Human Reproduction vol.7 no.7 pp.915-917. 1992

SHORT COMMUNICATION

Biochemical analysis of cervical mucus by nuclear magnetic resonance spectroscopy*

Gerhard Rohr2, Waltraud Eggert-Kruse1'4, Anja Pehlke, Ulrike Sahrbacher, Benno Runnebaum1 and Hans Robert Kalbitzer3 'Department of Gynaecological Endocrinology and Fertility Disorders, Women's Hospital, University of Heidelberg, VoSstr. 9, 6900 Heidelberg, 2Department of Internal Medicine IV, Mannheim, University of Heidelberg and 3Max-Planck-Institute for Medical Research, Heidelberg, FRG 4

To whom correspondence should be addressed

Biochemical evaluation of cervical mucus is difficult due to the characteristic rheological properties of this hydrogel. The application of high resolution nuclear magnetic resonance spectroscopy proved to be a valuable new method for differentiated biochemical analyses of human cervical mucus. A particular advantage is that it is non-destructive, that it can be applied to specimens of small volume and that no sample preparation, such as solubilization, is necessary. Key words: cervical mucus/nuclear magnetic resonance spectroscopy/cervical factor/sperm—mucus interaction

Introduction Cervical mucus is a multifactorially determined selection system regulating sperm transport to the site of fertilization and functions as a barrier protecting the upper female genital tract. Biochemical analysis of cervical mucus is difficult due to small amounts of material, heterogeneous composition, cyclical alterations of rheological properties, and its specific hydrogel-structure. Liquefaction, which is needed before conventional biochemical evaluation, possibly leads to artefacts (Schumacher, 1973a,b; Moghisshi, 1979; Ingerslev and Poulsen, 1980). In the sixties, Odeblad started to use the possibility of 'micro' nuclear magnetic resonance (NMR) to establish the 'classical' model of cervical mucus structure (Odeblad, 1966, 1968). Since that time, the methodology of high-resolution NMR spectroscopy has improved dramatically (see e.g. Wiithrich, 1986; Hausser and Kalbitzer, 1991). In the pilot study described here, NMR spectroscopy was evaluated as a method for qualitative and quantitative characterization of the properties of low- and medium-molecular weight components of human cervical mucus.

*Presented in part at the 7th Meeting of the European Society of Human Reproduction and Embryology (ESHRE), Paris, June 1991.

(£. Oxford University Press

Materials and methods Cervical mucus samples were obtained from women presenting with infertility at the Infertility Unit of the University of Heidelberg. After complete hormonal investigation as described previously (Eggert-Kruse et al., 1989), the hormonal influence on mucus properties was standardized by treatment with oestrogen. The patients were seen between the ninth and fourteenth day of the menstrual cycle after 5 days of sexual abstinence and after oral administration of ethinyl oestradiol (80 /tg/day) for at least 7 days before evaluation of cervical mucus. Any medication with a potentially negative effect on the rheological properties of the mucus, e.g. clomiphene citrate, was stopped one cycle previously. The cervix was exposed with a sterile speculum, cleaned of excess debris with a large cotton swab, and cervical mucus was carefully obtained from the endocervix by means of a special device (Aspiglaire® , IMV, L'Aigle, France). Samples contaminated with blood or with vaginal secretions were discharged. The microbiaJ colonization of the cervix was examined as described in detail previously (Eggert-Kruse et al., 1987, 1992) and the pH of the cervical mucus was controlled (Eggert-Kruse et al., 1990). All patients were free of symptoms of genital tract infection. For biochemical analysis with NMR, cervical mucus was used either fresh, directly after it had been obtained from the endocervix, or after freezing and storage in sterile plastic tubes at —20°C. Several approaches were made for NMR evaluation of cervical mucus: spectroscopy was performed on native endocervical mucus without pretreatment (only 50 /tl D2O added) or on mucus which was lyophilized and redissolved in D2O. Prepared mucus specimens were transferred to 5 mm NMR sample tubes (Wilmad, Buena, USA). One-(ID) and two(2D) dimensional 'H-NMR spectra were recorded with a Bruker AM-500 spectrometer operating at 500 MHz with a magnetic field of 11.4 Tesla (Bruker Comp., Rheinstetten, FRG). Chemical shifts were referred to trimethyl-silyl-perdeuteropropionic acid (TSP) used as internal standard. The water signal was suppressed by selective presaturation for 1 s. Typical recording times for ID spectra were 10 min, for 2D spectra 12 h. Results A typical one-dimensional NMR spectrum of endocervical mucus (obtained after treatment with oral oestrogens) is shown in Figure 1. Since water (H2O) represents > 95% of the midcycle cervical mucus content, its 'H-NMR signal is about 105 times more

