ARTICLE IN PRESS

American Journal of Obstetrics and Gynecology (2006) -, --

www.ajog.org

Premature rupture of the fetal membranes: Is the amnion the major determinant?
Michelle L. Oyen, PhD,a,b Steven E. Calvin, MD,b,c Daniel V. Landers, MDb,*
Department of Biophysical Sciences and Medical Physicsa and Division of Maternal-Fetal Medicine, Department of Obstetrics, Gynecology, and Womens Health,b University of Minnesota; Minnesota Perinatal Physicians, Allina Health System,c Minneapolis, MN
Received for publication August 22, 2005; accepted February 8, 2006

KEY WORDS
Fetal membranes Chorion Amnion Premature rupture of the membranes Preterm premature rupture of the membranes

Objective: The objective of the study was to evaluate the relative contributions of amnion and chorion to the strength of fetal membranes and to correlate these findings with gestational age. Study design: Fetal membranes from 78 pregnancies were tested for biaxial puncture force using a blunt, instrumented probe with a low-force load cell connected through a load cell conditioner to an oscilloscope. The average of 2 to 4 tests performed on independent regions of the membrane was recorded. Means and SDs were calculated through the gestational age ranges of less than 32, 32 to 36, or 37 weeks or longer. Linear regression analysis was performed across gestational age after grouping data by labor and mode of delivery. Results: There were trends toward decreasing puncture force with gestational age for both chorioamnion and amnion for both vaginal deliveries and cesarean sections. The trends were significant by linear regression for labored deliveries but not unlabored cesarean sections for both chorioamnion and amnion alone. There was no trend in chorion puncture force with either gestational age or delivery mode and the mean puncture force values were, on average, half those for the amnion. Conclusion: The amnion is significantly stronger than the chorion when subjected to biaxial strength testing. The amnion but not the chorion is significantly affected by the chemical and mechanical changes during gestation and the labor process. These data will help direct future studies on the effects of clinical and molecular modulators of inflammation on membrane rupture thresholds with special emphasis on the biochemical and structural changes in the amnion. 2006 Mosby, Inc. All rights reserved.

Preterm premature rupture of the fetal membranes (pPROM) is a well-recognized, leading cause of preterm birth.1 Although the signicance of pPROM has long
Supported in part by the Womens Health Fund at the University of Minnesota. * Reprint requests: Daniel V. Landers, Ob/Gyn Department (MMC 395), 420 Delaware Street SE, Minneapolis, MN 55455. E-mail: lande028@umn.edu 0002-9378/$ - see front matter 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2006.02.010

been recognized, the nature of the process leading to failure of the fetal membranes has yet to be delineated. Failure processes are intrinsically mechanical, but the mechanism by which biological and mechanical factors are associated with pPROM is poorly understood.2 An understanding of the baseline mechanical responses of the fetal membrane component layers, the amnion and the chorion, is a necessary foundation for investigation of premature membrane rupture as well as a necessary

