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Stereological studies on the true thickness of the villous membrane in human term placentae: a study of placentae from high-altitude pregnancies
Placenta (I985), 6, 249-258
Stereological Studies on the True Thickness of the Villous m e m b r a n e in Human Term Placentae: a Study of Placentae from High-altitude Pregnancies MOIRA R. JACKSON a, CAROLINE F. JOY a, T. M. MAYHEW a,c & J. D. HAAS b a Department of Anatomy, Marischal College, University of Aberdeen, Aberdeen AB9 1AS, UK b Division of Nutritional Sciences, Martha Van Rensselaer Hall, Cornell University, Ithaca, N Y z4853, USA To whom correspondence should be addressed
INTRODUCTION In the human haemochorial placenta, maternal and fetal blood spaces are separated by the villous membrane. This comprises layers of trophoblast and fetal capillary endothelium with a variable amount of connective tissue interposed. The membrane acts as a physical barrier to the transfer of respiratory gases and other metabolites. It has a considerable mass, accounting for roughly 12 per cent of total placental weight (Aherne and Dunnill, 1966; Laga, Driscoll and Munro, I973), and a high metabolic activity. For these reasons, the thickness of the villous membrane is of physiological interest. Arithmetic mean thickness is proportional to tissue mass and so reflects tissue oxygen requirements (Weibei and Knight, 1964). Harmonic mean thickness, based on reciprocal local thicknesses, accords greater weight to thinner regions, and is a critical determinant of total placental resistance to gaseous diffusion (Laga, Driscoll and Munro, 1973; Mayhew, Joy and Haas, 1984). Using microscopically thin sections, it is possible to measure 'minimum membrane thickness' (Aherne and Dunnill, I966 ) or 'maternofetal diffusion distances' (Sen, Kaufmann and Schweikhart, 1979). Unfortunately, these estimates represent neither true arithmetic nor true harmonic mean thicknesses. The problem lies in the fact that the apparent irregularity in thickness actually arises from two main sources of variation: (x) natural variation in real thickness, partly attributable to localized thinning at vasculo-syncytial membranes (Getzowa and Sadowsky, 195o, Sen, Kaufmann and Schweikhart, i979), and (z) sectioning artefact, introduced by chance variability in the angle at which the villous membrane is sectioned during microtomy (Weibel and Knight, 1964, Gundersen, Jensen and Osterby, I978 , Jensen, Gundersen and 0sterby, 1979). Both variations must be taken into consideration before true thicknesses can be estimated and before harmonic means can be substituted into appropriate diffusion equations (Mayhew, Joy and Haas, 1984). In this investigation, artithmetic and harmonic mean thicknesses are calculated using stereological methods previously applied to the alveolar air-blood barrier (Weibel and Knight, z49
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x964) and to the glomerular filtration barrier (Gundersen, Jensen and 0sterby, 1978). Values are then used to derive a thickness uniformity index for assessing the functional significance of vasculosyncytial membranes and syncytiaI knots in determining the diffusional resistance of the villous membrane. Results are based on analyses of randomized histological sections from term placentae delivered by women living at high altitude in Bolivia.
