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Amniotic fluid trypsin and premature rupture of membranes
CORRESPONDENCE The detection of facial anomalies with ultrasound To the Editors: I feel that the article by Pilu et al. (Prenatal diagnosis of craniofacial malformations with ultrasonography. AMJ OBSTET GvNECOL 1986;155:45-50) gives a falsely optimistic view of the sonographic prental diagnosis of craniofacial abnormalities, particularly those involving only the face. Thirteen of the 14 patients in whom facial abnormalities were diagnosed were associated with major, readily diagnosable cranial malformations. In all of these cases, the cranial findings were of primary importance. Both of the patients in whom there were facial abnormalities not associated with obvious abnormal cranial findings (cases 15 and 16) were missed, although technically adequate examinations of the fetal face were obtained. The sensitivity and specificity of sonographic examination in the diagnosis of fetal facial malformations remain to be demonstrated. The implication that its accuracy is currently well established is a disservice to both referring physicians and their patients, who may have unrealistic expectations. It also creates a dilemma for the consultant sonologist, who may be expected to deliver accuracy that may not be presently obtainable. William L. Koontz, M.D. Department of Obstetrics and Gynecology Division of Maternal-Fetal Medicine Ambulatory Care Building University of Louisville Louisville, Kentucky 40292
Reply To the Editors: In reply to Dr. Koontz's letter, we feel that an important distinction has to be made. In our study (AM J 0BSTET GYNECOL 1986;155:45-50), a selected obstetric population at high risk for fetal craniofacial malformation was considered. In each case a detailed and time-consuming ultrasound examination of the fetal face was performed. It is quite obvious that sensitivity and specificity of sonography would be much different in a low-risk obstetric population. With regard to this issue, the interested reader is referred to the very interesting article by Hegge et al. 1 that has been recently published. Our experience indicates that a targeted examination of the fetal face yields great accuracy in the early prenatal diagnosis of many craniofacial anomalies whether the risk factor is a positive familial history, exposure to teratogens, or an associated fetal anomaly. However, a distinction has to be made. Hypotelorism, hypertelorism, cleft lip, and cleft lip-cleft palate are usually 1316
readily identifiable. Some craniofacial malformations, such as micrognathia, can probably be diagnosed only late in gestation, and it is likely that isolated cleft palate is not amenable to prenatal diagnosis. The considerable experience that was collected at Bologna University since our original manuscript was submitted for publication is well correlated with our preliminary data. Although a study with a design similar to ours has not yet appeared in the literature, the increasing number of recent reports dealing with both sophisticated investigation of subtle details of the normal anatomy of fetal facial structures 2 and rather large series of welldocumented fetal craniofacial malformations diagnosed before birth 1· 3 seem to support our view. Gianluigi Pilu, M.D. Luciano Bovicelli, M.D. Section of Prenatal Pathophysiology Second Department of Obstetrics and Gynecology University of Bologna School of Medicine Via Massarenti 13 40138 Bologna, Italy REFERENCES l. Hegge FN, Prescott GH, Watson PT. Fetal facial abnor-
malities identified during obstetric sonography. J Ultrasound Med 1986;5:684. 2. Jeanty P, Romero R, Staudach A, eta!. Facial anatomy of the fetus. J Ultrasound Med 1986;5:607. 3. Saltzman DH, BenacerrafBR, Frigoletto FD. Diagnosis and management of fetal facial clefts. AM J OBSTET GYNECOL 1986;155:377.
