Increased activation of pelvic macrophages in infertile women with mild endometriosis

Increased activation of pelvic macrophages in infertile women with mild endometriosis JOUKO

HALME,

SUSANNE MARY

PH.D.

PH.D.

G. HAMMOND,

MADHWA

H.

SHAILAJA Chapel

M.D.,

BECKER, G. RAJ,

RAJ,

Hill,

North

M.D. M.D.

M.B.,

B.S.

Carolina

Pelvic fluid was collected from 66 women undergoing laparoscopic sttiratlon or diagnostic laparoscopy for evaluation of infertility. Cells consisting mainly of macrophages were separated, counted, and subjected to hi&chemical staining for acid phosphataae and myeloperotidase as markers of cell irritation. Pelvic fluid was analyzed for acid phoaphatase, neutrat protease, and extractable prostaglandin E2 and F,,. A higher proportlon (46% versus 15%) of the macrophages in the group with mild endometriosis exhibited positive stainlng for acid phosphatase as compared with the fertile group. Pelvic fluid from patients with mild endometriosis had higher acid phosphatase and neutral protease activity than that from fertile p&ems (p < 0.05, p < 0.01). The content of either prostaglandin was not significantly higher in the endometriosis group as compared with the fertile group. The results suggest that mild endometrlosis is assoctated with activation of macrophages and release of active substances into peritoneal ftuid that may be responsible for the associated infertility. (AM. J. OBSTET. GYNECOL. 145333, 1983.)

MILD ENDOMETRIOSIS is commonly found in women with primary or secondary infertility. Changes in the pelvic microenvironment caused by implants have been thought to lead to infertility n these patients.‘, * Recent studies have documented signs of increased irritation and inflammation in the pelvis of endometriosis patients, including increased concentration of peritoneal fluid prostaglandins and cellular components.‘, 2 However, signs of periodic pelvic inflammation have also recently been documented in normal fertile women.3 From the Divisions of Re@ductive Fertility and Gynecologic Oncology, Obstetrics and Gynecology, University School of Medicine.

Endocrinology and Department of of North Carolina

Supported by Grants l-RO-I-CA-21318-01 and PO-l -CA-29589-01 from the National Cancer Institute of the National Institutes of Health and partially by the Kenneth D. Dickinson Fund. Received

for publication

Revised August Accepted Reprint

July

12, 1982.

9, 1982.

September

I, 1982.

requests: Jouho Halme,

M.D.,

Ph.D.,

Department

of Obstetrics and Gynecology, 214 MacNiokr Building 202H, University of North Carolina School of Medicine, Chapel Hill, 0002-9378/83/090333+05$00.50/O

North

Carolina

27514.

@ 1983

The C. V. Mosby

Co.

In normal women with open fallopian tubes, the pelvic environment undergoes cyclic changes in its degree of irritation. During menstruation, very high numbers of these inflammatory cells are present in the pelvis, probably secondary to retrograde menstruation. Macrophages represent one line of response in the innate defense mechanism of the body. They originate in the bone marrow, circulate in blood as monocytes, and migrate to various body compartments where they undergo differentiation and possible activation. Passage of irritants through oviducts may lead to attraction of macrophages to the peritoneal cavity. This idea is further supported by the finding that the concentration of these cells is low in patients with obstructed fallopian tubes.3 Patients with endometriosis tend to have higher than normal numbers of pelvic macrophages. This may be secondary to local effects by the implants. The presence of higher numbers and/or more aggressive pelvic macrophages in endometriosis around the time of ovulation has been suggested as a mechanism by which this disease may cause infertility.3 Recent data by Muscat0 and associates4 has indicated that pelvic macrophages in endometriosis may in fact have higher capacity to phagocytize sperm. The pres333

334

Halme

February Am. J. Obstet.

et al.

I

n=26 Fertile

n= 18

Infertile with Endometriosir

n=IO Unknown InfertIlity

Fig. 1. The concentration of macrophages in samples. The bars indicate the mean macrophage tion, and the hwiwntal lines indicate 2SEM. The between groups are not statistically significant (p

pelvic fluid concentradifferences > 0.05).

ent study is an attempt to further characterize these cells in different patient populations by use of cytochemical methods. We also find chemical evidence in the pelvic fluid that suggests increased macrophage activation in mild endometriosis. Some of these macrophage-related products might also be responsible for adverse effects on fertility.