MATERIALS AND M E T H O D S Material was collected as part of a much larger prospective study of maternal adaptation to altitude during pregnancy and its consequences for subsequent growth and development of the neonate. Details of the larger project may be consulted in Haas (I98O). Briefly, Amerindian women and non-Indian women of European ancestry were selected on the basis of uncomplicated, full-term gestation and the availability of records on age, socioeconomic status, migratory and reproductive history, dietary intake, health, blood biochemistry and haematology, anthropometry for nutritional status, genetic markers and smoking habits. Present results are confined to those from placentae belonging to a randomized subsample of I5 European mothers drawn by lottery from the larger population samples (Haas, i98o ). This number of placentae provided standard errors of less than 6 per cent of each group mean for the three thickness variables under scrutiny (see below). This level of precision is usually considered adequate for statistical comparisons between groups. All mothers had been born and raised at an average altitude of 36oo m in the Bolivian capital of La Paz. In order to verify gestational ages estimated from the last menstruation, a clinical examination (Dubowitz, Dubowitz and Goldberg, 197o) was administered to all infants within 24 hours of delivery. Infants were between 266 and 282 completed days of gestation. All were weighted by an attending neonatologist within minutes of delivery. It is important when making measurements involving fetal capillaries within villi to standardize handling of the placenta, especially the timing of clamping of the umbilical cord. In this study, the cord remained unclamped and uncut for up to one minute from delivery of the infant. Clamps were applied at two sites, the nearer being about io cm from the umbilicus, and the cord was cut between them. The clamp on the maternal side was subsequently released for roughly 15 sec for the purpose of sampling cord blood. It was thenre-applied until expulsion of the placenta. Following expulsion, the cord was immediately clamped and trimmed to within 5 mm of the fetal aspect of the placenta. Placentae were weighed after removing attached membranes and blood clots. Gestational ages, birthweights and trimmed placental weights are provided in Table i. Tissue s a m p l i n g Systematic samples of tissue from two quadrants per placenta were removed, fixed in isotonic formal saline and embedded in paraffin wax. From each of four to five blocks of tissue per placenta, a single arbitrary section (thickness c. 3/~m) was cut and stained by the Masson trichrome method. Light microscopical fields of view were obtained from the sections by systematic random sampling procedures (Weibel, I979; Mayhew, i983) and prepared as colour transparencies. Sets of 2o transparencies per organ were projected onto a flat, white cardboard screen for stereological evaluation. The final magnification ( x 2ooo) was standardized with a stage micrometer calibration scale.
Human Villous Membrane Thickness
25I
Table I. Gestational age, newborn birthweight and trimmed placental weight for a group z5 subjects from 36oo m altitude in Bolivia
Subject number IO17 IOI8 X037 xo5o Io54 1o65 I077 HI7 Ill 9 I132 3oi0 4oi2 4o47 407~ 4o97
Gestational age (days)
Weight of newborn (kg)
Weight of placenta (g)
268 266 274 274 274 269 282 273 272 277 267 278 272 274 278
2.93 2"50 2.82 3.22 2.80 2.93 3.29 2A3 2.8O 3.30 3.03 3.22 3.00 2.50 3.oo
466 335 368 49I 383 479 588 377 393 494 586 597 435 429 383
Stereological m e t h o d s All projected images were analysed by the same person, who had no prior knowledge about the placentae or their origins. In order to estimate arithmetic mean thickness (Ta) and harmonic mean thickness (Th), the projection screen was ruled with a set of equidistant, parallel, straight lines. Where these test lines randomly intercepted profiles of villous membranes, intercept lengths from the trophoblastic surface to the luminal surface of capillary endothelium were measured by ruler (see Figure i). Altogether, 47-57 intercept lengths per placenta were sampled. As far as it was possible to identify them, stem villi were excluded from these measurements. Their histological and morphometric characteristics suggest that stem villi play a
/5 Figure i. Section through villus illustrating chance encounters between test lines and profiles of villous membranes.
The positions and lengths of five random intercepts are indicated (L1-L~). cl = capillary lumen; fc = fetal capillary endothelium; st = stromal tissue; tr = trophoblast.
