FERTILITY AND STERILITY Copyright © 1979 The American Fertility Society

Vol. 32, No.3, September 1979 Printed in U.SA.

THE HUMAN UTEROTUBAL JUNCTION: CONTRACTILE PA'ITERNS OF DIFFERENT SMOOTH MUSCLE LAYERS AND THE INFLUENCE OF PROSTAGLANDIN E 2 , PROSTAGLANDIN F 2 a, AND PROSTAGLANDIN 12 IN VITRO*

LENNART WILHELMSSON, M.D.t BO LINDBLOM, M.D. NILS WIQVIST, M.D., PH.D. Department of Obstetrics and Gynecology, University ofGoteborg, S-413 45 Goteborg, Sweden

The three different smooth muscle layers of the human uterotubal junction, i.e., the external spiral-shaped (uterine) layer, the intermediate circular layer, and the internal longitudinal layer, were mechanically separated using a stereomicroscope. Small strips from these layers dissected along the fiber direction were suspended in organ chambers for recording of spontaneous contractile activity. The spontaneous activity differed in that the longitudinal layer had a significantly higher frequency of contractions than the other two layers and that the duration of contractions was significantly longer in the uterine layer as compared with the two tubal layers. The administration of prostaglandin (PG) F 2a and PGI2 in vitro at a concentration of 100 ng/ml induced a stimulatory response in all three types of specimens and in all phases of the menstrual cycle. On the other hand, the addition of PGE 2 (100 ng/mlJ elicited differentiated responses, i.e., inhibition of the spontaneous rhythmic activity in the circular layer and a stimulation of the uterine layer. In the innermost longitudinal specimens, PGE 2 caused inhibition in all phases of the cycle except in the periovulatory period, during which there was a clear-cut stimulation. The functional significance of these experimental observations is discussed in relation to relevant physiologic aspects of reproduction. Fertil Steril32:303, 1979

Studies on the time course of ovum transport through the human fallopian tube have shown that the ovum is delayed within the ampulla for 2 to 3 days followed by rapid passage through the isthmic portion of the tube,1-3 indicating that this segment acts as a functiohal sphincter. Earlier morphologic investigations4-6 have revealed certain anatomical distinguishing characteristics in the arrangement of the smooth muscle fibers in the proximal part of the isthmus, i.e., the uterotubal junction (UTJ) , and in the distal end, i.e., the

ampullary-isthmic junction (AIJ). Although there are minor differences in the description of the fiber direction and in the distinction of these muscle layers, they are easily recognized and can be separated by the use of a stereomicroscope. Recent experiments in our laboratory have shown that the circular and longitudinal muscle layers at the AIJ have different spontaneous contractile patterns and respond differently to prostaglandin E2 (PGE2), thus suggesting that the proposed sphincter mechanism may have a complex functional arrangement. 7 Furthermore, in the middle and proximal parts of the isthmus there is an additional muscle layer, the inner longitudInal layer, which increases in thickness towards the UTJ; parallel to this there is a narrowing of the tubal lumen. s It has been suggested that the contractile pat-

Received March 2, 1979; revised April 16, 1979; accepted April 18, 1979. *Supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Medical Research Council (Grant MFR 17X-02019-13C), GOteborgs Llikaresallskap, and the Faculty of Medicine, University of GOteborg. tTo whom reprint requests should be addressed.

303

September 1979

WILHELMSSON ET AL.

304

UJJ --......._-.-----------_. __110--·------------_.

FIG. 1. Schematic muscular arrangement of the human uterotubal junction: the external spiral-shaped uterine layer, the intermediate circular layer, and the internal longitudinal tubal layer.

terns of the UTJ may be of importance not only in the control of ovum transport but also in the transport of spermatozoa and in the retrograde passage of noxious material from the uterine cavity.5 Nevertheless, no data are available on the contractile activity of the different smooth muscle layers in this region. In the present investigation an attempt was made to elucidate the contractile patterns of the human UTJ by using an in vitro technique that allowed a distinction of the spontaneous activities of the three smooth muscle layers of this segment: the external spiral-shaped uterine layer, the intermediate circular layer, and the innermost longitudinal layer (Fig. 1). Since there is evidence that prostaglandins may be involved in the physiologic control of oviductal and uterine motility, the influepce of prostaglandins (PGE 2 , PGF 2 a, and PGI2 ) on the contractile activity of the different muscle specimens were investigated and related to the phase of the menstrual cycle. MATERIALS AND METHODS

