. J Francis Borgio, grant number: 2017-100-IRMC). The authors thank the Institute of Research and Medical Consulting (IRMC) for the equipment and facilities.
Dr. J Francis Borgio reported that the patent pending in the US Patent Office has not yet been submitted (a multiplex PCR method for identifying the sex of early pregnancy samples). The author declares that there are no other conflicts of interest in this work.
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