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dimensional NMR spectroscopy which allows identification of complete spin systems. As an example a TOCSY (total correlation spectroscopy) spectrum of the same sample is depicted in Figure lb. Although the primary assignment (which needs to be done only once) is best performed on a sample of mucus dissolved in D2O, it is also possible to record high-quality spectra of untreated mucus (Figure 2). In this spectrum, two additional signals can be observed which correspond to the CH2- and CH3-protons of ethanol.

intense than the signals of the dissolved substances. The water signal is usually attenuated by selective presaturation. The highest spectral quality can be obtained by replacing H2O with D2O as was done with the sample in Figure 1. A large number of 'H-NMR signals can be resolved in the spectrum which correspond to low-molecular weight constituents in the mucus. Some of these signals could be assigned and are indicated in Figure 1. The primary assignment was done from the chemical shifts and multiplet patterns and supported by two-

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Fig. 1. 'H-NMR spectra of human cervical mucus in D2O. (a) One-dimensional spectrum: lower trace complete spectrum; upper trace enlarged sections of the same spectrum, (b) TOCSY spectrum of the same sample. The resonances of phenylalanine (Phe), tyrosine (Tyr), glucose (Glc), lactate (Lac), creatinine (Crea), glycine (Gly), choline (Cho), taurine (Tau), citrate (Cit), succinate (Sue), acetate (Ac), alanine (Ala), valine (Val), leucine (Leu), proline (Pro), serine (Ser), tryptophan (Trp), and of a small tyrosine-containing peptide (Tyr-Pep) are labelled.

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Fig. 2. 'H-NMR spectrum of fresh, untreated cervical mucus The signals of ethanol (Eth) which is present in this mucus specimen, are labelled.

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NMR evaluation of cervical mucus

Discussion The cervix is the main barrier regulating sperm transport to the upper female genital tract. Therefore, cervical mucus occupies a key position in human fertility. A comprehensive biochemical evaluation with special regard to influencing factors is needed further to clarify the clinical significance of the 'cervical factor' in infertility investigation and treatment and to examine the mechanisms involved in sperm —mucus interaction. The hydrogel-structure of cervical mucus makes an analysis by conventional biochemical methods difficult and time-consuming for sample preparation, sometimes impossible. Previous reports concerning the composition of cervical secretions and its cyclical alterations have been obtained after solubilization of this viscous material using different approaches: mostly after pretreatment with proteinases, centrifugation or ultrafiltration (Schumacher, 1973a,b; Wolf, 1977a,b; Moghissi, 1979; Ingerslev and Poulsen, 1980; van Kooij et al., 1983; Jager et al., 1984). However, the chemical alterations caused by the sample preparation might lead to artefacts. Another problem is the small amount of endocervical secretions that are available even in the periovulatory period, thus not allowing multiple biochemical analyses to be performed in parallel. High-resolution NMR spectroscopy offers the possibility of a comprehensive evaluation of smaller molecules of the mucus. It can be applied to specimen volumes ^500 /*1. A particular advantage in analysing such viscous material as cervical mucus, compared to conventional methods, is that samples can be used in their native state and that no solubilization is necessary. Furthermore, the results of the present study demonstrate, that with this modern technology, a more differentiated analysis could be performed than with conventional methods, including highperformance liquid chromatography (HPLC) (Gladdines et al., 1988). NMR spectroscopy also permits all (expected and unexpected) constituents containing a certain nucleus (e.g. 'H, 13C, 3IP) to be observed in a single spectrum, not only just the substance one is looking for. An example is seen in Figure 2, where the signals of ethanol are visible, a constituent which was not expected before. An additional advantage of NMR spectroscopy is that it gives direct information about the chemical structure of the compounds observed since multiplet patterns in ID spectroscopy and coupling patterns in 2D spectroscopy are closely related to the chemical structure. The evaluation of subtle changes in mucus composition in certain cases of infertility under hormonal treatment, the impact of different solubilization procedures before laboratory investigations, as well as the influence of certain microorganisms colonizing the cervical os on the mucus quality, is currently being investigated in our laboratory by means of highresolution NMR spectroscopy. A comprehensive biochemical analysis of cervical mucus under different conditions will render more insights into the aetiology of cervical factor infertility and, on the other hand, might have implications for the development of new cervical contraceptives.