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M. R. Jackson, C. F. Joy, T. M. Mayhew, J. D. Haas
minimal role in maternofetal gas diffusion (Laga, Driscoll and Munro, I973; Sen, Kaufmann and Schweikhart, i979; Mayhew, Joy and Haas, I984). Given that the encounters between test lines and villous membrane profiles are random in position and orientation, it has been shown (Weibel and Knight, i964; Gundersen, Jensen and 0sterby, i978 ) that the mean intercept length ( L ) is related to T by the simple equation Ta = LJ2 and, taking the mean of reciprocal intercept lengths, that the harmonic mean intercept length (Lh) is related to T h by T h -- 2Lh/3 The dimension T is a measure of the volume (and hence the mass) of villous membrane associated with a unit area of trophoblastic surface (Weibel and Knight, i964). This dimension therefore influences the relative amount of oxygen that is consumed in the villous membrane as the gas is diffusing from the maternal to the fetal erythrocytes. Being based on reciprocal intercept lengths, T h places greater emphasis on thinner regions of the villous membrane: that is, on precisely those regions (vasculosyncytial membranes) considered to be best suited to facilitating passive transfer by diffusion. From these considerations it is clear that T equals T h and that the ratio ( T J T h ) equals I when the membranes are uniform in thickness. However, for membranes that, like the villous membrane, are irregular in thickness, ( T J T h ) > I. Therefore, the ratio T J T h affords a convenient index of thickness uniformity which we denote by the symbol Itu. D a t a handling Values of Ta, T h and Itu were calculated for each placenta after averaging random intercept lengths and reciprocal intercept lengths measured on all transparencies from all histological sections. The estimates were corrected for tissue shrinkage, the linear correction factors for these preparations being between 1.25 and 1.5o. Subsequently, intercept length size-frequency distributions were constructed for each placenta and employed to determine the overall percentage frequency distribution. Knowing the group mean T a for this observed distribution allowed us to calculate the expected distribution for a villous membrane of uniform thickness (see Gundersen, Jensen and 0sterby, 1078). Observed and expected distributions of intercept lengths were compared using a chi-squared test (Bailey, I972). Individual placental values of T , T h and Itu were later used to calculate group means and standard deviations (s.d.). As measures of the observed variability between placentae, and of intercept-to-intercept variability within placentae, coefficients of variation (c.v. = ioo x s.d./mean) were also calculated. All these calculations were handled using pre-recorded BASIC programs run on a Hewlett-Packard HP85 personal computer.
RESULTS The results are presented in Tables 2 and 3 and in Figure 2. Our qualitative observations support previous impressions. The thickness of the villous membrane is not equal throughout in microscopically thin sections. It is clear that part of the lack of uniformity reflects the fluctuating geometrical relationships between the trophoblast, connective tissue stroma and fetal capillaries. The latter tend to have a sinuous course and sometimes appear dilated, particularly those near the periphery of villi where they are separated from the trophoblast by minimal amounts of
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Human Villous Membrane Thickness Table 2. Estimates of arithmetic mean, reciprocal mean and harmonic mean intercept lengths for villous membrane profiles
Placenta number IOI 7 IOI8 IO37 ]05 ~ lO54 lO65 1~ I I 17 I119 1132 3oio 4o12 4o47 4o7~ 4o97
Intercept length (#m) L
Reciprocal intercept length (/~m 1) L, 1
Harmonic mean (,um) Lh
IO.O1 + 7.25 (72.6) a 12.55+8.4o(67.o ) 7-34 + 5-76 (78-4) 8.05 + 4.82 (59.9) 9.6o+6.55(68.3) 8.73 + 5-45 (62.3) 5.65 + 4.74 (84.0) 8.1o + 5.68 (7o.I) 7"5~ ) 7.47+4.93(66.1) 9.25 + 8.78 (95.o) lO.55+7.7o(73.o ) 9.14 + 7 .26 (79.5) 9.55 + 5.66 (59.5) 9.68 + 5.86 (6o.5)