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2 step in the development of interventions aimed at restoring membrane function following early rupture. The existing data on chorioamnion (CA) membrane mechanical behavior come from studies using 3 types of mechanical testing: (1) uniaxial tensile testing,3-5 (2) biaxial ination burst testing,6,7 and (3) biaxial puncture testing.8-11 Our group has performed extensive biomechanical testing on fetal membranes using a wide range of testing techniques and emphasizing biomechanical best practices for studying the strength and deformation characteristics of membrane materials, including the nonlinear and viscoelastic behavior.5 We recently published data from controlled biomechanical testing on amnion in uniaxial tension and in planar equibiaxial tension testing using dierent testing types to examine the viscoelastic response of amnion.12,13 In addition, we utilized biaxial drumhead puncture testing to characterize the puncture resistance of the membranes.8 These studies demonstrated that a handheld puncture testing device, which can be brought into a clinical setting for routine mechanical characterization of CA membranes, gives puncture force values comparable with those reported by previous investigators10,11 using the same probe geometry but obtained with a mechanical testing machine. The measurement of puncture force is related to both the modulus of elasticity and intrinsic membrane strength.14 The eect of gestational age on the puncture force of the whole CA membrane was examined by Pressman et al.10 In a separate series of studies, the contributions of the component layers (amnion and chorion) to the full CA response were examined in term labored and unlabored specimens using tensile strength testing.3,4 Previous studies have examined the CA bilayer as a single entity and have not isolated the mechanical response of the amnion and chorion component layers as a function of gestational age. The purpose of the current study was to evaluate the relative contributions of amnion and chorion to the puncture resistance of the fetal membranes. The puncture test methodology was used to examine the mechanical response of both monolayer component and bilayer membrane specimens at a wide variety of gestational ages and under various clinical circumstances. Oyen, Calvin, and Landers labored cesarean section, or unlabored cesarean section), and mechanism by which membrane rupture occurred (rupture prior to the onset of labor, spontaneous rupture during labor, or articial rupture). The sample was convenient for this observational study; patients were not selected according to characteristics dened a priori, and as such, there was an uneven sample number among groups (Table I). The study population was predominantly white and the mean maternal age was 30.1 G 6.3 years (for the 64 of 78 subjects for whom age was recorded). All samples for testing were obtained away from the clinical rupture site. Intact free membranes were excised from the placental margin. One piece of each membrane was retained as intact CA, whereas additional pieces were separated into amnion (Am) and chorion using gentle traction. The placental amnion reection (Ap) was separated from the placental margin inward to the base of the umbilical cord and then excised. In the case of multiple gestations, the amnion from the dividing membrane was sampled as well and is denoted as the dividing amnion (DA). The DA was examined only in subjects in whom it could be unambiguously identied; in subjects in whom the individual amniotic sacs were separated during the delivery process, no DA tests could be performed. Tests were performed on specimens held between parallel at-plate gripping surfaces with a 2-cm diameter circular opening. A portable testing device was used to test the samples for biaxial puncture force.8 The device included a blunt metal probe with a one eighth inch (3.2 mm) diameter and a spherical tip. The probe was instrumented with a low-force load cell (Sensotec, Columbus, OH), which was connected through a load cell conditioner to an oscilloscope (Fluke, Everett, WA). The peak force level (Fmax) was recorded as force was applied at the center of the specimen until membrane rupture. Each test required a few seconds to perform, minimizing the potential eect of membrane tissues drying in air and limiting errors because of viscoelastic deformation. Two to four puncture tests (depending on amount of available tissue) were performed on independent regions of each membrane component from each sample, and an average of these tests was taken for each tissue type for each individual specimen. Linear regression (Microcal Origin, OriginLab, Northampton, MA) was used to establish the presence or absence of trends for failure force with gestational age (GA) using the raw data and with GA as a continuous variable. For the linear regression analysis, each tissue layer (CA, amnion, C, Ap, DA) was examined individually after grouping data by delivery mode (labored [L], including both vaginal or section deliveries, or unlabored [U]) for a total of 10 individual regressions. Averages and SDs were calculated for membrane puncture force for data grouped in gestational age ranges of less than 32 weeks, 32 to 36 weeks, or 37 weeks or longer for presentation in table form.

Material and methods

Fetal membranes from 78 pregnancies ranging in gestational age from 21 to 41 weeks were tested within 12 hours of delivery as part of an ongoing study of membrane mechanical behavior. The Institutional Review Board of Abbott-Northwestern Hospital/Allina Health System approved this study. Consent for placental evaluation was obtained from each patient. Clinical data were collected for each patient including gestational age, mode of delivery (labored vaginal,

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Oyen, Calvin, and Landers
Table I Specimen numbers for puncture testing experiment, by number of fetuses, delivery mode, and gestational age Singles (n = 60) Labored vaginal (n = 27) Less than 32 wk 32-36 wk 37 wk or more 2 5 20 Labored section (n = 3) 2 1 0 Unlabored section (n = 30) 7 4 19 Multiples (n = 18) Labored vaginal (n = 3) 0 1 2 Labored section (n = 5) 0 4 1 Unlabored section (n = 10) 0 7 3