Specimens were obtained from 25 women of reproductive age with regular menstrual cycles who were undergoing sterilization by bilateral tubal resection or hysterectomy because of uterine myomas. Besides the menstrual history the phase of the cycle was evaluated by histologic examination of an endometrial biopsy and by macroscopic examination of the ovaries at operation. In most cases preoperative blood samples were obtained for determinations of 17,a-estradiol, progesterone, and luteinizing hormone by radioimmunoassay. Routine premedication and general anesthesia were given in all instances. Upon opening the ab-

domina I wall the uterine cornua were excised without clamping and immediately placed in chilled, oxygenated Krebs-Ringer bicarbonate buffer of the following composition (millimolar): NaCI, 122; KCI, 4.7; CaCI2 , 2.5; MgCI 2 , 1.19; NaHC0 3 , 15.5; and KH2 P0 4 , 1.19. A 5-mm section of the oviduct at the site ofinsertion of the tube into the uterine wall was considered to represent the UTJ. The luminal diameter of this segment as measured under the microscope at x40 magnification was found to vary between 0.5 and 1.5 mm. By the use of a pair of small tweezers and a pair of microsurgical scissors, the different muscle layers were carefully separated under a stereomicroscope equipped with a transluminous light, and the fiber direction of the muscle layers was checked histologically. From each layer, small muscle strips approximately 4 mm long and having a cross-sectional area of 1.0 to 1.5 sq mm were dissected along the muscle fiber direction. The strips were suspended in an organ chamber filled with 50 ml of the buffer solution fortified with 10.0 mM d-glucose. The organ chamber was continuously aerated with 96% O2-4% CO 2 at 37° C. One end of each muscle strip was connected to a metal hook and the other end to a force transducer (Grass model FT 03). The muscle activity was recorded isometrically, and the signals were amplified and registered on a polygraph (Grass model 7 D). The strips were allowed to accommodate for a period of 60 minutes under a passive tension of approximately 5 mN (Fig. 2). During the following experimental period of 20 minutes the frequency and duration of contractions were measured and statistically analysed. Chemicals. Highly purified crystalline PGE 2 and PGF2 were dissolved in small volumes of lX

Polygraph

Amplifiers

2

Organ bath

37" C

Buffer solution -_--...::...

FIG. 2. Schematic illustration ofthe in vitro system with two muscle strips suspended in the organ chamber and connected to the force transducers.

Vol. 32, No.3

THE HUMAN UTEROTUBAL JUNCTION

UTERINE LAYER

Ju\lLJ~

CIRCULAR LAYER

LlilliUUillillWJJJv~~

LONGITUDINAL LAYER

JRUllhlllimnmmmJIDll

I

4 mN

Smin

FIG. 3. Spontaneous activity of the three different layers at the human UTJ.

99.5% ethanol and stored in the freezer as stock solutions for 1 to 2 months. Immediately prior to each experiment these solutions were diluted in buffer 30 to 500 times to give the appropriate concentrations. A sodium salt of methylated PGI 2 was stored at -20 C and diluted with buffer immediately before use. The maximal concentration of ethanol in the organ bath was 0.03%, which is far below the concentration at which ethanol per se can influence the spontaneous activity. Statistical Procedure. Mean values ± standard error are given. Analysis of variance with one criterion of classification was used when more than two groups were compared. 0

RESULTS

Spontaneous Activity. At the end of the accommodation period, spontaneous phasic activity appeared in 80% of the uterine strips and in 90% of the circular and longitudinal "tubal" strips (Fig. 3). The activity generally had a rhythmic character, but in a few instances the uterine strips developed "bursts" of contractions. The onset of phasic activity in the uterine strips was frequently ac-

