Human hydrosalpinx

Human hydrosalpinx Histologic study and chemical composition

AMNON

DAVID,

CELSO-RAMON BERNARD Philadelphia,

of fluid

M.D.* GARCIA,

M.D.

CZERNOBILSKY,

M.D.**

Pennsylvania

A histologic study of 9 hydrosalfiinges and chemical analysis of their @id content were performed. In most of the cases the wall was fibrotic; the epithelial lining in the main was impaired although in scattered areas it appeared normal and probably well functioning. Electrolyte values were similar to those found in the serum of the patients. Proteins and bicarbonate values were very low. Correlation between these low values and the functional potential of the hydrosalpinx should be considered, since it may be related to the degree of tubal damage. These asfiects are discussed and the potential use of chemical study of hydrosalpinx fluid is reviewed. Consideration is given for use of the findings in evaluating functional prognosis prior to tuboplasty.

MANY OF T r-r E procedures recommended for correction of human infertility are basically empirical, because of the uncertainty regarding the physiologic mechanisms of reproduction, Fundamental research in this area has not kept pace with the needs of this important human problem. The success of tuboplasty, for examply, is often less than optimal, probably because of a lack of basic understanding of the complicated intracacies of the Fallopian tube. A patent tube is often assumed to function merely as a simple conduit for fertilized eggs. In reality, the ovum and its further development are dependent upon the intraFrom the Division of Reproductive Biology, Department of Obstetrics Gynecology, and the Laboratories Pathologic Anatomy, Department Pathology, School of Medicine, of Pennsylvania. *Ford Foundation Fellow in Refiroductive Biology, Address: Hashomer Hospital, Tel-Aviv School, Tel-Aviv, Israel. **Address Czernobilsky, Philadelphia,

tubal environment which is controlled by location and function of the ciliated cells, secretory cells, and the complex activities of the tubal wall. Recent investigations have revealed that oviductal physiology is more complex than previously entertained.lp 2 The mechanical and secretory activities of the oviduct are delicately balanced by the specific actions of various hormones. 3, 4 As a result, appearances can be deceiving and thus significant gross morphologic distortions of the oviduct may be associated with relative functional integrity, while morphologically normal-ap pearing oviducts may lack adequate motility and secretions. Hydrosalpinx is characterized by an obliteration of the fimbriated OSand subsequent distention of the oviduct by an accumulation of variable amounts of fluid, usually of serous nature. The etiology and functional prognosis of this condition are still undear. This probably explains the use of numerous therapeutic approaches as well as the extreme variation in response to these treatments. The sole classification of hydro-

and of of

University TelMedical

reprint requests to Dr. 3400 S ruce Street, Pennsy Qvania 19104.

400

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105 3

Human

salpinges, based on gross anatomic configuration, has been that given by Shirodkar.5 He divides human hydrosalpinges into three groups, dependent upon thk degree of distentio:n of the oviduct, and correlates the outcome of surgical repair with the degree of tubal distention. Shirodkar also considered the appearance of the fimbrial apparatus and that of the muscular wall after terminal salpingostomy and emptying of the hydrosalpin,Tes as significant in predicting the outcome of operation for distal occlusion. However, other factors as well should be considered to evaluate the outcome of these complex operations. Tissue evaluation from biopsy material is difficult without compromising oviductal function. Thus if a relationship between the chemical composition of the hydrosalpinx fluid and the histology of the oviduct can be established, a more reliable prognostic conclusion might be entertained prior to operation. This study presents preliminary findings concerning the chemical components of human hydrosalpinx fluid and tentative conclusions concerning the possible relationship between the chemistry of the fluid and the history of the oviducts. The potential and application of the chemical analysis of hydrosalpingeal fluid for evaluation of functional prognosis prior to operation are discussed. Material

and

methods

Fresh specimens of hydrosalpingeal tubes were obtained from women undergoing elective hysterectomy primarily for nontubal disease. Six of the nine specimens included in the present study were obtained from patients in the reproductive age, ranging in age from 29 to 45. Endometrium for histologic study was available in each case, thus the phase of their menstrual cycle at the time of operation was established.s Two patients, 56 and 68 years old, were postmenopausal for 12 to 20 years, respectively, and their endometria were inactive. The last specimen was obtained from a 47-year-old patient in the premenopausal phase, who had a bilateral ligation performed 15 years prior l:o hysterectomy. The endometrium at

