Year : 2010 | Volume
: 21 | Issue : 2 | Page : 165--168
Biochemical evaluation in human saliva with special reference to ovulation detection
S Alagendran1, G Archunan2, S Velayutha Prabhu2, B Enrique-A Orozco3, Rosalinda Guevara Guzman3,
1 Department of Animal Science, Bharathidasan University, Tiruchirappalli - 24; Department of Biotechnology, Nehru Memorial College (Autonomous), Puthanampatti, Tiruchirappalli, India; Departamento de Fisiología,Sensorial Fisiología Laboratorio, Faculty of Medicine UNAM, Ave. Universidad C.P - 04510, México D.F
2 Department of Animal Science, Bharathidasan University, Tiruchirappalli - 24, India
3 Departamento de Fisiología,Sensorial Fisiología Laboratorio, Faculty of Medicine UNAM, Ave. Universidad C.P - 04510, México D.F
Department of Animal Science, Bharathidasan University, Tiruchirappalli - 24, India
Aim : The aim of the present study was to investigate the level of salivary sialic acids and glycosaminoglycans with reference to salivary hormones during the normal menstrual cycle.
Settings and Design: Fifty women volunteers were selected for the present study.
Materials and Methods : Saliva was collected from 50 women and ovulation was detected in women with normal menstrual cycles through basal body temperature (BBT), ultrasound and salivary ferning. Samples were divided into five categories, as prepubertal (6-9 years), pre-ovulatory phase (6-12 days), ovulatory phase (13-14 days), postovulatory phase (15-26 days) and menopause (above 45 years). Each sample was subjected to evaluation of the sialic acids and glycosaminoglycans along with salivary hormones.
Results : The result revealed that the ovulatory phase has increased sialic acid and glycosaminoglycans during the menstrual cycle when compared with that of the other phases. Consequently, an increased level of hormones such as luteinizing hormone and estrogen during the ovulatory period when compared to that of the pre-ovulatory and postovulatory periods appeared to be noteworthy. Statistically, analysis was performed using one way-ANOVA (LSD; post hoc method) to determine the significance as P < 0.001, 0.01, 0.05 in between the reproductive phases of the menstrual cycle.
Conclusion : This study concluded that saliva-specific carbohydrates in the ovulatory saliva make the possibility to develop a biomarker for detection of ovulation by non-invasive methods.
|How to cite this article:|
Alagendran S, Archunan G, Prabhu S V, Orozco B E, Guzman RG. Biochemical evaluation in human saliva with special reference to ovulation detection.Indian J Dent Res 2010;21:165-168
|How to cite this URL:|
Alagendran S, Archunan G, Prabhu S V, Orozco B E, Guzman RG. Biochemical evaluation in human saliva with special reference to ovulation detection. Indian J Dent Res [serial online] 2010 [cited 2021 May 18 ];21:165-168
Available from: https://www.ijdr.in/text.asp?2010/21/2/165/66625
The cyclic physiologic changes are mainly brought about by the ovarian hormones estrogen and progesterone, the levels of which show variation during the menstrual cycle. Identification of the period of ovulation in humans is critical in the treatment of infertility. Success in in vitro fertilization and embryo transfer has been associated with the exact time of ovulation. In the recent years, attention has been paid to the noninvasive method in ovulation detection. Recent reports shows that the saliva is a very good source of both hormones and biochemicals and that their levels change in accordance with the menstrual cycle. Carbohydrates are the major diet for many mammalian species. Galef  and Rameshkumar  reported that the nature of the feeding habit would have a major impact on the excretion of biomolecules. This may be the reason for a considerable release of carbohydrates in the saliva. As ovulation approaches, the high level of circulating estrogens stimulates the breakdown of glycogen and other materials into glucose  in mammals. This glucose will be utilized for energy release in the cell. The metabolic process is broadly concerned with three major classes of macromolecules, i.e. protein, carbohydrates and lipids. All the three biologically originated supramolecules are very much essential for the energy production growth and maintain a normal physiological and biochemical status in each and every cell of an organ and organisms.