Amniotic fluid trypsin and premature rupture of membranes To the Editors: We read the article, "The relationship between amniotic fluid trypsin activity and premature rupture of membranes," (AM J 0BSTET GYNECOL 1986;155: 1043-8) by Kanayama et al. with interest and admiration. Nevertheless, we suggest that: (1) the principal conclusion is inaccurately expressed; (2) the case for amniotic fluid trypsin as the principal culprit in premature rupture of membranes remains unproved; and (3) other factors and information should be considered. The authors write that they demonstrated trypsin activity in amniotic fluid. Rather, they have measured "trypsin-like" enzymatic activity, since the chromogenic substrate used (N-benzyl-L-isoleucyl-L-glutamyl-glycylL-argenine-p-nitroanilide) demonstrates proteolytic activity by a number of other serine proteases. Interestingly, this same substrate is used to assay coagulation factors II (prothrombin) and X (Stewart-Prower factor).1· 2 A nearly identical synthetic substrate is also used to measure plasminogen activator, plasmin, and urokinase activity.'· 1 Similarly, u 1-antitrypsin is not specific
Correspondence
Volume 157 Number 5
1317
Table I. Comparative measures of the tensile strength of human amniochorion after 24 hours at 37° C, 6% C0 2 incubation with various enzyme and control solutions; note that trypsin demonstrated the least impairment of membrane strength of enzymes measured N
Buffer control Collagenase, I 0 fLg/ml Collagen, 20 fLg/ml Trypsin, I 0 fLg/ml Trypsin, 20 fLg/ml Elastase, 20 ·fLg/ml
Pseudomonas aeruginosa (collagenase-producing)
and inhibits a number of trypsin-like proteases. 5 Therefore the effects of other serine proteases and antiproteases were likely also measured in these tests. One of these enzymes measured was probably plasmin. Burgos et al. 6 and Jenkins et al. 7 demonstrated plasminogen/plasmin in amniochorion and suggested that focal concentrations of this protease participate in the pathogenesis of premature rupture of membranes. It is likely that the 2-week gestational age difference between the premature rupture of membrane (31 weeks) and control (33 weeks) groups influenced the results. Pocknee and Abramovich 8 demonstrated that amniotic fluid trypsin (determined by radioimmunoassay) decreased rapidly near term. Milwidsky et a!." could find no differences in amniotic fluid or fetal membrane acid protease activity among women with or without premature rupture of membranes. Other information suggest that trypsin is not as important in premature rupture of membranes as hypothesized: (1) Since trypsin-like activity is dispersed in amniotic fluid, it would act to "cover the waterfront" and diffusely weaken amniochorion. Studies performed during the past century do not demonstrate overall weakening of prematurely ruptured membranes.10 (2) Furthermore, Lavery and Miller 11 noted that meconium exposure decreased strength and elasticity of fetal membranes. They suggested that intrauterine exposure to fetal gastrointestinal enzymes, prominently including trypsin present in meconium, predisposes to premature rupture of membranes. However, meconium staining and premature rupture of membranes could not be correlated in a large clinical review. 11 Previous studies consistently show localized inflammation and/or necrosis adjacent to the rupture site. 12, 13 Membrane thinning reported here is much more dramatic than that measured by Artal et a!. 10 In two earlier studies, Polishuk et al. 1"· 15 noted great variability but no consistent evidence of fetal membrane thinning in premature rupture of membranes. While examining the possibility that ascending, transcervical infection and/or inflammation may focally impair membrane integrity, wei" have assessed the effects of various bacterial and mammalian proteases on the physical properties of fresh human amniochorion. Although bacterial collagenase (Sigma Chemical Co.,
5
5 5 5 5 5 5
Mean%
100 29 11 92 86 <10 <10
SD% ± 12
±19 ±30 ± 15 ±26
St. Louis, Mo.), porcine elastase (Sigma), and collagenase-producing Pseudomonas aeruginosa markedly reduced measurements of fetal membrane tensile strength, bovine pancreatic trypsin preparations (Sigma) did not (Table I). Preliminary work shows that human protease-containing neutrophil lysate also reduces membrane strength and elasticity, whereas lymphocyte lysate and other control preparations do not. Sbarra et al. 17 induced similar membrane weakening with direct application of phospholipase A 2 to fetal membranes, which suggests that other enzymatic systems are also involved. We suggest that highly localized fetal membrane impairment by bacterial-, maternal-, and/or fetal-derived proteases and other substances are probably more important in the pathogenesis of premature rupture of membranes than are trypsin-like substances dispersed in amniotic fluid. Certainly, focal destruction of protease inhibitors could allow damage by a number of proteolytic enzymes. James A. McGregor, M.D.C.M. Janice I. French, C.N.M. David Lawellin, Ph.D. University of Colorado H eatlh Sciences Center 4200 East Ninth Avenue Denver, Colorado 80262
REFERENCES 1. Board PG. The use of ftuorogenic peptide substrates for the detection of coagulation factors II and X after electrophoresis. Ann Hum Gene~ 1982;46:293. 2. Egberg N, Heedman PA. Simplified performance of amidolytic X factor assay. Thrombosis Res 1982;25:437. 3. Nieuwenhuizena W, Wijngaards G, Groeneveld E. Fluorogenic peptide amide substrates for the estimation of plasminogen activators and plasmin. Analytical Biochem 1977;83:143. 4. Barlow GH, Marder VJ. Plasma urokinase levels measured by chromogenic assay after infusions of tissue culture as urinary source material. Thrombosis Res 1980; 18:431. 5. Beatty J, Bieth J, Travis J. Kinetic of association of serine proteases with inactive and oxidized alpha 1 proteinase inhibitor and alpha 1 antichymotrypsin. J Bioi Chern 1980;256:3931-4. 6. Burgos H, Hsi B-L, Yeh C-JG, eta!. Plasminogen binding by human amniochorion. A possible factor in premature
1318 Correspondence
7.