hydrosalpinx

401

the time of operation was inactive. Two normal tubes and two pyosalpinges served as histologic controls. These tubes were removed from patients of reproductive age. Grossly the hydrosalpinx specimens were dilated (from 2 to 13 cm. in greatest dimension) with a closed fimbriated end and a nondilated proximal portion of varying lengths. The transition between the dilated and normal appearing portions of the tubes was abrupt in all cases. A luminal communication between the dilated and nondilated parts could be identified in each specimen. Fluid content in these tubes varied from 3 to 80 ml. and was water clear to pale yellow in 8 cases, while in one case it was tinged with blood, requiring centrifugation. Upon excision of the specimens, fluid was collected in vacuum tubes or with syringes sealed with mercury so as to minimize contact with air which might interfere with bicarbonate determination. This fluid was examined immediately for electrolytes, bicarbonate, total proteins, albumin, and globulin and subjected to electrophoresis. Sodium and potassium were determined with the flame photometer (Model 143) ; chloride and CO, content for bicarbonate were determined by microanalysis using the AutoAnalyzer (Technicon) . Total proteins were determined by the modified biuret method and electrophoresis was performed on an ultrafiltration apparatus. pH was determined using a pH meter (Corning, Model 12) before each set of chemical determinations. Comparable multiple sections were taken from each of these oviducts. Tissues processed for sectioning were fixed in Bouin’s solution and stained with Delafield’s hematoxylin-eosin, Masson’s trichrome, Mayer’s mucicarmine, Mallory’s Alcian blue stains, and McManus periodic acid-Schiff (P.A.S.) before and after diastase digestion. The latter stain was also performed on tissue fixed in Carnoy’s solution. Results

Histology. The histologic features are described in detail below and summarized in Table I.

402

David,

Table

Garcia,

I. Summary

and

Czernobilsky

of histologic

findings

in hycirosalpinx

Epithelium

and

lamina

propria

Tubal

1. Foci of normal-appearing prominent in remaining

ciliated and nonciliated epithelium, plicae (Figs. 6, 7, and IO)

1. Fibrosis

2. Numerous

flat

2. Focal

cuboidal

and

epithelial

cells

(Fig.

3. Thin, dilated blood vessels remaining plicae (Fig. 5)

in lamina

propria,

-4. Scant

in lamina

propria

round

cell

Fig. 1. Low left

infiltration

power view side of microphotograph.

of hydrosalpinx (Hematoxylin

8) prominent

edema

wall

3) (Fig.

4)

3. Atrophy and (Fig. 2)

distortion

of muscle

4.

subserosal

blood

showing fibrotic wall and eosin. x25.)

and trichrome-stained sections from the dilated portions of the hydrosalpinx specimens showed a thin wall in which the muscle layers appeared atrophic or were partly or completely replaced by fibrous connective tissue (Figs. 1 and 2). The fibrous replacement was most prominent in the thinnest portions of the wall (Fig. 3). There were focal areas of edema present separating and distorting direction of muscle fibers (Fig. 4). Inflammatory cells were scant and consisted mostly of lymphocytes. In the previously ligated tube fibrosis of the wall was minimal, but edema and focal chronic inflammation were Hematoxylin-eosin-

in

(Fig.

Thick

with

walled

few

remaining

plicae

fibers

vessels

near

present. Thin-walled dilated blood vessels representing arterioles, venules, and capillaries were scattered in the wall of the tubes but were most numerous in the stroma of the few remaining papillary projections (Fig. 5), The majority of the subserosal vessels were thick-walled arteries and veins of medium-sized caliber. A very few bloodless thin-walled endothdial lined structures, probably representing lymphatics, were identified in the wall, especially in the lamina propria of the plicae. The degree of fibrosis of the waH was proportionately related to the paucity of vascular channels, being devoid of the latter in many instances.

