*2.1.3 Placental Messenger RNA (mRNA)*

The big breakthrough came with the promising technique of detection of **Placental mRNA** for beta -hCG & hPL by RT-PCR in the bloodstains from pregnant women [10]. A lot of research was done in the 21st century with respect to this.

**What is mRNA?** mRNA is the step in between protein-encoding DNA translation and the proteins production by ribosomes [11]. Placenta expressed genes based mRNA transcripts easily detected in maternal plasma prove that the source of fetal nucleic acid release is placenta [12]. Only placental trophoblasts express hPL and beta hCG mRNA which is detected in maternal plasma.

**Method of Detection of mRNA**: In order to detect presence of these mRNA, RT-PCR is the method of choice. Advantages of the RT-PCR technique is manifold. First it is highly specific and sensitive. The second advantage is that it can be designed to be human specific, proving to be advantageous over the immunological tests of pregnancy which have high chances of false positivity [13].

The controversy was that mRNA was thought to be highly unstable and was considered to be degraded quickly making its use as a possible detection tool in old stains questionable. But few studies have refuted this dogma and there are instances wherein the mRNA has been detected in bloodstains as old as 16 years [14].

An ideal mRNA-based test should be in detectable quantities early in pregnancy as well as throughout pregnancy with rapid clearance after delivery. Out of the known 11 genes which were reported to have pregnancy-specific expression patterns, only 2 genes have been thoroughly studied - β-subunit of the hCG and human placental lactogen [13]. Let us look at both the molecules closely to determine which is ideal.

**Human Placental Lactogen**: The various parts of the hPL mRNA is depicted in **Figure 1**. As per the demonstration, RT-PCR detection of hPL was noted throughout pregnancy increasing until delivery and it was undetectable within 24 hours following delivery, which makes it an ideal test [13]. The added advantage is that, when there is a mixed sample, it can be designed to be human specific [10].

**Figure 1.** *Human placental lactogen mRNA nucleotide (source: [15]).*

#### **Figure 2.**

*Method describing the quantification of mRNA using RT PCR technique.*

#### **Method of RT PCR for quantification of mRNA** [15]:

As in **Figure 2**, the exact methodology of quantification of mRNA using RT PCR technique is described.

Advantages of hPL mRNA: A study done by Gauvin et al. demonstrated the detection of transcripts of hPL in as little as 0.25 cm2 of dried bloodstain, showing that this test has a high sensitivity. The other advantage noted was that the hPL transcript was demonstrated in bloodstains as old as 56 days at room temperature. All these factors- stability and high sensitivity imply that RT-PCR hPL mRNA assay is an ideal marker for pregnancy related forensic diagnostics [10].

**β-subunit of the hCG:** With respect to the other marker – βhCG, it was noticed that the mRNA concentration reduced as pregnancy advances [16]. The RT-PCR of βhCG mRNA levels are detected in the first trimester where their levels are the highest. After which, the levels decreases as pregnancy advances making its detection difficult. So to conclude, by itself βhCG mRNA may not be reliable as a biomarker when you have to consider the entire duration of pregnancy [13].

Also research has been done in the area to estimate the gestational age from the bloodstains. A study based on the rationale that the use of time-wise reverse expression intensity pattern of the hPL and βhCG transcripts could predict the period of gestation from the pregnant woman's bloodstains. Gauvin et al. tested this hypothesis and found that there was a significant positive relation in women with gestational ages between 8 and 20 weeks. But the biggest disadvantage faced was that the RT-PCR assay for βhCG is less sensitive when compared to hPL.

#### **3. Obstetric markers in substance abuse**

#### **3.1 Obstetric markers in alcohol abuse**

Alcohol abuse in the mother is a problem which affects the fetus drastically. The teratogenic effects of alcohol on the fetus are well known causing Fetal Alcohol Syndrome, a severe form of affection of the Fetal Alcohol Spectrum Disorder. Also the pregnant woman can commit crimes under the influence of alcohol.

The biomarkers specific to alcohol abuse in pregnancy are ethyl glucuronide (EtG), fatty acid ethyl esters (FAEEs) and ethyl sulphate (EtS) which are nonoxidative direct ethanol metabolites. These remain positive in maternal serum and urine for FAEEs for up to 24 h in serum and EtG in urine up to 5 days. These are promising. Also carbohydrate-deficient transferring (CDT) and phosphatidylethanol in blood of the mother are being evaluated.

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*Obstetric Markers as a Diagnostic Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.97670*

**3.2 Obstetric markers In drug abuse**

Drug abuse in pregnancy is not uncommon. But unfortunately, the teratogenic effect of drugs can prove disastrous on the fetus. Use of cocaine prenatally causes preterm labour, abruption, congenital anomalies and low birth weight babies. Cannabis metabolites use causes difficulty in memory and learning. Opiate exposure prenatally results in withdrawal symptoms in neonates. Evaluation of these drugs in hair of the mother and meconium of the neonate can be done by standard chromatography methods. Information based on maternal hair depends on its length, while the exposure during pregnancy results in these drugs getting accumulated in the meconium, which needs to be analyzed as soon as its passed once fetus is born [17]. The forensic implications are that these pregnant women on drug use exhibit depression, anxiety, psychological struggle, can commit crimes under the drug influence and are liable for arrest and can be tried in the court according to the

Paternity testing in the prenatal period could be required in cases of pregnancy resulting from rape. Earlier, the paternity testing depended on invasive procedures such as chorionic villus sampling and amniocentesis. These procedures could pose a risk to the wellbeing of the mother and fetus. In order to find a relatively safer testing method, non-invasive methods using the cell free fetal DNA (cffDNA) were

Initially the genetic markers investigated were short tandem repeat (STR) loci. But, the increased stutter amount hindering allelic assignments and decreased size of DNA fragments- maternal & fetal proved to be a hindrance [20]. Also only Y-chromosome STRs (Y-STRs) could be used which restricted the application to male fetuses only and not in female fetuses. The chances of false paternity exclu-

Then the Single nucleotide polymorphisms (SNPs) were investigated. A recent study done by Tam et al. developed a systematic SNPs selection procedure which reduced the number of target-SNPs for sequencing analysis to an average of 148 effective SNPs to calculate the probability of paternity. But the possible drawback is that in order to perform the test, a large number of loci is required [22]. But this is

With the number of SNPs to be tested on an average being 148, it can be cumbersome. So research is on to find a better genetic marker. Hence, the use of microhaplotypes is being researched. Microhaplotypes are the regions of ~200 bp containing two or more SNPs and at least three different haplotypes. Microhaplotypes with only 15 regions and with admixtures of DNA are being researched to determine

Obstetric markers is emerging as an important aspect in the forensic diagnostics. The pregnant women may face crimes like rape, physical harm against them. While, they may undergo criminal abortions, illegal feticide or have a substance abuse issue for which they can be held liable. The various obstetric markers- use of mRNA in bloodstains, use of biomarkers in substance abuse or the noninvasive genetic

prevailing laws of the countries where they are prosecuted [18].

the mainstay for noninvasive paternity testing as of now.

paternity in a non-invasive manner [23].

**5. Medicolegal implications**

**4. Obstetric markers in paternity suits**

investigated [19].

sions were increased [21].
