Fallopian tube ciliary beat frequency in relation to the stage of menstrual cycle and anatomical site

R.A. Lyons1, O. Djahanbakhch1,3, T. Mahmood1, E. Saridogan1, S. Sattar1, M.T. Sheaff2, A.A. Naftalin1 and R. Chenoy1

1 Academic Department of Obstetrics and Gynaecology and 2 Department of Histopathology, St Bartholomew's and The Royal London Hospital School of Medicine and Dentistry, Whitechapel, London E1 1BB, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The cyclical changes in ciliary structure and motion within the human Fallopian tube are well documented. Previous investigators have studied ciliary beat frequency (CBF) in relation to menstrual cycle and anatomical site, but with conflicting results. METHODS: Using a technique that records variations in light intensity, we have studied the changes in CBF in relation to the menstrual cycle and anatomical site. Fallopian tubes were collected from 26 women who underwent hysterectomy for benign conditions. Menstrual history, hormone profile and endometrial biopsy results were used to determine the stage of the cycle. Fourteen women were in the proliferative phase, and 12 women in the secretory phase. RESULTS: Mean CBF for all subjects was 5.3 ± 0.2 Hz. There was no significant difference in CBF in relation to anatomical site. In the fimbrial region the ciliary beat was faster in the secretory (5.8 ± 0.3 Hz) as compared with the proliferative phase (4.9 ± 0.2 Hz), P < 0.02. CONCLUSIONS: It is possible that this increase in fimbrial CBF may contribute to ovum retrieval and transport after ovulation. However, the reproductive significance of the changes in CBF in relation to the menstrual cycle needs further investigation.

Key words: cilia/ciliary beat frequency/Fallopian tubes/fimbrial region/menstrual cycle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The first description of a distinct oviductal cycle in women was made by Novak and Everett (Novak and Everett, 1928Go). In common with the rest of the female reproductive tract, the Fallopian tube undergoes cyclical changes under the influence of ovarian steroids during the menstrual cycle (Critoph and Dennis, 1977aGo).

Both ciliated and secretory epithelial cells are low in height during the early follicular phase and increase to reach their greatest height in the periovulatory period, at which time secretory activity is maximal (Patek, 1974Go). Receptors for estradiol and progesterone have been identified within the Fallopian tube epithelium, and the expression of these receptors varies according to the stage of the menstrual cycle (Pollow et al., 1981Go; Amso et al., 1994Go). Ovarian steroids are known to affect the tubal mucosa: oestrogen stimulates epithelial cell hypertrophy, secretion and ciliogenesis; atrophy and deciliation are associated with high concentrations of serum progesterone (Verhage et al., 1979Go; Donnez et al., 1985Go).

There are conflicting reports on the changes in ciliary beat frequency (CBF) throughout the menstrual cycle. Critoph and Dennis detected a significant increase in the ciliary beat frequency of the isthmus and ampulla after ovulation (Critoph and Dennis, 1977bGo). In contrast, Westrom et al. found no cyclical or anatomical variation in beat frequencies (Westrom et al., 1977Go).

This discrepancy may be due to differences in the techniques used to measure CBF. A variety of in-vitro systems to assess ciliary beat have been described. These include the use of a stroboscope, laser light-scattering spectroscopy and the photoelectric method (Ballenger and Orr, 1963Go; Lee and Verdugo, 1977Go; Kennedy and Duckett, 1981Go). In the latter, variations in light intensity are recorded by a photomultiplier unit and converted into an electrical signal. This was the method used by Westrom et al. (Westrom et al., 1977Go), whilst Critoph and Dennis (Critoph and Dennis, 1977bGo) used a photographic method to record CBF by means of a high-speed camera. There are limitations in the accuracy of all the above-mentioned procedures. Observer error in the photographic technique can be marked (Toremalm, 1961Go), whereas the photoelectric method measures only an average CBF across several ciliated cells and is inaccurate when the cilia are in asynchronous rhythm (Naitoh and Kaneko, 1973Go; Kennedy and Duckett, 1981Go).

We employed a light intensity technique with analogue contrast enhancement. This has the advantage of being easy to use and highly reproducible (Devalia et al., 1990Go). The microscopic image is transferred onto a television screen and the contrast intensified electronically. The changes in light intensity due to ciliary motion are analysed by specially designed computer software. As the differences in light intensity are computed directly into Hertz and are not subject to any additional manual or mathematical manipulation, the likelihood of error is minimized.