Material and methods. Subjects. The subjects were 28 infertile women undergoing diagnostic laparoscopy as part of an infertility evaluation and 38 fertile patients undergoing laparoscopic sterilization. Of the latter group, ten were using combination oral contraceptives and 28 were using no medication. All fertile patients had regular cycles and were multiparous. Each of the 28 infertile patients had been infertile for a minimum of 2 years and had undergone routine evaluations, consisting of physical and pelvic examinations, semen analysis, serum prolactin and testosterone determinations, thyroid panel, midluteal progesterone determination, 3 months of basal body temperature, and hysterosalpingography. All of these studies were normal. Laparoscopic procedures were done between days 15 and 21 of the menstrual cycle. Eighteen patients were found to have endometriosis, which was graded according to the American Fertility Society classification.5 All 18 patients were classified as having mild disease as defined by this classification. Fluid collection and analysis. Peritoneal fluid was aspirated under direct visual control from the posterior cul-de-sac and anterior uterovesical compartment through a laparoscopic silicone rubber catheter or a

I, 1983 Gynecol.

Veress needle by way of a second puncture. Aspiration was done before the patient was placed into the Trendelenburg position in order to be sure that all peritoneal fluid was collected. The fluid volume was recorded, and the sample was immediately placed in ice-cold tubes containing heparin. The tubes were centrifuged at 300 X g and the cells in the pellet were counted in a hemacytometer and thereafter subjected to morphologic analysis. If the fluid contained an excess of erythrocytes, it was underlayered with Ficoll-Hypaque (LSM, Litton, Bionetics, Lanexa, Maryland) and centrifuged for 10 minutes at 400 x g. The interface was then analyzed for macrophages. Identification of macropbages. Macrophages were identified by morphologic features and by nonspecific esterase stain6 on cytocentrifuge preparations. Cytochemical stains. Cytocentrifuge preparations on peritoneal cells were stained for nonspecific esterase according to the method of Yam and colleagues,6 for myeloperoxidase according to that of Kaplow,? and for acid phosphatase according to that of Burst0ne.s Peritoneal fluid supernatants were analyzed for protein content by Lowry9 with crystalline bovine serum albumin used as a standard. Fluid acid phosphatase activity was measured by the method of Musson and coworkerslo and neutral protease with azocoll as substrate at pH 7.4 as described by Saheki and Holzer.” Prostaglandins Ez (PGE2) and F,, (PGF,,) were extracted and measured with specific radioimmunoassays as described by Jaffe and Behrman.r2 Briefly, 1 ml of peritoneal fluid was extracted with 3 ml of petroleum ether, and the upper ether layer was discarded. The aqueous layer was again extracted with 3 ml of a solvent consisting of petroleum ether : isopropyl ethanol : 1M hydrochloric acid (3 : 1: 1). Phases were separated by addition of 2 ml of ethyl acetate plus 3 ml of water, followed by low-speed centrifugation. The organic phase was evaporated to dryness at 50” C in a nitrogen atmosphere. Two different-size samples in duplicates were then used in radioimmunoassays with specific antisera for PGF,, or PGE, in order to document the dose-response linearity in the assay. The intra-assay variation was 8% and interassay variation was 15%. The cross-reaction between PGEz and PGF,, was less than 10%. Statistical analysis. The statistical analysis of the data was done with the unpaired Student’s t test and Wilcoxon’s rank sum test in comparison of different groups of variables. A p value of ~0.05 was used as an indication of a significant difference between groups. Re9UltS

The majority (about 90%) of the cells in peritoneal fluid were macrophages in all patients studied. How-

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Pelvic macrophages and endometriosis

I

Fertile

Sndometriosis

335

Unknown lnfsrtilitv

. 0 . 0

. . :

AP

MP

AP

0 0 t

AP

MP

MP

Fig. 4. The percentage of positive macrophages for acid phosphatase (MP)

(API and myeloperoxidase in pelvic fluid cell pellets. The solid circles = AP and the open circles = MP.

ever, heterogeneity was obvious in each macrophage population. Some samples contained mainly small monocyte-type macrophages, while others had large histiocyte-like macrophages. The majority of the samples contained a mixture of macrophages representing different stages of maturation. The endometriosis samples tended to have macrophages of larger size. Cytoplasmic vacuolization and content of phagocytosed erythrocytes and granulocytes was variable and not confined to one group of patient samples. The mean concentration of macrophages in pelvic fluid of fertile patients was 0.7 million per milliliter (Fig. 1). Macrophage concentration was slightly higher in patients with endometriosis (0.99 million per milliliter) or unexplained infertility (2.1 million per milliliter). These differences were not statistically significant. Cytochemical analysis of two enzyme markers, acid phosphatase and myeloperoxidase, revealed that, as a group, patients with endometriosis had higher proportions of macrophages with acid phosphatase activity (mean = 45.1%) than did the group of fertile patients (mean, 15.4%), and this difference is significant (p < 0.01) by Wilcoxon rank sum test (Fig. 2). The differences between other groups of patients were not significant. The proportion of myeloperoxidase-positive macrophages was not significantly different in any of the infertile patient groups as compared with fertile women. This is a marker for recently arrived monocyte-type peritoneal macrophages. Some individual samples from normal patients without evidence of endometriosis could be highly acid phosphatase positive and low in myeloperoxidase activity, indicating a wide variation in macrophage characteristics even in normal women around the postovulatory period. The analysis of pelvic fluid composition revealed