8. 9.
10.
11. 12. 13. 14. 15. 16. 17.
November 1987 Am J Obstet Gynecol
rupture of membranes. AM J 0BSTET GYNECOL '1982; 143:958. Jenkins DM, O'Neill M, Mattar M, et a!. Degenerative changes and detection of plasminogen in fetal membranes that rupture prematurely. Br J Obstet Gynecol 1983; 90:841. Pocknee RC, Abramovich DR. Origin and levels of trypsin in amniotic fluid throughout pregnancy. Br J Obstet Gynecol 1982;89: 14'2. Milwidsky A, Hurwitz A, Eckstein L, Mayer M, Gutman A. Proteolytic enzymes in human fetal membranes and amniotic fluid. Enzyme 1985;33: 188-96. Artal R, Sokol RJ, Neuman M, eta!. The mechanical properties of prematurely and non-prematurely ruptured membranes: methods and preliminary results. AM J OsSTET GYNECOL 1976;125:655. Lavery JP, Miller CE, Johns P. Effect of meconium on the strength of chorioamniotic membranes. Obstet Gynecol 1980;56:711. Knox IC, Hoerner JK. The role of infection in premature rupture of membranes. AM J 0BSTET GYNECOL 1950; 59:190-4. Bourne G. The human amnion and chorion. Chicago: Year Book Medical Publishers, 1962:175-92. Polishuk WZ, Kahane S, Peranio A. The physical properties of fetal membranes. Obstet Gynecol 1962;20:204. Polishuk WZ, Kahane S, Hadar A. Fetal weight and membrane tensile strength. AM J 0BSTET GYNECOL 1964; 88:247. McGregor JA, French JI, Lawellin D, et al. Bacterial protease-induced reduction of chorioamniotic membrane strength and elasticity. Obstet Gynecol 1987;69:167. Sbarra AJ, Selvaraj RJ, Cetrulo CL, et al. Infection and phagocytosis as possible mechanisms of rupture in premature rupture of the membrane. AM J 0BSTET GYNECOL 1985;153:38.
-+-TRYPSIN
CONTROL
«1 (ill)
Reply To the Editors: I thank Dr. James A. McGregor and his colleagues for their interest in this report. We also agree with their result that the main cause of premature rupture of membranes is latent chorioamnionitis. Premature rupture of membranes was caused by latent chorioamnionitis in about 60% of the cases in our study. However, chorioamnionitis is not recognized in all fetal membranes with premature rupture of membranes. For example, patients have no pathogenic bacteria or inflamed cells in or around the cervix, no uterine contractions, and their cervixs are closed. Furthermore, the microscopic observations of amniochorion do not contain chorioamnionitis. Even such cases sometimes produce premature rupture of the membranes. What is the mechanism of causation in such cases? We supposed that trypsin in amniotic fluid had proteolytic power according to the circumstances. Trypsin activity in such patients was higher than in controlled groups. We measured trypsin activity with S2222(KabiVitrum Co., Sweden), which is specific for trypsin. As shown in Table I, there is little possibility in measuring thrombin, factor Xa, and plasmin by 82222. Moreover, we obtained data opposite to theirs, namely, that type III collagen in amnion was degraded by trypsin in amniotic fluid (bovine) and meconium in amniotic fluid (human) (Fig. 1). Thus under what circumstance~ does trypsin modify its proteolytic activity in amniotic fluid? We recently measured trypsin levels
-+- MECONIUM
crl(ill)
«1 (V) crl( I)
Fig. I. Electrophoresis (7.5% polyacrylamide gel) of collagen in amnion. (Left lane = Normal amniotic collagen; middle lane = amniotic collagen (100 mg) treated with trypsin [10 mg] at 35° C for 6 hours; right lane = amniotic collagen [100 mg] treated with meconium [10 mg] at 35° C for 6 hours.) The migration of type I, Ill, and V a-chains is shown. Note the marked reduction of type Ill collagen in the middle lane and the slight reduction of that in the right lane.