o.163 +o.i35 (82.6) a o.126+o.o94(74.2) o.224 + o. I72 (76.5) 0.2o9 + 0.188 (9o.2) o. 182 + o.I6O (87.9) o.167 + o.t23 (73.8) 0-313 "4-o.225 (71.7) o.225 +0.188 (83.4) ~ o.2o6"I-o.144(69.7) o.2oo + o. 15o (75.4) o.17o+o.158(93.1 ) o.220 + o.2o3 (92.2) o. 156 ___o. I Io (7o.3) o. 152 + o. lO2 (67.0)
6.i 4 7.92 4.46 4.79 5.5~ 5.99 3-19 4.44 4.43 4-85 5-oi 5.89 4.54 6.42 6.57
a Mean + s.d. (c.v. per cent). Table 3. Estimates of true arithmetic mean thickness, harmonic mean thickness and uniformity index for the villous membrane
Placenta number IOI 7 IOI8 IO37 105~ 1054 IO65 I O77 I 117 I I t9 1132 3OlO 4o12 4o47 4o7o 4o97 Group mean s.d. c.v. (per cent)
Arithmetic mean thickness (pm) T
Harmonic mean thickness (/lm) Th
Thickness uniformity index Ta/T h
5.00 6.27 3.67 4.O2 4.80 4.36 2.82 4.05 3.75 3.74 4.62 5.28 4.57 4.77 4.84
4.00 5.28 2.97 3.t9 3.67 4.OO 2.13 2.96 2.96 3.23 3.34 3.93 3.o3 4.28 4.38
1.22 I. 19 1.23 !.26 X.3X I .O9 1.33 1.37 1.26 1.15 1.39 1.34 t.5I 1.12 t.tl
4.44 o.817 i8. 4
3.56 0.777 21.8
1.26 o.118 9-4
s t r o m a l tissue o r o n l y b y basal l a m i n a . A t s u c h sites, t h e t r o p h o b l a s t is u s u a l l y a t t e n u a t e d a n d f o r m s r e c o g n i s a b l e v a s c u l o s y n c y t i a l m e m b r a n e s . A t o t h e r sites, t h e t r o p h o b l a s t t h i c k e n s i n t o s y n c y t i a l k n o t s , t h e capillaries are n a r r o w e r a n d m o r e c e n t r a l l y located, a n d i n c r e a s e d a m o u n t s o f s t r o m a l tissue i n t e r v e n e b e t w e e n t h e t r o p h o b l a s t a n d capillary e n d o t h e l i u m . T h e s e o b s e r v a t i o n s a p p l y to all r a m i f i c a t i o n s o f t h e villous tree, t h e d i f f e r e n c e s b e t w e e n v a r i o u s classes o f villi b e i n g q u a n t i t a t i v e r a t h e r t h a n qualitative. N o a t t e m p t was m a d e to
=54
M. R. Jackson, C. F. Joy, T. M. Mayhew, J. D. Haas
30-
Cr
20"
O~ 0= e= 0 r n
10-
Ta; L aI 0
'~ 0
TI 10
210
30
410
I 50
Intercept length, jam Figure 2. Observed frequencydistribution of random intercept lengths pooledfor 15 placentae. Mean intercept length
(L,) and the arithmetic meanthicknessof the actual membrane(Ta) are indicated. The distribution differssignificantly from that expectedfor a membraneof uniformthicknessthroughoutfor whichno randomintercept can be less than T a in length. characterize terminal villi and intermediate villi separately. Therefore, the following quantitative findings refer to the villous membrane on these ramifications collectively. Individual random test line intercepts varied in length from less than 2/am to more than 5o/am. Despite this large intra-placental variation (c.v. 6o to 95 per cent), mean intercept lengths per placenta varied only between 5.6/am and 12.6/am, the corresponding range for harmonic mean intercept lengths being 3.2 to 7-9/am (Table 2). The overall size-frequency distribution of observed intercept lengths was calculated and plotted from the percentage frequency distributions of the 15 placentae. This histogram (Figure 2) comprises ten size classes each representing an equal increment of intercept length based on the largest distance recorded (i.e., 53.2/am). The observed distribution (c.v.c. 73 per cent) differed significantly from the expected distribut/on for a uniformly thick villous membrane (chi-squared value, 6o.4; degrees of freedom, 9; P < o.ooi). Estimated group means (c.v.) for the three membrane thickness variables were 4.44/am 0 8 per cent) for T , 3.56/am (22 per cent) for T h and 1.26 (9 per cent) for Itu (Table 3). In spite of the differences in T a and T h between placentae, and the su bstantial differences in intercept and reciprocal intercept lengths within placentae, the observed c.v. for Itu was comparatively small. In other words, the uniformity index was relatively constant from one placenta to the next.
DISCUSSION This investigation has sought to provide more reliable information on the true thickness of the villous membrane in human term placentae by deriving estimates of its arithmetic and harmonic
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mean thicknesses. Before comparing present estimates with previous findings, or discussing their functional significance, it is worth considering the errors influencing them.