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Fig. 2. Wall of hydrosalpinx trichrome. x130.)

showing

fibrosis,

The epithelium lining the distended portion of the tube showed marked variations. While in some segments tall columnar ciliated, secretory, and peg cells were seen, adjacent areas presented low cuboidal and completely flattened cells (Figs. 6, 7, and 8). The latter were predominant in the most dilated and thinnest portions of the hydrosalpinx while the normally encountered cells were most common in the remaining plicae (Figs. 9 and 10). In addition, glandlike spaces formed by invagina.ting epithelium were scattered in the wall of the dilated tubes. These glandular structures were also lined by a variety of cells, some normal, others flat. The lamina propria showed varying degrees of round cell infiltration. The morphology of the normal ciliated and nonciliated epithelial cells in the hydrosalpinges corresponded to the phase lof the menstrual cycle in the patients in the reproductive era, while the epithelium of the premenopausal and postmenopausal patients showed a mixture of ciliated and nonciliated cells, some of the latter demonstrating secretion. Epithelial mitoses could not be demonstrated. The ciliated and nonciliated cells con-

atrophy,

and

distortion

of muscle

hydrosalpinx

fibers.

403

(Masson’s

Fig. 3. Portion of hydrosalpinx wall completely replaced by fibrosis with flattened epithelial lining. (Hematoxylin and eosin. x140.)

404

David,

Garcia,

and

Fig. 4. Severe edema epithelium. (Hematoxylin

Fig. 5. Surface part dilated blood vessels,

Czernobilsky

separating muscle fibers and eosin. x120.)

of one of few remaining lined by cuboidal epitheiium.

tamed P.A.S.-positive, diastase-resistant material, mostly concentrated in the cilia and near the luminal borders. Small amounts of P.A.S.-positive diastase digested granules were present in some of the ciliated cells. The cuboidal cells had only small irregular

in hydrosalpinx

specimen

plicae in hydrosalpinx (Hematoxylin and

lined

with large, eosin. x250.)

with

flattened

thin

walled

foci of P.A.S. material while the flat cells showed none. Small amounts of P.A.S.-positive, diastase-resistant material were also present within the lumen of the tubes. MUScle fibers in the wall were P.A.S. positive, diastase digestible. Alcian blue-positive ma-

Volume Number

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:105 3

Fig. 6. Area of normal-appearing toxylin and eosin. x350.)

ciliated

Fig. 7. Secretory cells with dome-like postmenopausal patient. (Hematoxylin

hydrosalpinx

cells with scattered peg cells in hydrosalpinx.

cell extremities extruding and eosin. x350.)

terial vras present in the cilia and in the luminal borders of ciliated and nonciliated cells. Somewhat lesser amounts were seen in cuboidal cells while flat cells had none to very minimal Alcian blue material. Intraluminal secretion was Alcian blue positive.

405

(Hema-

into lumen in hydrosalpinx

of

There were patchy areas in the dilated fibrous wall which stained strongly with Alcian blue. Mucicarmine was negative except in one hydrosalpinx specimen in which some secretory epithelial cells and intraluminal material showed a positive reaction.

406

David,

Garcia,

and

Czernobilsky

Fig. 8. Cuboidal and compIetely and eosin. x250.)

flattened

Fig. 9. Well-formed plicae protruding toxylin and eosin. x25.)

epithelial

cells lining

into lumen of markedly

The nondilated, less distorted part of the hydrosalpinges showed normal distribution of muscle layers in the wall, without evidence of fibrosis. Thick-walled subserosal blood vessels and scarce thin-walled lamina propria vessels, as well as normal epithelial lining corresponding to the phase in the

hydrosalpinx.

(Hematoxylin

distended hydrosalpinx.

(Hema-

menstrual cycle of these patients, were : observed. The epithelium of the pyosalpinx s#pecimens was markedly abnormal. Ciliated cells were scarce and the lining epithelium consisted of mainly a mixture of columnar nonciliated and low cuboidal cells with nu clear

Volume Number

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105 3

Pig. 10. appearing remaining

High-power view of vacuolated ciliated wall of hydrosalpinx

epithelium cells lining to the left.

of oviduct plicae to (Hematoxylin

Fig.

shown in Fig. 9, demonstrating the right, and low cuboidal and eosin. x300.)