Reply To the Editors: In reply to Dr. Koontz's letter, we feel that an important distinction has to be made. In our study (AM J 0BSTET GYNECOL 1986;155:45-50), a selected obstetric population at high risk for fetal craniofacial malformation was considered. In each case a detailed and time-consuming ultrasound examination of the fetal face was performed. It is quite obvious that sensitivity and specificity of sonography would be much different in a low-risk obstetric population. With regard to this issue, the interested reader is referred to the very interesting article by Hegge et al. 1 that has been recently published. Our experience indicates that a targeted examination of the fetal face yields great accuracy in the early prenatal diagnosis of many craniofacial anomalies whether the risk factor is a positive familial history, exposure to teratogens, or an associated fetal anomaly. However, a distinction has to be made. Hypotelorism, hypertelorism, cleft lip, and cleft lip-cleft palate are usually 1316
readily identifiable. Some craniofacial malformations, such as micrognathia, can probably be diagnosed only late in gestation, and it is likely that isolated cleft palate is not amenable to prenatal diagnosis. The considerable experience that was collected at Bologna University since our original manuscript was submitted for publication is well correlated with our preliminary data. Although a study with a design similar to ours has not yet appeared in the literature, the increasing number of recent reports dealing with both sophisticated investigation of subtle details of the normal anatomy of fetal facial structures 2 and rather large series of welldocumented fetal craniofacial malformations diagnosed before birth 1· 3 seem to support our view. Gianluigi Pilu, M.D. Luciano Bovicelli, M.D. Section of Prenatal Pathophysiology Second Department of Obstetrics and Gynecology University of Bologna School of Medicine Via Massarenti 13 40138 Bologna, Italy REFERENCES l. Hegge FN, Prescott GH, Watson PT. Fetal facial abnor-
malities identified during obstetric sonography. J Ultrasound Med 1986;5:684. 2. Jeanty P, Romero R, Staudach A, eta!. Facial anatomy of the fetus. J Ultrasound Med 1986;5:607. 3. Saltzman DH, BenacerrafBR, Frigoletto FD. Diagnosis and management of fetal facial clefts. AM J OBSTET GYNECOL 1986;155:377.
Amniotic fluid trypsin and premature rupture of membranes To the Editors: We read the article, "The relationship between amniotic fluid trypsin activity and premature rupture of membranes," (AM J 0BSTET GYNECOL 1986;155: 1043-8) by Kanayama et al. with interest and admiration. Nevertheless, we suggest that: (1) the principal conclusion is inaccurately expressed; (2) the case for amniotic fluid trypsin as the principal culprit in premature rupture of membranes remains unproved; and (3) other factors and information should be considered. The authors write that they demonstrated trypsin activity in amniotic fluid. Rather, they have measured "trypsin-like" enzymatic activity, since the chromogenic substrate used (N-benzyl-L-isoleucyl-L-glutamyl-glycylL-argenine-p-nitroanilide) demonstrates proteolytic activity by a number of other serine proteases. Interestingly, this same substrate is used to assay coagulation factors II (prothrombin) and X (Stewart-Prower factor).1· 2 A nearly identical synthetic substrate is also used to measure plasminogen activator, plasmin, and urokinase activity.'· 1 Similarly, u 1-antitrypsin is not specific
Correspondence
Volume 157 Number 5
1317
Table I. Comparative measures of the tensile strength of human amniochorion after 24 hours at 37° C, 6% C0 2 incubation with various enzyme and control solutions; note that trypsin demonstrated the least impairment of membrane strength of enzymes measured N
Buffer control Collagenase, I 0 fLg/ml Collagen, 20 fLg/ml Trypsin, I 0 fLg/ml Trypsin, 20 fLg/ml Elastase, 20 ·fLg/ml
Pseudomonas aeruginosa (collagenase-producing)