Results
Of the 78 membrane specimens examined, 30 were from vaginal deliveries, 8 from cesarean sections following active labor, and 40 from unlabored cesarean sections. Puncture force data for tissue layers from labored vaginal deliveries and unlabored sections are compared in Table II (the labored cesarean sections were excluded from the table because of the small number of available specimens). Puncture forces were consistently larger for cesarean section deliveries, compared with vaginal deliveries (Table II). There were trends (by linear regression) toward decreasing puncture force (Fmax) with increasing GA for both CA and amnion, and for both vaginal and section deliveries. The trends were statistically signicant (P ! .05) by linear regression for labored deliveries but were not signicant for unlabored cesarean section deliveries for both CA and amnion (linear regression plots not shown; R2 values were 0.1 for CA for both L and U; R2 ! 0.1 for amnion-U, and 0.3 for amnion-L). There was no trend in C puncture force with gestational age (P O .05; R2 ! 0.1 for both L and U) and chorion puncture force values were, on average, approximately half those for CA or amnion. Scatter plots are shown in the Figure for the puncture force of amnion layers from 3 dierent locations within the sac as a function of gestational age (a continuous variable). The amnion split from the CA showed a sharp, monotonic decrease in puncture force with GA from 30 weeks to full term; prior to 30 weeks gestation, the puncture force appeared to be increasing. However, the small number of samples with GA less than 30 wks does not allow for a conclusive evaluation of the behavior in this regime. The placental amnion and DA showed no trend in puncture force with gestational age (P O .05 for all combinations of Ap and DA, L and U). Mean values for puncture force for the DA and Ap were statistically dierent from A values from the same pregnancy by paired t tests (P ! .02 for DA and P ! .01 for Ap) and in both cases the A puncture force values were smaller than those for the DA or Ap. Membranes ruptured prior to the onset of labor (premature rupture of the membranes [PROM]) in 7 labored vaginal cases and spontaneously during the course of labor in 9 labored vaginal cases (the remainder

Table II Fmax for CA membranes and the component chorion (C) and amnion (Am) layers for singleton L vaginal or U section deliveries as a function of GA GA ! 32 wk, N CA-L CA-U Am-L Am-U C-L C-U 4.61 5.80 4.69 4.31 1.60 2.71 G G G G G G 0.10 1.77 0.92 1.22 0.76 1.11 GA 32-36 wk, N 3.92 4.66 3.55 4.05 2.27 2.61 G G G G G G 1.41 1.28 1.25 0.76 1.89 1.32 GA R 37 wk, N 3.47 4.10 2.89 3.62 1.87 2.18 G G G G G G 1.57 1.62 1.08 1.03 0.61 0.81

Data are presented as mean G SD, and all Fmax is reported in newtons (N).

was articially ruptured). Puncture force data for these 16 membranes are shown in Table III; the average gestation was slightly longer for the spontaneous labor specimens. Average puncture force for these membranes are shown in Table III for CA, amnion, chorion, and placental amnion. These puncture forces did not dier signicantly for any tissue between the 2 dierent membrane rupture mechanisms by unpaired t tests and if anything were slightly greater in the PROM specimens because of the smaller GA.

Comment
The mechanical response of the fetal membranes is a combination of the mechanical responses of the individual chorion and amnion components. Results from the current study have demonstrated that the chorion does not seem to be signicantly aected by the chemical and mechanical changes that occur during gestation and delivery, whereas the amnion is quite sensitive to these changes. The implications of these ndings are 2-fold. First, the chorion is providing a mechanical buer to the membrane, preventing the whole CA from degrading as quickly as the amnion degrades, particularly in the case of labored specimens. This has implications in clinical practice. Promoting chorion integrity near the cervix may help prevent premature membrane rupture. Second, the chorioamnion as a whole is less sensitive to dierent obstetrical conditions than the isolated amnion. In vitro experiments designed to manipulate

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Table III Fmax for vaginal singleton deliveries, in which the membranes ruptured prior to the onset of labor (OOL) or spontaneously during labor Rupture prior to OOL n = 7 mean GA = 34.9 wk, N Chorioamnion Amnion Chorion Placental amnion 3.98 3.90 2.64 4.61 G G G G 1.44 0.76 1.61 1.73 Spontaneous rupture n = 9 mean GA = 38.2 wk, N 3.88 2.87 1.70 4.87 G G G G 1.75 1.17 0.52 1.50

Data are presented as mean G SD with the number of individual specimens given for each group, and all puncture forces are reported in newtons (N). There was no statistical difference between the 2 rupture modes for any tissue layer by unpaired t test (P O .05 for all comparisons). OOL, Onset of labor.