305

companied by an increased basal tone which persisted during the observation period. In the absence of in vitro administration of drugs the spontaneous activity continued for several hours, but subsequently the frequency and amplitude of contractions decreased. The spontaneous activity of strips from the uterine layer was characterized by a low frequency and long duration of contractions, whereas strips from the circular tubal layer had a shorter duration and exhibited a higher frequency. Longitudinal tubal specimens were distinguished by the shortest duration and the highest frequency of contractions (Fig. 4). The amplitude of spontaneous contractions varied between 2 and 4 mN, roughly corresponding to a variation in crosssectional area (approximately 1.0 to 1.5 sq mm). There was no significant difference in mean amplitude and cross-sectional area between strips from the different muscle layers. PGE2 • The addition ofPGE 2 to the organ bath in a final concentration of 100 ng/ml caused a stimulation of all uterine strips in terms of elevation of tone and increased contractional frequency. Strips from the circular layer reacted with a weak inhibition during the periovulatory period but with a more pronounced inhibition during the other phases of the cycle. The internal longitudinal tubal specimens exhibited a distinct increment in amplitude and frequency of contractions at about the time of ovulation, whereas during the other phases of the cycle the administration of PGE 2 resulted in a clear-cut inhibition of the longitudinal strips in terms of decreased amplitude and frequency of contractions (Fig. 5). PGF2 a. The addition ofPGF 2" (100 ng/ml) caused stimulation in all three types of specimensmarkedly increased tone, frequency, and ampliSEC 80

50 CONTRACTIONS 40 PER 10 MIN 30

30

20

20

10

10 0

0 UT

CIRC

LONG

UT

CIRC

LONG

FrG.4. Histogram showing the mean values offrequency and duration of contractions. Note the significantly higher frequency ofthe longitudinal layer as compared with the two others (P < 0.01) and the significantly longer duration of contractions of the uterine layer in contrast to the two tubal layers (P < 0.01).

306

WILHELMSSON ET AL.

September 1979

u u

~~WvVVWff t

~

c

't ' ,

1_\

_1-

c

_ _ I4mN

L

t

-

peri ov.

3min

L

FIG. 5. Tracings illustrating the effect of PGE 2 , 100 ng/ml (arrows), at about the time of ovulation (right panel) as compared with the remaining phases of the cycle (left panel). U, Uterine spiral-shaped layer; C, intermediate circular layer; L, inner longitudinal layer.

tude (Fig. 6). These effects were observed during all phases of the menstrual cycle. PGI2 • The administration of PGI 2 (100 ng/ml) induced in all types of strips stimulatory responses qualitatively similar to the effects of PGF 2" (Fig. 7). However, experiments including a quantitative comparison of the contractile response to PGF2 " and PGI 2 revealed that PGF 2 " administration induced stimulation at lower concentration and resulted in greater maximal response, i.e., PGF 2 " possessed both greater potency and efficacy than PGI 2 • The threshold dose for PGF 2 " is approximately 1 ng and that for PGI 2 10 ng/ml. The in vitro effects caused by PGE 2 , PGF 2 a, and PGI 2 on

u t

c

3min FIG. 7. Stimulating effects of PGI2 (100 ng/mD in muscle strips from the uterine and the tubal layers of the UTJ.Arrows indicate the addition ofPGI2 • U, Uterine spiral-shaped layer; C, intermediate circular layer; L, inner longitudinal layer.

the various types of muscle strips are summarized in Table 1. DISCUSSION

The existence of a physiologic sphincter function at the level of the UTJ has not yet been convincingly demonstrated. Interestingly, cinematographic studies using metallic spheres as ovum surrogates indicate that the UTJ rather than the AIJ is the absolute limit for the movements of an ovum during the "arrest" period. 8 The relatively narrow lumen of the tube at the human UTJ has made intraluminal pressure devices for recording contractile activity difficult to apply and the results obtained difficult to interpret. Results based on studies using gas or fluid insufflation are also difficult to evaluate because of the high pressures applied and the possible interference of the contractility within the entire uterine wall. 9 The present study describes an in vitro technique for selective recording of contractile activity in the different muscle layers at the UTJ, and this ap-