and inhibits a number of trypsin-like proteases. 5 Therefore the effects of other serine proteases and antiproteases were likely also measured in these tests. One of these enzymes measured was probably plasmin. Burgos et al. 6 and Jenkins et al. 7 demonstrated plasminogen/plasmin in amniochorion and suggested that focal concentrations of this protease participate in the pathogenesis of premature rupture of membranes. It is likely that the 2-week gestational age difference between the premature rupture of membrane (31 weeks) and control (33 weeks) groups influenced the results. Pocknee and Abramovich 8 demonstrated that amniotic fluid trypsin (determined by radioimmunoassay) decreased rapidly near term. Milwidsky et a!." could find no differences in amniotic fluid or fetal membrane acid protease activity among women with or without premature rupture of membranes. Other information suggest that trypsin is not as important in premature rupture of membranes as hypothesized: (1) Since trypsin-like activity is dispersed in amniotic fluid, it would act to "cover the waterfront" and diffusely weaken amniochorion. Studies performed during the past century do not demonstrate overall weakening of prematurely ruptured membranes.10 (2) Furthermore, Lavery and Miller 11 noted that meconium exposure decreased strength and elasticity of fetal membranes. They suggested that intrauterine exposure to fetal gastrointestinal enzymes, prominently including trypsin present in meconium, predisposes to premature rupture of membranes. However, meconium staining and premature rupture of membranes could not be correlated in a large clinical review. 11 Previous studies consistently show localized inflammation and/or necrosis adjacent to the rupture site. 12, 13 Membrane thinning reported here is much more dramatic than that measured by Artal et a!. 10 In two earlier studies, Polishuk et al. 1"· 15 noted great variability but no consistent evidence of fetal membrane thinning in premature rupture of membranes. While examining the possibility that ascending, transcervical infection and/or inflammation may focally impair membrane integrity, wei" have assessed the effects of various bacterial and mammalian proteases on the physical properties of fresh human amniochorion. Although bacterial collagenase (Sigma Chemical Co.,
5
5 5 5 5 5 5
Mean%
100 29 11 92 86 <10 <10
SD% ± 12
±19 ±30 ± 15 ±26
St. Louis, Mo.), porcine elastase (Sigma), and collagenase-producing Pseudomonas aeruginosa markedly reduced measurements of fetal membrane tensile strength, bovine pancreatic trypsin preparations (Sigma) did not (Table I). Preliminary work shows that human protease-containing neutrophil lysate also reduces membrane strength and elasticity, whereas lymphocyte lysate and other control preparations do not. Sbarra et al. 17 induced similar membrane weakening with direct application of phospholipase A 2 to fetal membranes, which suggests that other enzymatic systems are also involved. We suggest that highly localized fetal membrane impairment by bacterial-, maternal-, and/or fetal-derived proteases and other substances are probably more important in the pathogenesis of premature rupture of membranes than are trypsin-like substances dispersed in amniotic fluid. Certainly, focal destruction of protease inhibitors could allow damage by a number of proteolytic enzymes. James A. McGregor, M.D.C.M. Janice I. French, C.N.M. David Lawellin, Ph.D. University of Colorado H eatlh Sciences Center 4200 East Ninth Avenue Denver, Colorado 80262
REFERENCES 1. Board PG. The use of ftuorogenic peptide substrates for the detection of coagulation factors II and X after electrophoresis. Ann Hum Gene~ 1982;46:293. 2. Egberg N, Heedman PA. Simplified performance of amidolytic X factor assay. Thrombosis Res 1982;25:437. 3. Nieuwenhuizena W, Wijngaards G, Groeneveld E. Fluorogenic peptide amide substrates for the estimation of plasminogen activators and plasmin. Analytical Biochem 1977;83:143. 4. Barlow GH, Marder VJ. Plasma urokinase levels measured by chromogenic assay after infusions of tissue culture as urinary source material. Thrombosis Res 1980; 18:431. 5. Beatty J, Bieth J, Travis J. Kinetic of association of serine proteases with inactive and oxidized alpha 1 proteinase inhibitor and alpha 1 antichymotrypsin. J Bioi Chern 1980;256:3931-4. 6. Burgos H, Hsi B-L, Yeh C-JG, eta!. Plasminogen binding by human amniochorion. A possible factor in premature
1318 Correspondence
7.