As the available literature shows conflicting results, the purpose of our work was to re-evaluate previous studies using the analogue contrast enhancement technique. Particular emphasis was placed on accurate staging of the menstrual cycle, as measured by endocrine and histological assessment. We have therefore studied firstly the variation of ciliary beat frequency in relation to different anatomical sites within the Fallopian tube and secondly the effect of the menstrual cycle on the ciliary beat frequency.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue collection
Normal Fallopian tubes were collected from patients undergoing hysterectomy for benign conditions after obtaining written consent and local ethical committee approval.

Fallopian tubes were collected into ice-cold minimum essential medium (MEM) with Earle's salts and L-glutamine and supple-mented with heparin (CP Pharmaceuticals, Wrexham, UK; 1.8 IU/ml), streptomycin (Evans Medical, Surrey, UK; 50 µg/ml), penicillin (Glaxo Laboratories, Middlesex, UK; 100 IU/ml) and HEPES (Life Technologies, Paisley, UK; 10 mmol/l). Fallopian tubes were rinsed several times to remove all visible evidence of blood and cut into isthmic, ampullary and fimbrial portions. Small (1–2 mm) pieces of tissue were dissected free from each of the three portions of the Fallopian tube and placed into Petri dishes containing 1 ml of medium without heparin and HEPES.

Measurement of CBF
Tissue samples were allowed to stand immersed in medium to reach room temperature. Experiments were performed in a tissue culture room where a constant temperature is maintained. CBF was measured under an Olympus inverted microscope. Readings were taken randomly over a 1 min period from several areas of each tissue, so that between 50–100 readings of CBF were made on each tissue explant and the average taken. The recordings were analysed using a CE-1 contrast enhancer (Brian Reece Scientific Instruments, Newbury, UK), which increased the contrast of the video signal so that it was possible to analyse the cilia on a television monitor. After obtaining a sharply focused image, a cross hair light-detecting sensor was placed over the real time image of individual cilia beating on the television screen. As the cilia beat, they produce changes in light intensity on the screen; these changes are detected by the sensor at its cross point and computed into frequencies (in Hz) by the use of a computer incorporating a PCX 1 video digitizer card (version 7.1) and cell track 8 software (Brian Reece Scientific Instruments).

Fallopian tubes were collected from 26 premenopausal women. All had regular menstrual cycles and no woman had used hormonal medication within 3 months of surgery. The hormonal status of patients was determined by assaying serum FSH, LH, estradiol and progesterone on venous blood samples taken immediately prior to surgery. Plasma was immediately centrifuged and stored at –70°C. The samples were analysed in batches. All samples were measured using direct chemiluminescence: FSH and LH were detected using a two-site sandwich immunoassay, and estradiol and progesterone by a competitive immunoassay. The inter- and intra-assay coefficients of variation for the hormone assays were respectively as follows: FSH 7.1 and 1.7%; LH 6.3 and 4.5%; estradiol 8.5 and 4.0%; progesterone 8.1 and 3.9%. Histological examination of the endometrium was performed using the Noyes method to assess menstrual phase (Noyes et al., 1950Go). Only women in whom all these investigations correlated with stage of cycle as determined by the menstrual history were included in the study. Fourteen women were in the proliferative phase of their menstrual cycle and 12 in the secretory phase.

Statistics
Between 50 and 100 measurements of CBF were made on each tissue explant. The readings appeared normally distributed (Figure 1Go). The mean CBF was calculated for each patient, and statistical comparisons were made between the isthmus, ampulla and fimbria using one-way analysis of variance. The CBF within Fallopian tubes taken from women in the proliferative and secretory phases was compared using a two-tailed unpaired Student's t-test.



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Figure 1. The distribution of ciliary beat frequency. The columns refer to the total number of readings of CBF taken from all anatomical sites on every tube. A normal curve is superimposed using SPSS Version 10 software.

 

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 Materials and methods
 Results
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 References
 
The mean ciliary beat frequency of the study population was 5.3 ± 0.2 Hz (range 3.4–8.7 Hz). There was no significant difference in CBF between the isthmus, ampulla and fimbria (Table IGo).


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Table I. Mean ciliary beat frequency (in Hz) in the different anatomical sites of the human Fallopian tube (n = 26)
 
Although ciliary beat frequency in the secretory phase tended to be faster in the ampulla and isthmus than in the proliferative phase, this did not reach significance. However, in the fimbrial region, there was a significant increase in CBF in tubes in the secretory phase of the menstrual cycle when compared with the proliferative phase (P < 0.02) (Figure 2Go). Figure 3Go is a scatter diagram of fimbrial CBF showing the distribution of results according to the day of the menstrual cycle. This illustrates the increase in CBF in the fimbria in the secretory phase.