t

L

~~~I4mN I

3min

I

t

FIG. 6. Tracings showing the stimulatory effect ofPGF2", 100 ng/ml (arrows), typical of all muscle layers of the UTJ. U, Uterine spiral-shaped layer; C, intermediate circular layer;L, inner longitudinal layer.

TABLE 1. Qualitative Responses to Different Prostaglandins in the Smooth Muscle Layers of the Human UTJa

Uterine layer Circular layer Longitudinal layer

PGE,

PGF,,,

POI,

+

+ + +

+ + +

-/+

aAt about the time of ovulation the inhibitory response ofthe longitudinal layer to PGE 2 was reserved to stimulation. In the circular layer, PGE z constantly elicited inhibition of contractility.

Vol. 32, No.3

THE HUMAN UTEROTUBAL JUNCTION

proach offers the possibility of distinguishing between the different contractile patterns of the three muscle layers described. The most striking feature of these patterns was the long duration and low frequency of contractions in the uterine layer as compared with the two tubal layers. A similar slow motility pattern has been observed by recording uterine contractility in vivo. Taken together, these observations indicate that the high frequency of contractions in the tubal muscle layers are not conducted to the uterine musculature. Another interesting observation in the present study was the differentiated response to PGE2 by the uterine layer in comparison with the two tubal layers, and the reversed response of the inner longitudinallayer to this compound at about the time of ovulation, suggesting a steroid hormone-dependent shift in receptor sensitivity. Since morphologic studies in this laboratory and those by others have shown that the longitudinal muscle extends into the mucosal folds, 5 it is tempting to speculate that the PG-induced contractions of the innermost longitudinal layer might result in an effective closure of the isthmic lumen at ovulation and thus prevent premature ovum transfer. If the present in vitro results hold true in the in vivo situation, not only the AIJ but also the UTJ (or possibly the entire isthmus) may serve as a sphincter that controls the proper timing of ovum entry into the uterine cavity. However, the hormonal and neuronal mechanisms which might be responsible for the initiation of such a change in prostaglandin responsi veness are still poorly understood. Acknowledgment. Prostaglandins were kindly supplied by ICI Ltd., Pharmaceutical Division, Manchester, England.

307 REFERENCES

1. Cheviakoff S, Diaz S, Carril M, Patritti N, Croxatto HD, Llados C, Ortiz ME, Croxatto HB: Ovum transport in women. In WHO Symposium, San Antonio, Texas, 1975, Edited by MJK Harper, CJ Pauerstein, CE Adams, EM Coutinho, HB Croxatto, DM Paton. Copenhagen, Scriptor, 1976, p 146 2. Dellepiane G, Costanzo A, La Placa A, Pala A: Evaluation of rate of ovum transport in the human oviduct. In Proceedings of the Vth European Congress on Fertility and Sterility, Venice 1978. In press 3. ArefI, Hafez ESE: Utero-oviductal motility with emphasis on ova transport. Obstet Gynecol Survey 28:679, 1973 4. David D, Czernobilsky B: A comparative histological study of the uterotubal junction in the rabbit, rhesus monkey and human female. Am J Obstet Gynecol101:417, 1968 5. Hafez ESE: Anatomy and physiology of the mammalian uterotubal junction. In Handbook of Physiology, Edited by RO Greep, EB Astwood, Sect 7: Endocrinology, Vol 2: Female Reproductive System, Part 2. Washington DC, American Physiological Society, 1973, p 87 6. Vasen LCLM: The intramural part of the fallopian tube. Int J Fertil 4:309, 1959 7. Lindblom B, Hamberger L, Wiqvist N: Differentiated contractile effects of prostaglandins E and F on the isolated circular and longitudinal smooth muscle of the human oviduct. Fertil Steril 30:553, 1978 8. Diaz J, Vasquez J, Diaz S, Diaz F, Croxatto HB: Transport of ovum surrogates by the human oviduct. In WHO Symposium, San Antonio, Texas, 1975, Edited by MJK Harper, CJ Pauerstein, CE Adams, EM Coutinho, HB Croxatto, DM Paton. Copenhagen, Scriptor, 1976, p 404 9. Stabile A: Interpretation of manometric oscillations observed during uterotubal insufflation. Fertil Steril 5:138, 1954