The present study was performed to evaluate the changes in salivary sialic acid and glycosaminoglycans (GAGs) in the regular menstrual cycle. Thus, the presence of saliva-specific carbohydrates in the ovulatory saliva makes the possibility to develop a biomarker for the detection of ovulation by noninvasive methods.
Materials and Methods
Collection of samples
The saliva of women was collected as per the spitting method , and the sample was processed in preweighed ice-chilled tubes, the collection period being about 10 min. The saliva was collected from 50 different women volunteers during various periods, viz. pre-ovulatory (6-12 days; n = 10), ovulatory (13-14 days; n = 10), postovulatory phases (15-26 days; n = 5) and also from pre-pubertal (7-9 years; n = 10) and menopause stages (above 45 years; n = 15). The ovulatory phase was confirmed by ferrning pattern in saliva.  Collection of saliva using cotton or a polyester roll (Salivette, Sarstedt) may seem attractive in some instances because of convenience and ease of use. The volunteers were instructed to refrain from smoking and drinking during the study period. Saliva samples for steroid measurement were stored for up to -20C. Sample collection from women volunteers and the procedure adapted in the present study were approved by the ethical committee of the Bharathidasan University, Trichy.
Salivary GAG was determined as described elsewhere.  The resulting clear blue solution was measured in 1-cm microcuvettes with a UV-spectrophotometer at 620 nm. The glycosaminoglycan contents of the saliva samples were determined by reference to a calibration curve constructed using chondroitin-4 sulfate as standard. The results were conveniently expressed as mg/ml of saliva.
Thiobarbituric acid assay of sialic acids was assed by the method of Warren.  Optical densities of the organic phase were determined at 549 nm. The units were measured in mg/ml.
The sample was extracted with sodium azide to keep it stable for longer than a week at room temperature. Samples were ice covered soon after collection so as to break down the mucopolysaccharides. Samples (1 ml) were centrifuged at 4,000 rpm for 10 min at 4oC. The clear supernatant was concentrated and used for determination of estrogen , and progesterone,  analyzed using a radioimmunoassay kit purchased from Diagnostics Systems Laboratories, Sinsheim, Germany. The sensitivities of luteinizing hormone (LH), estrogen and progesterone were 14 mIU/L, 1-4 pg/ml and 0.7-1.8 ng/ml, respectively. The intraassay variations for LH, estrogen and progesterone were 4.5 mIU/L, 5.0 ng/ml and 1.6 pg/ml, respectively. All samples were run in one assay to avoid interassay variation.
Data are mean ± standard error of the mean (SEM) of six replicates. Data are analyzed by one-way analysis of variance (ANOVA) using SPSS Software package version 5.0 (SPSS Inc., Chicago, IL, USA). A probability of P P P<0.01 [Figure 1]a. GAGs in normal young women presented a biphasic pattern, with higher concentration values during the first half of the menstrual cycle. To ascertain whether this difference was statistically significant, the mean value of the preovulatory and postovulatory periods was recorded. The values are expressed as mean ± SEM (17.41 ± 2.63 mg/ml; 11.39 ± 1.74 mg/ml). Comparison of these two mean values showed that average salivary GAG concentration values might not have been valid for all baseline values. Furthermore, the GAG values showed significant variation in the ovulatory phase when compared to that of the other two phases [Figure 1]b. In the prepubertal and menopausal phases, the average of GAG and sialic acid was decreased when compared to that of the ovulatory phase.
However, sialic acid showed the maximum significant variation during the ovulatory phase (42.15 ± 2.72 mg/ml) as compared to that of the preovulatory (10.94 ± 1.17 mg/ml) and postovulatory (8.66 ± 1.25 mg/ml) phases. In the prepubertal and menopausal phases, the values were not significant. The mean concentration of LH increases remarkably during the onset of the ovulatory phase; however, estrogen increase was noticed in the pre-ovulatory phase. The increase of progesterone levels was noticed from pre-ovulatory to postovulatory phases. The comparison between sialic acid and GAGs [Figure 1]a and b oscillates in the human saliva, and may be due to the circulation of ovarian hormones.