8. 9.
10.
11. 12. 13. 14. 15. 16. 17.
November 1987 Am J Obstet Gynecol
rupture of membranes. AM J 0BSTET GYNECOL '1982; 143:958. Jenkins DM, O'Neill M, Mattar M, et a!. Degenerative changes and detection of plasminogen in fetal membranes that rupture prematurely. Br J Obstet Gynecol 1983; 90:841. Pocknee RC, Abramovich DR. Origin and levels of trypsin in amniotic fluid throughout pregnancy. Br J Obstet Gynecol 1982;89: 14'2. Milwidsky A, Hurwitz A, Eckstein L, Mayer M, Gutman A. Proteolytic enzymes in human fetal membranes and amniotic fluid. Enzyme 1985;33: 188-96. Artal R, Sokol RJ, Neuman M, eta!. The mechanical properties of prematurely and non-prematurely ruptured membranes: methods and preliminary results. AM J OsSTET GYNECOL 1976;125:655. Lavery JP, Miller CE, Johns P. Effect of meconium on the strength of chorioamniotic membranes. Obstet Gynecol 1980;56:711. Knox IC, Hoerner JK. The role of infection in premature rupture of membranes. AM J 0BSTET GYNECOL 1950; 59:190-4. Bourne G. The human amnion and chorion. Chicago: Year Book Medical Publishers, 1962:175-92. Polishuk WZ, Kahane S, Peranio A. The physical properties of fetal membranes. Obstet Gynecol 1962;20:204. Polishuk WZ, Kahane S, Hadar A. Fetal weight and membrane tensile strength. AM J 0BSTET GYNECOL 1964; 88:247. McGregor JA, French JI, Lawellin D, et al. Bacterial protease-induced reduction of chorioamniotic membrane strength and elasticity. Obstet Gynecol 1987;69:167. Sbarra AJ, Selvaraj RJ, Cetrulo CL, et al. Infection and phagocytosis as possible mechanisms of rupture in premature rupture of the membrane. AM J 0BSTET GYNECOL 1985;153:38.
-+-TRYPSIN
CONTROL
«1 (ill)
Reply To the Editors: I thank Dr. James A. McGregor and his colleagues for their interest in this report. We also agree with their result that the main cause of premature rupture of membranes is latent chorioamnionitis. Premature rupture of membranes was caused by latent chorioamnionitis in about 60% of the cases in our study. However, chorioamnionitis is not recognized in all fetal membranes with premature rupture of membranes. For example, patients have no pathogenic bacteria or inflamed cells in or around the cervix, no uterine contractions, and their cervixs are closed. Furthermore, the microscopic observations of amniochorion do not contain chorioamnionitis. Even such cases sometimes produce premature rupture of the membranes. What is the mechanism of causation in such cases? We supposed that trypsin in amniotic fluid had proteolytic power according to the circumstances. Trypsin activity in such patients was higher than in controlled groups. We measured trypsin activity with S2222(KabiVitrum Co., Sweden), which is specific for trypsin. As shown in Table I, there is little possibility in measuring thrombin, factor Xa, and plasmin by 82222. Moreover, we obtained data opposite to theirs, namely, that type III collagen in amnion was degraded by trypsin in amniotic fluid (bovine) and meconium in amniotic fluid (human) (Fig. 1). Thus under what circumstance~ does trypsin modify its proteolytic activity in amniotic fluid? We recently measured trypsin levels
-+- MECONIUM
crl(ill)
«1 (V) crl( I)
Fig. I. Electrophoresis (7.5% polyacrylamide gel) of collagen in amnion. (Left lane = Normal amniotic collagen; middle lane = amniotic collagen (100 mg) treated with trypsin [10 mg] at 35° C for 6 hours; right lane = amniotic collagen [100 mg] treated with meconium [10 mg] at 35° C for 6 hours.) The migration of type I, Ill, and V a-chains is shown. Note the marked reduction of type Ill collagen in the middle lane and the slight reduction of that in the right lane.