Sources o f error Generally speaking, these are of two types, viz. random (or accidental) error and systematic error or bias (Mayhew, i983). Random errors are introduced by the natural 'biological variation' between placentae and by variation within organs imposed by the experimental sampling design. Within-organ variation therefore includes differences between tissue blocks within placentae, between fields of view within blocks, between villi within fields and between intercepts within villi. Some of this variation is also attributable to the precision of the method of measuring intercept lengths. Fortunately, it has been demonstrated repeatedly that within-organ errors often have little influence on the overall precision ofmorphometric estimates (see Shay, 1975; Gupta et al, 1983; Mayhew, 1983). By means of pilot studies, we confirmed for ourselves that analysing a reasonably large number of organs was the most economical way of minimizing these errors. The fact that we elected to sample fewer blocks of tissue per placenta than in some earlier morphometric studies (Aherne and Dunnill, 1966; Lags, Driscoll and Munro, 1973) is of little significance from the viewpoint of determining the precision of our final estimates of thickness. Indeed, a good indication of the precision of final estimates is afforded by calculating their coefficients of error ( = standard error/group mean). The values were 4.8 per cent (Ta), 5.6 per cent (Th) and z.4 per cent (Ira) when expressed as percentages. Systematic errors are introduced by the sampling design, by various technical limitations and by the constraints of the geometric model on which the pertinent stereological relationships are based (Weibel and Knight, 1964; Gundersen, Jensen and0sterby, 1978; Weibel, 1979; Mayhew, 1983). The methods we have used rely on the fact that the length of a random test line intercept across a membrane is determined solely by local membrane thickness and by the angle between the test line and the normal to the membrane (Weibel and Knight, 1964; Gundersen, Jensen and 0sterby, 1978). The main condition to be fulfilled is that of random orientation between membranes and test lines. Since this is exclusively a sampling problem, we have tried to meet this condition by randomized sampling at all stages of the selection process (tissue blocks, sections, fields of view, intercepts). The methods are robust enough to be only minimally affected by systematic errors due to variations in the surface curvature and thickness distribution of the villous membrane. However, technical biases from shrinkage and section thickness effects do influence the validity of present estimates. We have tried to compensate for shrinkage in order to compare our results with those obtained in other laboratories. Estimates have not been corrected for section thickness. Approximate corrections are available (e.g., Weibel, 197o), but it should be borne in mind that in thicker parts of the villous membrane the effects of section thickness are diminished in comparison to thinner regions. Suitable corrections therefore require knowledge of the distributions of membrane thickness (Gundersen, Jensen and 0sterby, 1978) which are, in any case, best reconstructed from semi-thin sections for light microscopy or ultra-thin sections for electron microscopy. Vasculosyncytial membranes, the thinnest regions, are estimated to account for only IO to z6 per cent of the total surface area of terminal and intermediate villi in such sections (Aherne and Dunnill, 1966; Sen, Kaufmann and Schweikhart, I979). In view of the sectioning artefact due to chance encounters between membranes and section planes, these percentages are probably underestimates.
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M. R. Jackson, C. F. Joy, T. M. Mayhew, .7. D. Haas
C o m p a r i s o n with previous findings Direct comparisons are not possible because of the lack of adequate information on the measuring convention. The term 'minimum membrane thickness' (Aherne and Dunnill, I966 ) probably refers to the apparent thickness of vasculosyncytial membranes measured at right angles to the trophoblastic surface. Such distances may overestimate true arithmetic mean thickness at these sites by about 27 per cent (see Jensen, Gundersen and 0sterby, t979, P- 22, formula 9)- Therefore, the value of 3. 5 pm for minimum membrane thickness could represent an arithmetic mean thickness of 2.7/~m for vasculosyncytial membranes. The term 'maternofetal diffusion distance', as used by Sen, Kaufmann and Schweikhart (I979) , is misleading. It, too, seems to be an orthogonal mean intercept length, calculated for the entire villous membrane including stem villi. Since it is not derived from reciprocal intercept lengths, the dimension is not a harmonic mean thickness and so cannot be interpreted as an effective diffusion distance. If their value of 5.94 pm is an orthogonal intercept length, it would correspond (Jensen, Gundersen and 0sterby, 1979) to a true arithmetic mean thickness of 4.67 pm. This figure is reasonably close to our estimate of 4.44 pm which is based on thicker sections and excludes stem villi. Our estimate of 3.56 ~m for the harmonic mean thickness of highland placentae compares with one of 4.08 pm for a group of low altitude placentae from Bolivia (Mayhew, Joy and Haas, 1984). These findings suggest that the harmonic mean thickness of io/~m adopted by Laga, Driscoll and Munro (i973) is far too high for substituting into diffusion equations. Indeed, their estimates appear to be informed guesses relying on mean villous diameter, central rather than peripheral fetal capillaries, and values of Itu more appropriate to the lung than to the placenta (see below).