/ n=26 Fertile

n=l6 Infefttte with Endcmetriosis

n=IO Unknown Infertility

Fig. 3. Activity of acid phosphatase and neutral protease in the pelvic fluid supernatant in different patient populations. The hatched bars indicate mean acid phosphatase activity (fertile versus endometriosis, p < 0.05).OpenbQ7sindicate mean neutral protease activity (fertile versus endometriosis, p < 0.01). Vertical lines indicate 2 SEM.

protein concentration to be very similar in all patient groups. The mean (+-SEM) protein concentration in the fertile population was 41.5 + 6.0 mglml, that in the endometriosis patients was 37.3 + 6.3 mg/ml, and that in the unexplained infertility patients was 45.3 t 9.0 mg/ml. In order to gain further information of macrophage activation in pelvic fluid environment, pelvic fluid supernatants were also assayed for markers implicated in this activation. Fluid acid phosphatase activity was significantly higher in patients with endometriosis

Volume Number

Correspondence

145 3

stated that their data showed a marked increase in the incidence of neural tube defects (NTDs) in insulindependent diabetic patients which can best be detected by routine a-fetoprotein (AFP) screening in this group. All but one (87.5%) of their affected infants had anencephaly, a condition which is best diagnosed by ultrasound examination. Furthermore, the authors made no mention of the false negative rate for this test of an NTD. They have previously reported that up to 12.5% of patients with a NTD will have a normal serum AFP level.’ It is regrettable that the authors plotted only three of the eight values from affected pregnancies. Two of these three values lie very close to the upper limit for the normal population and give little comfort to the clinician faced with a patient with unsure dates. This is particularly disturbing as the authors pointed out that the curve for the fetus of the diabetic patient may be retarded by 2 weeks compared to the curve for the nondiabetic population. Furthermore, we are left without any information on five of the affected fetuses. We believe the authors would agree that it is possible that one of these patients may have had a normal maternal serum AFP level. There are several other bothersome issues. The data base used to establish the importance of amniocentesis in the diabetic patient with a single abnormal serum AFP screening test was not mentioned. The authors failed to provide this important information about the accuracy of a single abnormal maternal serum AFP level in the diabetic pregnancy. Finally, although the authors stated that they may have had some selection bias, there are too few data points for affected fetuses for a general prediction to be made about incidence rates and for recommendations to be given for the implementation of the screening program in diabetic patients. Finally, how do Milunsky and associates explain their high incidence of NTDs in infants of diabetic patients? Lawrence Steven Department of Obstetrics and Gynecology Division of Maternal-Fetal Medicine Los Angeles CountylUniversity Southern Medkal Center 5K40, Women’s Hospital 1240 North Mission Road Los Angeles, California 90033

qf

D. Platt, H. Go&,

M.D. M.D.

California

REFERENCE

1. Milunsky, A., Alpert, E., Neff, R. K., and Frigoletto, D.: Prenatal diagnosis of neural tube defects. IV. Maternal serum alpha-fetoprotein screening, Obstet. Gynecol. 55: 60, 1980.

Reply to Drs. Pkttt end Golde To the Editors:

We are pleased to be able to reply to the letter of Drs. Platt and Golde. We regard our paper on maternal