membrane strength via degradation or enhancement of the collagen network may need to be carried out on isolated amnion, not just because it is the strongest part of the membrane15 but also because its mechanical response is most sensitive to manipulation. Spontaneous failure of the chorioamniotic membrane in vivo occurs via a mechanical process that is aw controlled: the membrane will fail at a point at which the largest aw or defect exists. For this reason it is perhaps not surprising that we and others16,17 have found no great dierences between the mechanical behavior of membranes that ruptured spontaneously during labored delivery and those that ruptured prior to the onset of labor. From a clinical perspective this is good news: the membrane is not degrading throughout but in a very localized manner. Examining the problem of localized collagen degradation in vivo, and the production of localized aws in the membrane that reach a critical size for failure, will likely add insight to the problem of pPROM and provide information associated with the potential to restore localized degraded membrane regions to the greater mechanical properties of the surrounding membrane. The amnion from the CA membrane demonstrated a monotonic decrease in puncture force as a function of gestational age from 30 weeks gestation to full term. Although gestational age categories were somewhat arbitrarily chosen on the basis of available data (Table II), this is a continuous variable wherein small changes in GA correspond to clear decreases in puncture force (Figure 1). These data for amnion puncture force seem to correspond well with similar plots of amnion collagen
Figure Fmax as a function of GA for amnion from the CA membrane (Am), the placental surface (Ap), and the dividing membrane (DA). There is a signicant trend toward decreased puncture force with GA for amnion (Am) but no trend for Ap or DA. Closed squares, U; open circles, L.

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Oyen, Calvin, and Landers content versus gestational age.18 The puncture force and collagen content demonstrate a similar total decrease over this GA range (a decrease of approximately a factor of 2 to 3 for GA from 30 to 40 weeks), and this provides motivation for future combined mechanicalbiochemical studies of the amnion tissue samples. The absence of changes with GA in the mechanical response of placental surface amnion and dividing membrane amnion raises 2 interesting points. First, the mechanism of amnion weakening with gestational age is localized, in that it does not occur in amnion in all locations of the same gestational sac. (This conclusion is in agreement with the apparently localized eect in PROM as well, as discussed above.) Second, these tissues are particularly attractive to serve as a controls for in vitro analysis of amniotic membrane degradation or repair. Future studies utilizing scanning electron microscopy will aim to examine the dierences between the dierent amnion layers that are leading to the dierent trends in behavior with GA. The Fmax as measured in this study is not an intrinsic material property, but it is directly related to both the intrinsic failure strength (sF) and elastic modulus (E) as well as extrinsic geometrical factors of tissue thickness (h) and probe radius (R)14: Fmax Z6psF 2 Rh=E There are several additional important features of this relation. First, in the absence of any changes in the tissues intrinsic properties, a thicker membrane will exhibit a greater puncture force. In addition, a membrane whose intrinsic properties are degraded but whose thickness is increased (eg, in chorioamnionitis) may not exhibit any degradation in puncture force, compared with an intrinsically stronger, but thinner, membrane. This may help explain why it can be dicult to localize risk factors for PROM: 2 dierent competing mechanisms of change in the membrane can oset. For best understanding of the puncture test, membrane thickness data will be collected in future studies aimed at identication of the precise mechanism of weakening in cases such as chorioamnionitis in which both weakening and thickening eects may occur. Finally, the puncture test is an extensive force-based test and the thickness (h) of each layer (amnion, chorion) is important. Although the intrinsic properties of the chorion are approximately an order of magnitude less than those for the amnion3,4 because the amnion is a smaller portion of the total membrane thickness the puncture forces dier by about a factor of 2, much less than the order of magnitude dierences in the intrinsic properties of strength and elasticity. The circumstance of clinical membrane rupture in vivo is related to the whole membrane structure, such that the thickness-weighted puncture test more accurately reects 5 the bilayer membrane mechanical behavior in vivo, compared with tensile tests aimed at intrinsic strength and elasticity properties. The usefulness of puncture testing in sensing bacterial degradation of membranes has also been established.11 The current study was a preliminary eort aimed at understanding the mechanical response of individual layers of the chorioamnion membrane as a function of GA. This observational study is limited in that its sampling was based on convenience and there were not sucient data for multivariate statistical analysis. Future studies will incorporate a statistical design aimed at further examination of the key results found in the current study, namely the importance of GA and the dominance of the amnion layer in the overall CA response. Ongoing studies will also correlate strength data with ultrastructural (collagen bril) degradation in amnion and chorion and biochemical evaluation of membranes. The implementation of this reproducible mechanical model for membrane strength testing will also provide a valuable in vitro system for studying eects of clinical and molecular modulators of inammation on membranes rupture thresholds. Correlating the biochemical, ultrastructural, and mechanical force data that can be collected on membranes in various clinical settings may well lead to new information on the cascade of events that leads to pPROM.