GAG content showed a characteristic pattern of fluctuation during the normal menstrual cycle with a distinct peak at ovulation. This peak of maximal GAG concentration (33.43 ± 3.85mg/ml in saliva) was centered according to the day of the midcycle LH surge, which served as the reference point, designated day 0. All cycles are presumed to be ovulatory on the basis of salivary ferning, ultrasound screening and ovarian hormone assays in relation to ovulatory periods. Previous reports, including data on salivary GAG content during the normal menstrual cycle, differ appreciably with regards to their results. For example, Erickson et al. did not find consistent variations during the normal menstrual cycle. In contrast, significant variation in the urinary GAG concentration during ovulation was reported by Carranco et al.,  who suggested that this could be an efficient method for determining this event. However, in the later report, a dye-binding method for assaying GAG was also used. Therefore, the daily urinary or salivary GAG concentration could have been affected by factors such as water intake, environmental temperature and dehydration.
Carbohydrates are the major diet for many mammalian species. Galef  and Rameshkumar  reported that the nature of feeding habit would have a major impact on the excretion of biomolecules. This may be the reason for a considerable release of carbohydrates in the saliva. For example, alteration in diet changes the urinary odors of guinea pig  and mice.  A minor component of the urinary protein complex of the house mouse revealed a glycoprotein containing N-linked oligosaccharide;  however, its importance in chemosignaling is not yet known. Further, the changes in carbohydrate content during the reproductive cycle are probably due to the changes in the hormonal level. As ovulation approaches, the high level of circulating estrogens stimulates the breakdown of glycogen and other materials into glucose  in mammals. This glucose will be utilized for energy release in the cell.
The present study reveals a significant increase in salivary glycosaminoglycan and sialic acid in the first half of the cycle, which paralleled the normal increases in serum estrogen levels that occurs at this phase. In general, estrogen inhibits the synthesis of extracellular matrix molecules by many mesenchymal cell types, such as vascular smooth muscle cells.  Such inhibition would shift the normal proteoglycan turnover toward degradation, which could explain the increase in GAG salivary secretion that we found in the first half of the cycle. This variation implies modulation by estrogens and, consequently, it should be considered when comparing the GAG concentration in saliva samples from women of child-bearing age. Nevertheless, the available evidence is strongly suggestive that GAG determination in saliva is a reliable method for ovulation detection.
Decrease in sialic acid concentration during the pre-ovulatory phase has been observed both in human cervical mucus  and human whole saliva.  The present results show remarkable cyclic variation usually with the high concentration around the ovulatory period (sialic acid, 42.15 ± 2.72 mg/ml). The present result suggests that there is a remarkable cyclic variation in many salivary components during the menstrual cycle. The gonadal hormones not only play a role in excretion of biomolecules but also play a significant role in the behavior of women. The findings from the present study are consistent with the previous findings that a significant increase in circulating estradiol concentrations occurs ~1 week before the LH surge.  The increase is associated with the appearance of the dominant follicle, the production of estradiol is correlated with follicular surface area and estrogen-rich follicles are the source of ova most likely to undergo successful fertilization and ongoing pregnancy. This fact is of great importance because saliva samples are often used as a diagnostic aid in the treatment of oral diseases. This kind of cyclic variation largely prevents the listing of the so-called normal values of many salivary components. Subsequently, it seems to be a hindrance to estimate ovulation time from saliva samples. Data of the present study provide a noninvasive method for determining the stages of the menstrual cycle through chemical profiles. These profiles may be considered to develop salivary biomarkers for the prediction of ovulation in women.
The result of the present study seems to confirm that self-determination of salivary LH levels is a reliable way to determine ovulation. These methods, in association with other biochemical indices such as GAGs and sialic acid, minimize the invasive method of ovulation detection. Furthermore, although variation in saliva ferning characteristics and BBT has a certain degree of coincidence with ovulation, the length of the fertile period is overestimated with these methods. Saliva ferning test and carbohydrates, particularly sialic acids and GAG, was probably brought under the influence of cyclic variation of ovarian hormones. The present results showed that there are remarkable cyclic variations in GAGs during the menstrual cycle. In conclusion, GAGs and sialic acid may be used as a biomarker for the prediction of ovulation to overcome the other invasive method.
