Daily Saliva and Vaginal Mucins Co-vary in Protein-to-Carbohydrate Ratio

The currently reported study employed the curiously named (but simple) quantitative, nondestructive technique of Multiple Attenuated Internal Reflection InfraRed (MAIR-IR) spectroscopy to assess daily variations of both saliva and vaginal secretions from a panel of consenting volunteers. The project goal was to determine whether the protein-to-carbohydrate (starch) ratio, determined by MAIR-IR analysis of salivary and vaginal secretions is an objective measure of the co-variation of mucinous secretions with daily menstrual cycle. Overall, the spectral data were found to provide valid signatures of vaginal mucus chemical changes that also correlated with cyclic periods of presumed fertility, such periods exhibiting repeat times of 14-16 days and 27-29 days in the thoroughly analyzed records. At mid-cycle, associated with ovulatory events, the protein-to-carbohydrate ratio of vaginal mucin maximizes. Higher carbohydrate ratios are associated with stiffer cervical mucus plug formation, which usually prevents infection, spontaneous abortion, and pre-term birth, but may also be responsible for infertility. Mucin relaxation compounds, such as the FDA-approved oral rinse, delmopinol, may induce fertility in such cases. DOI : 10.14302/issn.2576-2818.jfb-18-2298 Corresponding author: Anne E. Meyer, PhD, Research Associate Professor, Department of Oral Diagnostic Sciences, State University of New York at Buffalo, Buffalo, NY 14214, 716-829-6244, aemeyer@buffalo.edu Running Title: Mucin Variations Reflect Fertility


Introduction
Self-reports of menstrual cycle symptoms have long been criticized, calling for more objective measures that could correlate better with physiological events. 1 It is obviously important that, if cervical mucus specimens are to be the criteria objectively evaluated, there must be a proof of their stability while introduced to the analytical methods. 2 In the mucin glycoproteins, poly-anionic groups (mainly sialic acid) are carried on oligosaccharides (short starch segments) of about 9-10 monosaccharide lengths, but the chemical and serological differences in oligosaccharide structure were not previously reported to have any biological function. 2 This generally pessimistic view is challenged by a plethora of prior studies of salivary mucins, very similar to cervical glycoproteins, that do illustrate structural and functional changes (some of which co-vary with menstrual cycle days) with modified compositions. 3,4,5,6,7,8,9,10,11,12,13 Although carefully controlled saliva sampling regimens do demonstrate this cyclic variation, normal oral activities, eating, and drinking can obscure the same correlation that may be demonstrated more reliably by vaginal mucin smears.
Such cyclical changes may serve as indicators of both shorter and longer term fluctuations in states of human fertility, health, and disease.

Experimental Procedure
This report describes investigative research into cyclical changes that occur in protein/starch (P/S) ratios of intraoral glycoproteins and vaginal mucins among humans, as characterized by Multiple Attenuated Internal Reflection InfraRed (MAIR-IR 14 ) spectroscopy.
In addition to observing P/S relationships over several menstrual cycles, the investigation repeated examination of ratios within 24-hour time-spans to determine whether the variance in sampling times altered the sample results and to identify the amount of change that normally occurs in the P/S ratio on a daily basis.
To obtain a broad sample group for the study, 15  Prior to participation in the study, each volunteer was advised of the nature of the project, its scope, the sampling techniques to be used and the confidentiality of the data to be provided, in accordance with directives from the approving institutional Human Subjects Committee. It was also determined that the sterile cotton swabs for taking the samples would not pose any significant health hazard to the project participants. All participants were required to sign waiver statements and consent forms and were assigned participant numbers to preserve the confidentiality of all collected data.

Materials and Methods
Each of the participants was given a supply of sterile, culture collection swabs. Each mucin sample was taken by inserting a just-opened sterile swab directly into the vagina or the mouth. After removal from the body cavity, the swab was returned to its original sterile tube and a sample label was prepared. The label identified the participant's number, as well as the date and time at which the sample was taken. also filled in on the corresponding spectrometer graph and the graph was included in a notebook file maintained for each participant. P/S ratios were determined from the IR spectra of the samples and were plotted as points on graphs representing from one to six months of sampling. Numerous spectral analytical procedures were then applied to seek periodicities.
To measure each specimen's P/S ratio, the printout of the sample spectrum was compared to the baseline spectrum of the prism, as a percentage IR transmission value, at both the starch and protein peaks. An example of a sample spectrum is given in Figure 1. The protein/baseline % transmission was then divided by or subtracted from the starch/baseline percent transmission, result in P/S or P-S ratios. Two independent assays of each specimen were recorded and the average of the replicate ratios was then plotted for each day.
Calibration procedures were carefully applied.
Throughout this investigation, several control experiments were set up to help identify the parameters of the study. Each experiment was intended to control one aspect of the study. Baseline measurements also were taken on a daily basis by analyzing the clean germanium prisms in the spectrometers prior to any sample testing.

Results
Over 2000 individual spectral records were obtained for mucin samples from approximately 20    It was also of concern, recognizing the general availability of only one specimen per subject per day, to

Discussion
It has long been suspected that salivary mucin properties, such as viscosity, 17 reflect the time of ovulation, but this (like the relative clarity or opacity of the actual cervical mucus) generally has had no prognostic significance. Still, it has long been considered probable that quantitative or qualitative alterations in mucin glycoproteins are under hormonal control, 18 and also probably involved in sperm capacitation. 19,20,21 The study reported here demonstrated that the