11. Pyosalpinx showing markedly abnormal flattened and lining. The lamina propria shows acute and chronic inflammatory capillaries. (Hematoxylin and eosin. x250.)

atypism but no mitoses. Secretion could not be demonstrated. Glandular-like invaginations were also present. The lamina propria was diffusely infiltrated with polymorphonuclear leukocytes, lymphocytes, and plas-

hydrosalpinx

partly desquamated cells and numerous

cells

407

normal lining

epithelial dilated

ma cells which were also scattered through the rest of the wall (Fig. 11) . Foamy macrophages in the lamina propria were present in one case. Muscle fibers were separated by edema. There were numerous dilated thin-

408

David,

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and

Czernobilsky

walled blood vessels throughout the wall, many with leukostasis. Lymphatic channels could not be identified. In the few remaining ciliated cells, P.A.S.-positive, diastase-resistant material and Alcian blue-positive staining was present. Mucicarmine-positive material could be demonstrated in focal epithelial cells as well as in the secretions of one of the cases. In the normal tubes, the epithelium was composed of ciliated, nonciliated cells, and peg cells and corresponded to the phase in the menstrual cycle. The normal muscle layers were present in the wall. Scattered thin-walled blood vessels were seen in the subserosal area. None of these were markedly dilated. Lymphatics were not prominent. There was P.A.S.-positive, diastase-resistant material in the luminal borders of the epithelium and P.A.S.-positive, diastase-digested granules in some of the ciliated cells. Positive Alcian blue-staining material was also present in the same locations. Mucicarmine was negative. Chemical analysis of hydrosalpingeal fluid. The results of the chemical analysis are given in Table II. The pH of the fluid varied from 7.28 to 7.7. Chloride levels

Table II. Chemical

components

of hydrosalpinx

fluctuated from 111 to 132 mEq. per liter.. with a mean of 121.1 mEc1. per liter i 6 ^’ sodium from 139 to 153 mEq. per liter, mean of 144.0 mEq. per liter .F 3.8; potassium from 3.8 to 4.9 mEq. per liter, mean of 4.2 mEq. per liter of: 0.3; and bicarbonate from 8 to 29 mEq. per liter, mean 20 mEq. per liter -t 7.0. Total protein varied from 0.078 to 2.50 Gm. per 10 ml., mean 0.97 Gm. per 100 ml. F 0.83; albumin from 0.20 to 1.60 Gm. per 100 ml., mean 0.78 Cm. per 100 ml. i: 0.55; and globulin from undetectable values to 0.90 Gm. per 100 ml., mean 0.31 Gm. per 100 ml. 1 0.29. Results of protein electrophoresis followed the genera1 pattern of the serum proteins electrophoresis, with a marked flattening of all the values which was more pronounced in the globulin fractions alpha-l, alpha-2, beta, and gamma. Most of the electrophoretic results showed a predominance in the albumin fraction compared with the various globulins fractions. No statistically significant differences in the chemical composition could be found between menopausal hydrosalpingeal fluid and that of patients in the menstruating age, except for a slight tlecrease in the bicarbonate values.

fluid .-..-

Reproductive Patient 1 (29)

Chemical components Chloride

age

Menopausal age group”

group”

Patient 2 (29)

Patient 1 (32)

Patient 6 (45)

112

127

120

120

129

119

143

139

145

143

143

145

Patient 7 (47)

Patient 8 (56)

Patient 9 (68j

Mean value + S.D.

120

111

132

121.11

i: 6.74

140

145

1.53

144.00

+ 3.77

4.26

k 0.29

20.00

T 7.22

(mEq./L.) Sodium

(mEq./L.) Potassium (mEq./L.)

4.5

Bicarbonate

-

4.2

4.3

20

28

3.8 20

4.3 -

4.2 29

4.2

4.9

15

8

4.0

(mEq./L.) Total proteins (Gm./lOO ml.)

