The field of mucosal immunology research has expanded significantly over the years and understanding biomarkers as correlates of risk for adverse sexual and reproductive health remains an important focus of research.

Genital inflammation is one of the biological correlates of HIV risk (Masson et al., 2015a) and may also be a biomarker for the risk of preterm birth (Park et al., 2020). Studies have demonstrated that high levels of pro-inflammatory cytokines were associated with intra-amniotic infection that causes preterm births (Son et al., 2016; Amabebe et al., 2018). Additionally, genital inflammation has been strongly correlated with bacterial vaginosis (BV), and cervical cancer (Masson et al., 2015b; Zhou et al., 2021; Mtshali et al., 2021; Ntuli et al., 2022). Studies have repeatedly shown increased risk for HIV acquisition in women with genital inflammation, likely mediated through immune activation and recruitment of susceptible target immune cells to the genital tract or disruption of the mucosal barrier (Masson et al., 2015b). Furthermore, genital inflammation was shown to undermine the efficacy of topical 1% tenofovir gel used as pre-exposure prophylaxis (PrEP) for HIV prevention in women in the CAPRISA004 trial (McKinnon et al., 2018).

The majority of immunological research has focused on quantifying cytokines and chemokines due to the central role genital inflammation plays in adverse sexual and reproductive health outcomes (Masson et al., 2015b). However, the quantification of cytokines and chemokines in vaginal samples may be influenced by various factors including the method of mucosal sampling, the order of sampling, and heterogeneity in the quality of samples obtained (due to participant-specific factors like mucus and the presence of blood or seminal fluids) and laboratory processing (Delany et al., 2008; Dezzutti et al., 2011; Archary et al., 2015; Jaumdally et al., 2018). These factors may affect the sensitivity and accuracy of measuring biomarkers, and may further affect the interpretation and comparison of data for assessing the milieu in the cervicovaginal environment. Conventional methods used to collect genital specimens from the female genital tract (FGT) include ophthalmic sponges, cervicovaginal lavage (CVL), and cervicovaginal swabs (Delany et al., 2008; John et al., 2001; Rohan et al., 2000; Boskey et al., 2003). Although the CVL collection method has the advantage of a larger sample volume than other methods. Some studies have shown that the dilution of sponges and swabs alike affects the data obtained. Sponges reconstituted in smaller volumes of phosphate-buffered saline for eg., yield higher concentrations of soluble proteins like cytokines compared to more diluted CVL specimens (Boskey et al., 2003; Quesnel et al., 1997; Snowhite et al., 2002; Belec et al., 1995). Similar to CVL, the swab as a sampling method for the genital specimen is also not standardized. Factors contributing to difficulty in comparing data across methods include varied volumes and sample diluents used in upstream collection or downstream processing (Jaumdally et al., 2018; Boskey et al., 2003; Fichorova et al., 2008; McKinnon et al., 2014; Mngomezulu et al., 2021).

The continued use of these sampling methods highlights a need to search for an optimal sampling method that will provide advantages for detecting biological markers in women, even in very low concentrations. This study compared Softcup® menstrual cup sampling to the vulvovaginal swab sampling method for the detection of cytokines in genital samples of HIV-uninfected pregnant women. We find that Softcup® or menstrual cup, represents a sampling technology for the genital tract that provides consistent and reliable detection of cytokines, even for some of the least concentrated cytokines compared to other methods (Jaumdally et al., 2018).