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Figure 2. Variation in ciliary beat frequency (CBF) in the isthmus, ampulla and fimbria of the human Fallopian tube during the proliferative (n = 14) and secretory (n = 12) phases of the menstrual cycle. Each column represents the mean CBF of all readings taken from each anatomical site (isthmus, ampulla and fimbria) of Fallopian tubes in either the proliferative or secretory phase of the menstrual cycle. The error bars represent the SEM.

 


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Figure 3. Fimbrial ciliary beat frequency in relation to day of menstrual cycle. Each square represents the CBF of the fimbrial region of each Fallopian tube, plotted against day of cycle as determined by LMP. Each square represents one sample. A key is included on the figure.

 

    Discussion
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 Materials and methods
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 References
 
The Fallopian tube plays an essential role in gamete transport, fertilization and early embryogenesis. Successful fertilization requires the gametes and embryos to be transported within a well-defined time frame (Hafez and Blandau, 1969Go). This precisely timed process is influenced by contractions of the tubal musculature, ciliary activity and the flow of tubal secretions (Jansen, 1984Go). Although the relative importance of ciliary activity is unclear, there are indications that women with the `immotile cilia syndrome' suffer from subfertility (Pederson, 1983Go; McComb et al., 1986Go; Lurie et al., 1989Go). Whilst successful pregnancies do sometimes occur in these patients, they may be explained by the fact that a subgroup of women demonstrate some dyskinetic ciliary action (Halbert et al., 1997Go).

Ciliated cells constitute >50% of the total epithelial cell population within the fimbria, but the percentage of ciliated cells falls progressively from fimbria to isthmus (Patek et al., 1972Go; Jansen, 1984Go; Crow et al., 1994Go). Cells possessing cilia are more abundant on the apices of the mucosal folds within the tubal epithelium (Patek et al., 1972Go). The cilia are ~7 microns long and are attached to a refractile row of basal granules beneath the cell membrane. The axoneme is arranged in the standard configuration of two central single and nine peripheral double microtubules. Outer and inner dynein arms connect these microfilaments and allow the microtubules to slide on one another, thus permitting movement of the cilia.

The appearance of the cilia in the human Fallopian tube changes during the menstrual cycle. During the early proliferative phase the cilia appear `droopy', becoming erect and vigorous during the late follicular phase. Around the time of ovulation the strokes of cilia are synchronized and oriented towards the uterus in humans (Gaddum-Rosse et al., 1973Go). There is partial deciliation in the late secretory phase, which can be prevented in vitro by the administration of oestrogen in high doses (Goldberg and Friedman, 1995Go).

Alterations in CBF following ovulation have been detected in other mammalian species. In rabbits the rate of ciliary beat in the oviducts increases by 20% on the second and third days after copulation, corresponding to the time of movement of the ova down the tubes (Borell et al., 1957Go). However, in human studies results have been inconsistent. Indeed, there is as yet no agreement even on the baseline rate of CBF, with a wide range of frequencies of between 5 and 20 Hz being reported in the literature (Paltieli et al., 1995Go: Westrom et al., 1977Go).

The reason for this discrepancy in the findings may be due to the different methods employed to assess CBF, as well as the different parameters used to determine the stage of the menstrual cycle. In our study we employed a technique that is reproducible with minimum observer error. At the same time we assessed the menstrual cycle using endocrine parameters and histological dating of the endometrium.

Many different techniques are employed to measure CBF. Critoph and Dennis recorded CBF using a high-speed camera (Critoph and Dennis, 1977bGo). The film was then played at a reduced speed and the ciliary beat counted directly off a screen. In order to calculate the CBF, the results were then multiplied, which would tend to magnify observer error. For this technique, observer error has been estimated to exceed ±40 beats/min (Toremalm, 1961Go).

Westrom et al. used a photoelectric method (Westrom et al., 1977Go). The specimen was illuminated and the beating of the cilia produced variations in reflected light. These were detected by a photomultiplier that converted the signal into voltage variations recorded electronically. The spatial resolution of this technique is equal to ~10 ciliary cells (i.e. 2000–3000 individual cilia) and so only provides average values for CBF across several cells (Kennedy and Duckett, 1981Go). Moreover, the technique is inaccurate when the cilia are in asynchronous rhythm (Naitoh and Kaneko, 1973Go) or the intensity of the illuminating source fluctuates (Mercke et al., 1974Go).