2.20

0.40

0.40

0.078

0.4

0.90

~~~

0.90

2.50

0.97

t 0.83

Albumin

1.60

0.40

0.20

-

0.20

0.70

-

0.80

1.60

0.78

-e 0.55

0.60

0.00

0.20

-

0.20

0.20

-

0.10

0.90

0.31 + 0.29

(Gm./lOO

ml.)

Globulin (Gm./lOO ‘Numbers

ml.) in

parentheses

represent

age

of

patients.

Volume Number

105 3

Comment Following corrective surgery, variations and disturbances in the metabolism of the oviduct may persist even after patency has been re-established. Such anomalies would impair the normal development of a fertilized ‘egg or the process of fertilization itself. These metabolic disorders may stem from abnormalities of the lining epithelium or the tubal wall or variations in the chemical composition of the secreted fluid. Our histologic study of the hydrosalpinges revealed marked pleomorphism of the tubal lining epithelium. Even in the most distended of the hydrosalpinges there were areas in which the lining epithelium consisted of tall columnar ciliated and nonciliated cells as well as cells showing active secretion. As mentioned by Novak and Wood,ruff,7 such normal cells were more commonly observed in the few remaining plicae than in the thinner parts of the wall which were usually lined with low cuboidal or completely flat cells. It is of interest that such foci of normal epithelial lining were also observed in the specimens from postmenopausal patients. 8 The cellular distribution rnay suggest at first that epithelial damage was secondary to a pressure effect. However, since the pressure of fluid in a closed cavity, such as a hydrosalpinx, is equally distributed on all its parts, the presence of a mixture of normal and flattened epithelial lining cannot be explained by pressure effect alone. Novak and Woodruff’ advanced a theory that hydrosalpinx is the result of previous pyosalpinx. In our cases of pyosalpinx, nearly all of the epithelial lining was severely damaged. Thus, one can theorize that in cases of hydrosalpinx some parts of the epithelial lining were more involved by the inflammatory process, leaving other areas intact. It is also possible that the plicae rnay have formed through a regenerative process. In all these hydrosalpinges the tubal wall was characterized by distortion, paucity, or absence of muscle layers, suggesting markedly impaired motility. This was due to edema and/or fibrosis of the tubal wall

Human

probably

hydrosalpinx

409

resulting from previous infection.

As a result of this fibrosis, obliteration of the normal lymphatic sinuses and plexuses located just below the epithelium may occur. In the same manner one can explain the relative paucity of the vascular bed in the fibrotic portion of the oviducts. In all these cases the most dilated portions were located in the distal part of the tubes while the isthmus and uterotubal junction were not dilated. This may be due to the fact that the circular muscle layers in the latter areas are more compact and wider than in the other portions.g Thus these areas may be less distensible. Furthermore, muscular defects in the distended tubal wall resulting in impaired motility and/or a sphincter-like action at the uterotubal junction may possibly explain the retention of fluid despite a probe patent lumen throughout. The origin of hydrosalpinx fluid is still controversial. It may be derived either from continuous epithelial secretion or transudation from the vascular system through the wall. Most probably both mechanisms are involved.1° It is difficult to imagine that such a large accumulation of fluid could be produced by the few scattered secretory epithelial cells found in the hydrosalpinx specimens. Therefore transudation and/or lack of reabsorption through the wall may be the major factor. Selective transudation or reabsorption through the impaired wall may explain the low protein values found in our cases. On the other hand, release of granules by the secretory cells has long been considered the main mechanism of luminal secretion in the normal human tube, since the secreted material shows the same staining characteristics as the granules.ll No secretory activity in the ciliated cells was demonstrated by electron microscopy.l’> I2 Therefore, one would tend to explain the low protein values in the hydrosalpinx fluid studied, which showed a slight predominance of the albumin fraction and a very flat curve in the globulins fractions, as a reflection of the impaired secretory activity of the epithelial lining. It should be stressed, however, that the normal chemical compo-

410

David,

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and