The study was partially supported by a grant from UGC-SAP, DST-FIST, New Delhi and Dr. SA was awarded by DGAPA Postdoctoral Research associate in Sensorial fisiologνa laboratorio. Faculty of Medicine UNAM, Ave. Universidad C.P 04510, Mιxico D.F has kindly acknowledged.
|1||Galef BG Jr, Smith MA. Susceptibility of artificially reared rat pups to social influences on food choice. Dev Psychobiol 1994;27:85-92.|
|2||Rameshkumar K. Chemical characterization of Bovine (Bos Taurus) Urine with special reference to reproductive behavior. Ph.D Thesis. Tiruchirappalli, Tamil Nadu, India: Bharathidasan Uzniversity; 2000.|
|3||Ma W, Clement BA, Klemm WR. Cyclic changes of volatile constituents of bovine vaginal secretions. J Chem Ecol 1995;21:1895-906.|
|4||Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci 1993;20:72-7.|
|5||Bosch JA, Brand HS, Ligtenberg TJ, Bermond B, Hoogstraten J, Nieuw Amerongen AV. Psychological stress as a determinant of protein levels and salivary-induced aggregation of Streptococcus gordonii in human whole saliva. Psychosom Med 1996;58:374-82.|
|6||S Alagendran, G Archunan, S Achiraman. Prediction of ovulation in women through the occurrence of salivary fern prototype. Icfai J Life Sci 2007;1:7-15.|
|7||Whiteman P. The quantitative determination of glycosaminoglycans in urine with Alcian Blue 8GX. Biochem J 1973;131:351-7.|
|8||Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959;234:1971-5.|
|9||Lemay A, Bastide A, Lambert R, Rioux JE. Prediction of human ovulation by rapid luteinizing hormone (LH) radioimmunoassay and ovarian ultrasonography. Fertil Steril 1982;38:194-201.|
|10||Worthman CM, Stallings JF, Hofman LF. Sensitive salivary estradiol assay for monitoring ovarian function. Clin Chem 1990;36:1769-73.|
|11||Bolaji II, Tallon DF, O'Dwyer E, Fottrell PF. Assessment of bioavailability of oral micronized progesterone using a salivary progesterone enzyme immunoassay. Gynecol Endocrinol 1993;7:101-10.|
|12||Erickson DR, Ordille S, Martin A, Bhavanandan VP. Urinary chondroitin sulfates, heparan sulfate and total sulfated glycosaminoglycans in interstitial cystitis. J Urol 1997;157:61-4.|
|13||Carranco A, Reyes R, Huacuja L, Guzmαn A, Delgado NM. Human urinary glycosaminoglycans as accurate method for ovulation detection. Int J Fertil 1992;37:209-13.|
|14||Beauchamp GK. Diet Influences the attractiveness of urine in guinea pigs. Nature 1976;263:587-8.|
|15||Schellinck HM, Rooney E, Brown RE. Odours of individuality of germfree mice are not discriminated by rats in a habituation-dishabituation procedure. Physiol Behav 1995;57:1005-8.|
|16||Mechref Y, Zidek L, Ma W, Novotny MV. Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides. Glycobiology 2000;10:231-5.|
|17||Barchiesi F, Jackson EK, Gillespie DG, Zacharia LC, Fingerle J, Dubey RK. Methoxyestradiols mediate estradiol-induced antimitogenesis in human aortic SMCs. Hypertension 2002;39:874-9.|
|18||Carlborg L, Johansson ED, Gemzell C. Sialic acid content and sperm penetration of cervical mucus in relation to total urinary oestrogen excretion and plasma progesterone levels in ovulatory women. Acta Endocrinol (Copenh) 1969;62:721-31.|
|19||Oster G, Yang SL. Cyclic variation of sialic acid content in saliva. Am J Obstet Gynecol 1972;15:190-3.|