The technique we employed involves transfer of the microscopic image of the specimen onto a television screen, followed by electronic enhancement of the contrast to allow visualization of the specimen beyond the limit of the optical microscope. Signals relating to changes in light intensity due to ciliary motion are detected by a cross-hair light sensing probe and analysed by specially designed computer software. The device is precise enough to detect the beat of a single cilium and so remains accurate even when the cilia are beating asynchronously. It is unaffected by changes in light source intensity. Also, the fluctuations in light intensity are computed directly into Hz and are not subject to any additional mathematical manipulation, thus minimizing the risk of error.

Our assessment of mean baseline CBF of 5.3 Hz is similar to that reported by us in previous studies: 6.4 Hz (Saridogan et al., 1996Go) and 5.5 Hz (Mahmood et al., 1998Go), and is close to the figure of 5.2 Hz derived by Paltieli et al. in the only in-vivo assessment of CBF to date (Paltieli et al., 1995Go). We have shown no variation in CBF in the different anatomical sites (isthmus, ampulla and fimbria) of individual Fallopian tubes. This appears to contradict the findings from our previous research, in which ampullary CBF was found to be significantly faster than CBF from the fimbrial region of the tube (Mahmood et al., 1998Go). However, this preliminary report was performed on a sample of nine tubes predominantly from the proliferative phase of the menstrual cycle. In the current study the proportion of tubes in the secretory phase is almost 50%. Since we have shown that fimbrial CBF is faster in the secretory phase, this may explain why we failed to show any significant difference in CBF between the ampulla and fimbria, in contrast to the earlier work, where ampullary CBF was greater.

We have shown an increase in CBF of the fimbrial section of the tube during the secretory phase. This result agrees with those of Critoph and Dennis (Critoph and Dennis, 1977bGo) in so far as there appears to be a variation in CBF according to the menstrual cycle, with an increase in the secretory phase. However, Critoph and Dennis detected this change in the ampullary and isthmic sections of the tube, whereas in the current study the increase was confined to the fimbria.

With the exception of the study by Paltieli et al. (Paltieli et al., 1995Go), all studies of CBF have been performed in vitro. Care must be taken when applying the results of these studies to the in-vivo situation. Removal of mucosal explants obviously disrupts the neuronal and circulatory network supplying the ciliated cells and could affect CBF. The cilia are no longer bathed in the physiological tubal secretions, which are rich in steroid hormones, particularly in the secretory phase, and which could also affect CBF. Therefore, the findings in this in-vitro study do not necessarily reflect what is occurring in vivo.

Fallopian tube CBF is influenced by several factors. In addition to the alteration in ovarian steroids, which could be responsible for this effect, we cannot exclude a contribution from other substances. In in-vitro studies we have shown that progesterone in high dosage inhibits CBF, and that this inhibition is reversed by the progesterone receptor antagonist, mifepristone (Mahmood et al., 1998Go). We have also shown that angiotensin II increases CBF (Saridogan et al., 1996Go). CBF is known to be increased in vitro by prostaglandins F2{alpha}, E1 and E2 (Verdugo et al., 1980Go). Prostaglandin E production by the fimbria increases in the secretory phase (Nabekura et al., 1994Go). As the oocyte matures, follicular fluid contains increasing concentrations of prostaglandins, which play a pivotal role in follicle rupture (Seibel et al., 1984Go). When prostaglandin-rich follicular fluid enters the tube at ovulation, this may stimulate the cilia to beat faster, which may facilitate transport of the ovum along the tube.

In summary, there are cyclical changes in ciliary activity within the human Fallopian tube. This study has shown an increase in CBF in the fimbria during the secretory phase of the menstrual cycle. The hormonal stimulus for this change is not well understood. The significance of increased CBF in the fimbria in relation to ovum transport needs further exploration. Gamete and embryo transport may be aided by increased ciliary activity and the information provided by this study may act as a basis for future work. Further knowledge of the mechanisms affecting ciliary motion would aid our understanding of the events surrounding conception and might even give an insight into future contraceptive possibilities.


    Notes
 
3 To whom correspondence should be addressed. E-mail: o.b.djahanbakhch{at}qmul.ac.uk Back

Submitted on 23 April 2001


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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accepted on November 3, 2001.