Over the years cervical mucus has been extracted in different ways, with glass tubes, plastic tubes, various types of forceps and swabs, etc. It was observed that large cotton swabs sucked considerable amounts of the liquid phase of mucus, including the electrolytes like NaCl which are major constituents of the crystals. However, a swab has a definite advantage over tubes and forceps because of its simplicity when handling it. Therefore swabs with successively smaller amounts of cotton were tried. They served well as adhesive material for mucus but sucked up a smaller amount of the liquid material. Finally, swabs with only one or a few layers of cotton fibres were designed, "miniswabs". It was also observed that just rolling the mini-swab on the surface of the endocervix and transferring the material in a similar way to the slide (Fig. 12A) gave rise to a "print" of the endocervical mucosa. Using a cotton coil of 6 mm length on a wooden stick, 2 mm in diameter, gave a print 6 mm by 6 mm in size. Using successive prints in known order, the whole endocervix could be "mapped".

Fig. 12. Illustrating how cervical printing with a mini-swab is made (A). The print can also be spread out over a larger surface (B) in order to obtain more detailed information.

The mapping procedure has several sources of error. A glance at Figure 1 makes it understandable that some crypts may be hidden under the folds and inaccessible to the sampling swab. Another problem is the difficulty of determining the intracervical position of the coil exactly. It is also difficult to rotate the coil without sliding it on the endocervical surface.

One of the most important sources of error is not to have a reproducible adhesion of the endocervical and crypt contents on the cotton coil. Figure 13 illustrates this problem. The efficiency of mucus recovery depends largely on the relative adhesion of mucus to the crypt and endocervical lining, to the swab and to the glass surface. If the strengths of adhesion are crypt < swab < glass, the printing is quite efficient. But if the strengths are crypt > swab > glass, very little, if any, material is transferred from the endocervix to the slide. One important step before sampling therefore is to carefully clean the swabs and glass surfaces in pure ethanol before use in order to remove hydrophobic materials, thereby increasing transfer efficiency.

Fig. 13. This drawing shows how mucus in a crypt adheres on a miniswab which is rolled over the endocervical surface. From this illustration it is evident that the capacity of the adhesion of the miniswab is important for moving the mucus.

The crypt typing is easily done from the crystal and cell patterns in various parts of the prints (Figs. 5-9). The basis for this typing is given in Table 1 in Odeblad (1994a). If performed properly, cervical printing and mapping can yield much important information, e.g. the general ordering of the various crypts in the cervix. Preliminary results indicate that the crypts are located in linear, transverse arrays, confirming in principle the results of Fluhman (1961) on the cleft-fold architecture.

The crypt sequence in these arrays most often is as follows: LLSLLSLL, i.e. S crypts mixed with L crypts. However, in a few cases of infertility with normal proportions between L and S mucus, abnormal crypt sequences have been found, e.g. LLLLLLLL in many clefts and a few clefts with SSSSSS or SSSLSSS. This may imply that the S mucus is not normally dispersed amongst the L mucus but clumped together in one or a few bulk volumes. This will probably not allow a normal sperm transport. Also the spread-out specimens gave similar indications but not so clearly visible as the prints.

In principle, the whole of the crypt system is accessible to mapping, but sometimes folds and also "grapes" may hide more or less of the assembly of crypts, so that only a few of them become visible in the prints.

It has turned out that combining the principles involved in methods (1), (2) or (3) can give useful results. Making a spread-out of a microsample helps to detect "contamination" of a crypt with mucus from another crypt. This has already been discussed. Combining spread-out and printing has shown itself to be rather fruitful (Fig. 12B). If a short cotton coil is used and the material is rapidly spread out over an area of 10 by 20 mm on a slide, the average thickness will be 5 µm for a 1 mg sample. This implies that single cells will separate from other cells and can be studied individually.

Microscopic investigations show that cells and mucus of most types and subtypes can be identified in the very thin specimens obtained with miniswabs, cotton ring swabs or spiral sewing cotton swabs (Fig. 14). The spaces between the cotton rings or spiral rings and between the wooden stick and glass surface (Fig. 14) are large enough so that the fragile cells are not crushed. The cells remain intact together with the mucus they are producing.

Fig. 14. Various types of cotton swabs are shown A, regular swab; B, miniswab; C, cotton ring swab; D, spiral sewing cotton swab; E, how material removed with a cotton ring swab is deposited on a slide (similar with a spiral swab).

Fig. 15. Top: a P_t filament about 120 µm long with mainly triagular pattems, magnified 940 x . Bottom: a P_t f'ilament about 300 µm (= 0.3 mm) long magnified 320 x .

Of all the mucus and cell types studied, the P_t cells and Pt mucus are far the most easy to identify and study. Therefore most studies have been performed on these objects. The P_t secretion is characterized by a remarkable pattern (Fig. 15) with triangular structures along a central filament. These filaments can occasionally be 0.1 mm long or even more, but are usually some 10-30 µm long, and usually the cells and their secretion must be studied at high magnification, This creates problems. Not only are the structures often at the limit of the light microscope, but also the specimens have a variable thickness, from 1 µm to ~5 µm, which makes focusing difficult. Also, no fixing, no staining and no mounting is used, because the structures are water-soluble, and no immersion optics can be applied. This explains the blurring and lack of sharpness in some of the microphotographs.