385

serum AFP screening in diabetic pregnancy as particularly important to patient care. The language used is precise and the comments of Drs. Platt and Golde reflect that they have not carefully read the article. We reply to their specific points as follows. 1. We made no claim that serum AFP screening is the best method of detecting NTDs. Two recent experiences with open spina bifida missed on ultrasound examination, performed in two excellent departments, confirm our view of the adjunctive need for both serum AFP screening and ultrasonographic studies between 16 and 18 weeks’ gestation. 2. Platt and Golde complain that we made no mention of the false negative rate but then immediately note that we have previously reported a figure of 12.5% and indicate in the introduction known limits of detection with serum AFP screening. From the data presented it is obvious that we detected ail three NTDs among the 322 diabetic women from whom we obtained serum samples. Hence in this studs there were no false negative results. 3. The complaint is made that we plotted only three AFP values from affected pregnancies. Since we did not receive serum samples on the other live affected pregnancies, obviously their values could not be plotted. Platt and Golde have misinterpreted the purpose of Fig. 1 when complaining that two of the three cases with NTDs have values which “lie very close to the upper limit for the normal population.” Our Fig. 1 demonstrates that the values for anencephaly lie at a considerable distance from the upper limit of normal for diabetic pregnancy. They seem to be missing the essential thrust that a different set of curves for the normal range is required for the evaluation of serum AFP in diabetic pregnancy. 4. They complain about no information on five of the affected fetuses. From the text and Fig. 1 it is obvious that the five cases in which no serum was obtained constituted four with anencephaly and one with spina bifida. Since the one infant with spina bifida had a large open lesion and the others had anencephalt,, we would disagree that the serum AFP level would have been normal in any of these patients. 5. We did not “establish the importance of amniocentesis in the diabetic patient with a single abnormal serum AFP ..” In discussion we said that “a good case could be made” and this will be pursued further in a subsequent publication. 6. We did not fail to provide “important information” about the accuracy of a single abnormal serum AFP level in the diabetic pregnancy. Once again. we did not miss any of the three NTDs, all having associated high maternal serum AFP levels in the first sample. Provided that reference is made to a diabetic pregnancy serum AFP curve, there should be no basis for expectation of a different rate of accuracy when compared to that of nondiabetic pregnancy. 7. No claim has been made about the general incidence of NTDs in diabetic pregnancy. We reported at

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REFERENCES

1. Drake, T. S., O’Brien, W. F., Ramwell, P. W., and Metz, S. A.: Peritoneal fluid thromboxane Bz and 6-ketoprostaglandin F,, in endometriosis, AM. J. OBSTET. GYNECOL. 140~401, 1981. 2. Haney, A. F., Muscato, J. J., and Weinberg, J. B.: Peritoneal fluid cell populations in infertility patients, Fertil. Steril. 35:696, 1981. 3. Halme, J.. Becker, S., Hammond, M. G., and Raj, S. G.: Pelvic macrophages in normal and infertile women: The role of patent tubes, AM. J. OBSTET. GYNECOL. 142:890, 1982. 4. Muscato, J. J., Haney, A. F., and Weinberg, J. B.: Sperm phagocytosis by human peritoneal macrophages: A possible cause of infertility in endometriosis. AM. J. OBSTET. GYNECOL. 144:503, 1982. 5. The American Fertility Society: Classification of endometriosis. Fertil. Steril. 33:633, 1979. 6. Yam, L. T., Lee, C. Y., and Crosby, W. H.: Cytochemical identitication of monocytes and granulocytes, Am. J. Clin. Pathol. 55:283, 1971. 7. Kaplow, L. S.: Simplified myeloperoxidase stain using benzidine dihydrochloride, Blood 26:215, 1965. 8. Burstone, M. S.: Histochemical demonstration of acid phosphatase activity in osteoclasts, J. Histochem. Cytothem. 7:39, 1959. 9. Lowry, 0. H., Rosebrough, N. J., Fort, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent,J. Biol. Chem. 193:265, 1951.

10. Musson, R. A., Shafran, H., and Henson, P. M.: Intracellular levels and stimulated release of lysosomal enzymes from human peripheral blood monocytes and monocyte-derived macrophages, J. Reticuloendothel. Sot. 28:249, 1980. 11. Saheki, T., and Holzer, H.: Comparisons of the tryptophan synthase inactivating enzymes with proteinases from yeast, Eur. J. B&hem. 42:621, 1974. 12. Jaffe, B. M., and Behrman, H. R.: In Jaffe, B. M., and Behrman, H. R., editors: Methods of Hormone Radioimmunoassay, New York, 1979, Academic Press, Inc., pp. 119-42. 13. Dmowski, W. P., Steele, R. W., and Baker, G. F.: Deficient cellular immunity in endometriosis, AM. J. OBSTET. GYNECOL. 141:377, 1981. 14. Koninckx, P. R., Ide, P., Vandenbroucke, W.. and Brosens, I. A.: New aspects of pathophysiology of endometriosis and associated infertility, J. Reprod. Med. 24:257, 1980. 15. Davies, P., and Bonney, R. J.: Secretory products of mononuclear phagocytes: A brief review, J. Reticuloendothel. Sot. 26:37, 1979. 16. Rock, J. A., Dubin, N. H., Ghodgaonkar, R. B., Bergquist, C. A., and Kimball, A. W. Jr.: Cul-de-sac fluid in women with endometriosis: Fluid volume and prostanoid concentration during the proliferative phase of the cycle (d 8-121, In Scientific Abstracts, Twenty-ninth Annual Meeting of the Society for Gynecologic Investigation, Dallas, Texas, March 24-27. 1982. (Abst. 4X.1