References
1. Mercer BM. Preterm premature rupture of the membranes. Obstet Gynecol 2003;101:178-93. 2. Polzin WJ, Brady K. Mechanical factors in the etiology of premature rupture of the membranes. Clin Obstet Gynecol 1991;34:702-14. 3. Oxlund H, Helmig R, Halaburt JT, Uldbjerg N. Biomechanical analysis of human chorioamniotic membranes. Eur J Obstet Gynecol Reprod Biol 1990;34:247-55. 4. Helmig R, Oxlund H, Petersen LK, Uldbjerg N. Dierent biomechanical properties of human fetal membranes obtained before and after delivery. Eur J Obstet Gynecol Reprod Biol 1993;48:183-9. 5. Oyen ML, Calvin SE, Cook RF. Uniaxial stress-relaxation and stress-strain responses of human amnion. J Mater Sci Mater Med 2004;15:619-24. 6. Lavery JP, Miller CE. The viscoelastic nature of chorioamniotic membranes. Obstet Gynecol 1977;50:467-72. 7. Lavery JP, Miller CE, Knight RD. The eect of labor on the rheologic response of chorioamniotic membranes. Obstet Gynecol 1982;60:87-92. 8. Oyen ML, Cook RF, Calvin SE. Mechanical failure of human fetal membrane tissues. J Mater Sci Mater Med 2004;15:651-8. 9. Schober EA, Kusy RP, Savitz DA. Resistance of fetal membranes to concentrated force applications and reconciliation of puncture and burst testing. Ann Biomed Eng 1994;22:540-8. 10. Pressman EK, Cavanaugh JL, Woods JR. Physical properties of the chorioamnion throughout gestation. Am J Obstet Gynecol 2002;187:672-5. 11. McGregor JA, French JI, Lawellin D, Franco-Bu A, Smith C, Todd JK. Bacterial protease-induced reduction of chorioamniotic membrane strength and elasticity. Obstet Gynecol 1987;69:167-74. 12. Oyen ML, Stylianopoulos T, Barocas VH, Calvin SE, Cook RF. Uniaxial and biaxial mechanical behavior of human amnion. In: Viney C, Katti K, Ulm F-K, Hellmich C, editors. Mechanical

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properties of bioinspired and biological materials. Warrendale (PA): Materials Research Society; 2005. p. 161-6. 13. Oyen ML, Cook RF, Stylianopoulos T, Barocas VH, Calvin SE, Landers DL. Uniaxial and biaxial mechanical behavior of human amnion. J Mater Res 2005;20:2902-9. 14. Begley MR, Mackin TJ. Indentation of free-standing axisymmetric membranes. J Mech Phys Solids 2004;52:2005-23. 15. Manabe Y, Himeno N, Fukumoto M. Tensile strength and collagen content of amniotic membrane do not change after the second trimester or during delivery. Obstet Gynecol 1991;78:24-7.

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16. Bourne GL. The foetal membranes: a review of the anatomy of normal amnion and chorion and some aspects of their function. Postgrad Med J 1962;38:193-201. 17. Artal R, Sokol RJ, Neuman M, Burstein AH, Stojkov J. The mechanical properties of prematurely and nonprematurely ruptured membranes. Methods and preliminary results. Am J Obstet Gynecol 1976;125:655-9. 18. Skinner SJM, Campos GA, Liggins GC. Collagen content of human amniotic membranes: eect of gestation length and premature rupture. Obstet Gynecol 1981;57:487-9.

Condensation Biaxial strength testing of fetal membranes demonstrated fetal membrane strength is derived from the amnion,

not the chorion and strength decreases with advancing gestational age.