The epithelial cells are those normally shed from the cervical epithelium. These cells, however, can not be alive for any length of time. Because they have lost their supply of necessary metabolites from the circulation they all die. It is known that cell death can occur in two ways, called apoptosis and necrosis. In apoptosis, which is related to "programmed cell death", the cell nuclei become small, dense and strongly light-refracting. The cells are taken care of by macrophages, and no inflammation occurs. Necrosis may be called accidental cell death and is the result of mechanical damage, infarction, radiation or other external factors. T'he organic compounds in the cell disintegrate, leading to an increased osmotic pressure. The cell accordingly sucks in water, swells, the plasma membrane ruptures and the cell disappears. Often an inflammatory reaction is elicited.

Both of these two ways of cell devitalization can be identified in the specimens (Fig. 16), and the secretion produced by these dying cells is abnormal. Often a type of grainy secretion is present (not to be mixed up with the normal Z granules containing mucolytic enzyme). Also, sometimes we see that the dying cells produce P_y secretion.

Fig. 16. Two secreting P_t cells which will not survive. The cell to the right shows signs of apoptosis, the nucleus is small and dense and highly light-refracting. The other cell has just "exploded", and only residues remain - it has undergone a necrosis. The secretion from the apoptosic cell is mainly P_t. The necrotic cell has produced some P_t, some P_y.

The important thing is that we can apparently distinguish which cells are still working normally and which cells are in an agonal condition. In the normal cells we can clearly distinguish various phases of mucus production (Fig. 17). We can see apparently resting cells, cells in which mucus biosynthesis is going on, cells which release their P_t secretion and cells which have apparently come to rest, just having expelled their secretion. This is part of the so-called BRAMS principle for the mucus "life span" -- B for biosynthesis, R for release, A for activity, M for mucolysis and S for sensation. The BRAMS principle is described in some detail in Odeblad (1996).

Fig. 17. Four normal cells ordered in a typical working sequence. Cell A is at rest. Cell B has biosynthetic activity going on, the primary mucus being stored in round vacuoles. Cell C is in a state of release of P_t secretion. Cell D has left P_t mucus around itself and is returning to a resting condition.

Future studies may involve electron microscopy and MRNA and DNA studies on P_t cells in various phases of mucus biosynthesis and release.

As regards the P_t mucus we still do not know about its biological function. Some indications exist that it may have an immunological role. Further research is needed to clarify this. Studies on the cellular mechanisms involved in the secretion of the other mucus types may elucidate both normal and pathological mucus production and mucus symptom and in this way contribute to more extended application of the Ovulation Method in the future, for example, to women with various types of congenital and acquired diseases affecting the reproductive system.

A paragraph explaining the non-inclusion of phosphorus in Table 2 has been added immediately preceding Table 2. Some null entries in Table 2 have been replaced with a limit value. The caption to Table 2 has been edited to clarify the meaning of the entries.

The caption on Figure 15 has been corrected. The caption to Figure 16 has been edited.

Two references have been updated to reflect publication.

Fluhman, C. F. (1961). "The Cervix Uteri and Its Diseases." [Saunders Co.: Philadelphia.]

Höglund, A., and Odeblad, E. (1977). Sperm penetration in cervical mucus: a biophysical and group-theoretical approach. In "The Uterine Cervix in Reproduction." Workshop Conference in Rottach-Egern. Edited by V. lnsler and G. Bettendorf. pp. 129-34. [G. Thieme Publ. Co. : Stuttgart.]

Menarguez, M. (1998). "Caracterization Morfológica de Diversos Tipos de Moco Cervical Humano Mediante Microscopía de Luz y Microscopía Electrónica de Barrido" ["Morphological Characterization of Different Types of Human Cervical Mucus with Light Microscopy and Scanning Electron Microscopy."] Thesis, Department of Cell and Molecular Biology, Faculty of Medicine, University of Murcia, Murcia, Spain.

Odeblad, E. (1964). Secretory function of single cervical glands. Annual Meeting, Swedish Medical Association. p. 165. Summary paper, in Swedish.

Odeblad, E. (1994a). The discovery of different types of cervical mucus and the Billings Ovulation Method. Bulletin of the Natural Family Planning Council of Victoria, 21 (3), 3-15.

Odeblad, E. (1994b). The spread-out technique, advantages, pitfalls, interpretation. In IV Symp. Int. sobre Regulación Natural de la Fertilidad, October 6-8, 1994 pp. 295-303. [Colegio Oficial de Medicos, Barcelona.]

Odeblad, E. (1997). Cervical mucus and their functions. J. Irish Colleges of Physicians & Surgeons, 26 (1), 27-32.

Odeblad, E., et al. (1983). The biophysical properties of the cervical-vaginal secretions. International Review of Natural Family Planning 7, 1-56.

Temprano. H. et al. (1994). Tipos de moco cervical e identificacion de elementos al microscopio electronico de barrido. Resultandos preliminares. Proc. IV Symp. Int. sobre Regulación Natural del la Fertilidad, October 6-8, 1994 pp. 304-5. [Colegio Oficial de Medicos, Barcelona].