sperm-mucus interaction in vivo and in vitro. Hum. Reprod., 2, 301-308. Eggert-Kruse,W., Leinhos,G., Gerhard.I., Tilgen.W. and Runnebaum,B. (1989) Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus. Fertil. Steril, 51, 317-323. Eggert-Kruse,W., Kohler.A., Schwalbach.B., Gerhard.I. and Runnebaum,B. (1990) Influence of endocervical pH on sperm—mucus interaction. VII World Congress on Human Reproduction. Helsinki, June 1990. Eggert-Kruse,W., Pohl,S., Naher,H., Tilgen,W. and Runnebaum,B. (1992) Microbial colonization and sperm—mucus interaction: results in 1000 infertile couples. Hum. Reprod., 7, 612-620. Gladdines,M.M., Ackermans,M.T. Everaerts,F.M., Linden van der,P.J.O., Vader.H.L. and Wiegerinck.M.A.H.M. (1988) Analysis of the aqueous phase of human cervical mucus by reversed-phase highperformance liquid chromatography and capillary isotachophoresis. J. Chromatogr., 431, 317-326. Hausser,K.H. and Kalbitzer,H.R. (1991) NMR in Medicine and Biology. Structure Determination, Tomography, In-Vivo Spectroscopy. Springer, Heidelberg. Ingerslev,H.J. and Poulsen,F. (1980) Bromelin for liquefaction of cervical mucus in sperm antibody testing: its effect on spermagglutinating immunoglobulin G. Fertil. Steril., 33, 61—63. Jager.S., KremerJ. and Wilde-Janssen de,I.W. (1984) Are sperm immobilizing antibodies in cervical mucus an explanation for poor postcoital test? Am. J. Reprod. Immunol., 5, 56—60. Kooij van,R.J., Kathman.G.A.M. and Kramer.M.E. (1983) Secretory piece and plasma proteins in human cervical mucus during the cycle. J. Reprod. Fertil., 68, 63-68. Moghissi,K.S. (1979) The cervix in infertility. Clin. Obstet. Gynecol., 22, 27-42. Odeblad,E. (1966) Investigations on the secretions from single glandular units in the human uterine cervix. Acta Obstet. Gynecol. Scand. (Suppl. 2), 55, 139-154. Odeblad,E. (1968) The functional structure of human cervical mucus. Acta Obstet. Gynecol. Scand. (Suppl. I), 47, 57-78. Schumacher,G.F.B. (1973a) Soluble proteins of human cervical mucus. In Elstein,M., Moghissi,K.S. and Borth,R. (eds), Cervical Mucus in Human Reproduction. Scriptor, Copenhagen, pp. 93-112. Schumacher,G.F.B. (1973b) Soluble proteins in cervical mucus. In Blandau.R.J. and Moghissi,K. (eds). The Biology of the Cervix. University of Chicago Press, Chicago, IL, pp. 201—210. Wolf.D.P., Blasco,L., Khan,M.A. and Litt,M. (1977a) Human cervical mucus. I. Rheologic characteristics. Fertil. Steril., 28, 41—46. Wolf.D.P., Blasco,L., Khan.M.A. and Litt.M. (1977b) Human cervical mucus. II. Changes in viscoelasticity during the ovulatory menstrual cycle. Fertil. Steril., 28, 47-52. Wuthrich,K. (1986) NMR of Proteins and Nucleic Acids. Wiley, New York. Received on March 23, 1992; accepted on April 24, 1992

References Eggert-Kruse,W., Gerhard,I., Hofmann,H., Runnebaum.B. and Petzoldt,D. (1987) Influence of microbial colonization on

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Biochemical analysis of cervical mucus by nuclear magnetic resonance spectroscopy.

Biochemical evaluation of cervical mucus is difficult due to the characteristic rheological properties of this hydrogel. The application of high resol...
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