In the current study, GAPDH was used as a reference gene due to the fact that its transcription level remains relatively constant in response to experimental manipulation in most tissues (Li et al., 2010). The same study instrument was adopted by other researches such as Bauer et al. (2008) and Hass et al. (2009), wherein GAPDH was detected in all body fluids including menstrual and peripheral blood.
Overall, HBA was detected in the entire study samples including menstrual and peripheral ones. This finding is consistent with previous studies where blood markers HBA and aminolevulinate synthase 2 (ALAS2) were expressed in both menstrual and peripheral blood (Nussbaumer et al., 2006; Hass et al., 2011; Jakubowska et al., 2014; Song et al., 2015).
This may be related to the fact that HBA and hemoglobin B (HBB), as subunits of hemoglobin, are in the meantime ideal indicators for the presence of blood and menstrual secretion. The latter is comprised of a complex mixture of different tissue types including blood (30–50%), degraded endometrial tissue and epithelial cells shed at the vaginal lumen (Lindenbergh et al., 2012; Zhao et al., 2017).
Our results supported the fact that the matrix metalloproteinases (MMP7 and MMP11) are involved in the cleavage of extracellular matrix proteins and in tissue remodeling. Thus, in the endometrium, they function to break down the extracellular matrix of the endometrial stroma (Farage & Maibach, 2016; Jakubowska et al., 2014).
Similar to other studies, MPP7&11 were detected in all menstrual blood samples with absolute absence from peripheral blood samples. However, this finding was in contrast to the study conducted by Roeder et al. (2013), who showed poor specificity of MMP7 and MMP11 for differentiating menstrual from blood samples. This difference can be attributed to the difference in RNA amount added to their reactions; nonspecific signals might have occurred as a result of too much RNA with subsequent fallacies. Such findings raise concerns about the importance of standardization of RNA amount involved in the corresponding reactions (Jakubowska et al.,2014).
In the current work, the difference in the mean levels of HBA in both peripheral and menstrual blood samples was extremely significant with higher mean value among peripheral blood samples. This could be related to insufficient quantity and quality of blood obtained from menstrual samples by that menstrual blood samples lacked blood in both quantity. Only 30% to 50% of samples are blood with high percentage of mRNA degradation, therefore, it is not to be expected that menstrual blood will show the same expression levels as peripheral one (Hass et al., 2009; song et al.,2015). In the same context, comparison between HBA mean values exhibited a significant descending pattern through 1st 5 days of menses (P = .0159). Variation in gene expression expected over the reported days of menstruation, especially towards the end of menstruation, as menstrual blood flow can vary significantly may be the factor that influenced such pattern (Hass et al., 2014).
In terms of metalloproteinases, the mean levels of MMP11 in menstrual blood had rising pattern reaching its peak on the 2nd day (0.94) and 3rd day (0.93) of menses then declined to its lowest values on 4th and 5th days, however this difference was not statistically significant. Although these results were consistent with previous work of Bauer and Patzelt, (2008) who reported reduced expression of MMP11 on day one (62%) compared to on days 2 and 3 (90–94%), however Hass and his colleagues (2014) could not confirm that, which might be due to the time point of sample taking and inter-individual differences.
In the same setting, expression of MMP7 was declined throughout the days of menses with the highest mean value was in the third day and the lowest mean value was in the fifth day, with no significant difference. It was in accordance with other studies which evaluated the variation in marker detection amongst the samples from day one to day four of menstruation and concluded that the expression of menstrual blood markers varies depending on the day that sampling has occurred (Hass et al., 2014; Patel and Peel 2008).
Based on their mean values, MMP7 and MMP 11 showed no significant difference throughout days of menses from day one to day five. This is consistent with other studies where MMP7 and MMP11 appeared equally useful in the determination of menstrual secretion although MMP7 seems to be more sensitive; as higher signals were obtained when compared to MMP11 throughout menstrual days (Hass et al., 2009; Song et al., 2015; Lindenbergh et al., 2012; Xu et al., 2014).
A former study conducted by Bauer and Patzelt (2008) employing Real Time PCR revealed that MMP7 expression is a strong evidence for the menstrual sample. Their findings were inconsistent with the results generated in our study wherein MMP11 expression was found to be more constant and stronger than MMP7 expression during menstruation. Similarly, Hass and his colleagues found the MMP7 marker to be more suitable than the MMP11 marker in delimiting the menstrual period (days 1–4), since some vaginal secretion samples (days 6–28) revealed high MMP11 expression (Hass et al., 2009).
This highlights the truth that if a forensic sample is recovered from the first day of menses where endometrial cells are completely or partially absent and mRNA from the MMP11 gene are still in low levels which cannot be utilized for forensic analysis (Bauer & Patzelt 2002). MMP7 is thought to be more relevant as a marker for mRNA amplification because it can be amplified by the use of easily acquired commercially produced testing kits for immediate use in a forensic laboratory. MMP7 has smaller amplicon size (101 base pairs bp) in comparison to MMP11 (455 bp) that makes MMP11 a less valuable marker for menstrual blood identification in a forensic laboratory (Counsil and McKillip, 2010). Moreover, despite the successful reduction of MMP11 amplicon size down to 200 bp in some studies it is still much larger than mRNA amplified from the MMP7 menstrual blood marker (Ferri et al., 2004).