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sition of human oviductal secretion is not yet available for comparison. In the primate (rhesus monkey) the protein values of normal oviduct fluid are known to be similar to those of the serum.l”, I’ Furthermore, the effect of a prolonged period of time during which the fluid is retained in the hydrosalpinx lumen, on its chemical composition is not known, Because of the large amount of fluid accumulating in the lumen of a hydrosalpinx, the most probable explanation of this phenomenon is that the wall plays the role of an abnormal semipermeable membrane inhibiting free passage. In the severely damaged areas this transudate from the vascular system is devoid of macromolecules similar to proteins, permitting passage only to water and electrolytes. Even if the normal areas where the epithelial cells are preserved had normal secretions of proteins, these would be diluted by the large volume of fluid present. The values of chloride, sodium, and potassium of the hydrosalpinx fluid in this series were similar or even higher than the serum values which supports the above explanation. Thus, one may suppose that a high protein content in hydrosalpinx fluid approximating the serum values of the patient may suggest improved functional results following corrective tubal surgery. Oviducts in these cases may have retained a more normal function permitting support of the early development of a fertilized egg.

Bicarbonan: irl the hlnnarr liydrosalpinw fluid studied seemed to be present in smali amounts when compared to lower mammals like the rabbit.‘“* Ifi No comparative figures arc available in the primate. These low values may be due to a number of factors such as: lo\\- provision of glucose and adenosine triphosphate across a fibrotic wall to the secretory cells, impaired metabolic activity of the epithelium lining with lack of carbonic anhydrase and/or abnormal anaerobic glycolysis of the endosalpinx,” and dilution of the bicarbonate in the large amount of hydrosalpingeal fluid. Whichever factor is responsible, the fact remains that low bicarbonate values may impair the future of the newly ovulated and fertilized eggI as well as the capacitation of sperm.” Conclusion In most cases of hydrosalpinx, attempts to restore the anatomic and functional integrity of the oviducts are probably illusory. Good surgical results are most likely when the physiologic properties of the oviduct are found to be near normal. Therefore chemical studies of hydrosalpingeal fluid through culdoscopic or colpotomy aspiration prior to operation may prove to be useful in determining the functional prognosis of the corrective procedure.

REFERENCES

1.

2. 3. 4. 5. 6. 7.

Blandau, R. J.: Gamete Transport, Comparative Aspects, presented at the Mammalian Oviduct: International Symposium, Washington State University-Pullman, August, 1967. Mastroianni, L.: Clin. Obst. & Gynec. 5:781, 1962. David, A., Brackett, B. G., and Garcia, C.-R.: Fertil. & Steril. 20: 250, 1969. Mastroianni, L., Beer, F., Shah, U., and Clewe, T. H.: Endocrinology 68: 92, 1961. Shirodkar, V. N.: Internat. J. Fertil. 11: 361, 1966. Noyes, R. W., Hertig, A. T., and Rock, J.: Fertil. & Steril. 1: 3, 1950. Novak, E. R., and Woodruff, J. D.: In Novak’s Gynecologic and Obstetric Pathology, ed. 6,

8. 9. 10. 11.

12. 13. 14.

Philadelphia, 1967> W. B. Saunders Company, p. 262. Novak, E., and Everett, H. S.: AM. J. OBST. & GYNEC. 16: 499, 1928. David, A., and Czernobilsky, B.: AM. J. OBST. & GYNECI. 101: 417, 1968. Bishop, D. W.: Am. J. Physiol. 187: 347, 1956. Fredricsson, B.: Histochemistry of the Oviduct, presented at the Mammalian Oviduct: International Symposium, Washington State University-Pullman, August, 1967. Clyman, M. J.: Fertil. & Steril. 17: 281, 1966. Marcus, S. L., and Savaris, Cl. A.: Fertil. & St&l. 16: 785, 1965. Marcus, S. L.: S. Forum 15: 381, 1964.

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15. David, A., Brackett, B. G., Garcia, C.-R., and Mastroianni, L.: J. Reprod. & Fertil. In press. 16. Hamner, C. E., and Fox, S. B. : Biochemir:try of Oviductal Secretions, presented at the Mammalian Oviduct: International Symposium, Washington State University-Pullman, August, 1967. 17. Mastroianni, L., Winter&z, W. W., and Lowi, N. P.: Fertil. & Steril. 9: 500, 1958.

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18. Stambaugh, R., Noriega, Cl., and Mastroianni, L.: J. Reprod. & Fertil. 18: 51, 1969. 19. Hamner, C. E., and Williams, W. L.: Proc. Sot. Exper. Biol. & Med. 117: 240, 1964. 3400 Spruce

Street

Philadelphia,

Pennsylvania

19104

Erratum

In the article, “Statistical standardization of amniocentesis data in erythroblastosis fetalis,” by Cohn Campbell, M.D., Robert B. Jaffe, M.D., and Bruce A. Work, M.D., in the June 15, 1969, issue of the JOURNAL, the last paragraph on page 558 (continued on 559) should read: The regression equation for Group I, . . . yielded an “a” coefficient of 0.29 and “b” coefficient of -fI.O071. The regression equation for Group II yielded an “a” coefficient of 0.39 and a “b” coefficient of -0.0092.