Biological implications Our interest in harmonic mean thickness stems from a desire to calculate placental diffusing capacities for oxygen by combining the structural dimensions and physicochemical properties of the maternofetal diffusion pathway (Laga, Driscoll and Munro, I973; Mayhew, Joy and Haas, i984). Such studies demonstrate that the resistance of the villous membrane is the principal contributor to total diffusional resistance. Since the resistance of this membrane depends on its harmonic thickness, reliable estimates of this dimension are necessary. Substituting 'minimum membrane thickness' into the pertinent equations would tend to overestimate, and 'maternofetal diffusion distance' to underestimate, oxygen diffusing capacity, the former leading to even larger discrepancies between physiological and morphometric diffusing capacities than exist already (Mayhew, Joy and Haas, 1984). The present study suggests that there may be a negative correlation between newborn birthweight and harmonic mean thickness of the villous membrane (refer to Tables I and 3). Though the correlation is not statistically significant (perhaps due to inadequate numbers, or to too narrow a range of birthweights), a negative correlation might be anticipated in view of the positive and significant correlation between morphometric diffusing capacity and birthweight which exists during the last trimester of pregnancy (Mayhew, Joy and Hass, I984). Our use of the uniformity index I~u warrants further comment. The value of 1.26 by itself conveys little information because it is difficult to imagine what this means in a physical sense. However, comparisons with values for other tissue barriers help. Thus the basement membrane of the human renal glomerulus--considered to be relatively uniform in thickness--has an Itu of about I. i in non-diabetics and in patients with diabetes mellitus between zero and nine years' duration (Gundersen, Jensen and0sterby, I978, p. ,to, table 2). Similar values are found in other mammals. In contrast, the alveolar-capillary membrane of the lung has an average Itu of 2.2 in
Human Villous Membrane Thickness
~57
the rat (Weibel and Knight, 1964). In this particular barrier, the thickness may vary from only o.~/am in extremely attenuated regions to about 8/am in the thickest regions. A better way of interpreting Itu is tO consider its functional significance. The ratio T a / T h provides an indication of the efficacy of the villous membrane in permitting gas transfer by diffusion. Thus the figure 1.26 can be regarded as indicating that the diffusing capacity of the villous membrane is 26 per cent more than it would be if the membrane was uniformly thick throughout. In the rat lung the corresponding increase would be i zo per cent, in accordance with the fact that the lung is a more effective gas exchange organ. In the case of the placenta, it is clear that the improvement is achieved by concentrating much of the tissue mass away from vasculosyncytial membranes and in thicker regions, particularly at syncytial knots. It is therefore of considerable interest that the placenta should respond to hypoxic stress by increased syncytial knotting (e.g., Fox, i964; Tominaga and Page, 1966; Chabes et al, i968 ). The low inter-placental variation in Itu estimates, despite c.v. values of 18 per cent and 22 per cent for T a and T h respectively, and despite all of the random errors mentioned above, may be a further sign of the functional importance of this index. We may envisage Itu as reflecting a balance which has to be struck between the metabolic activity of the trophoblast (which requires a certain tissue mass and arithmetic mean thickness) and the need of a thin barrier for diffusion of gases and metabolites (which requires a certain harmonic mean thickness). Though T and T h may vary according to the needs of the individual fetoplacental unit, present results suggest that the structural compromise is quantitatively similar in different individuals. The real lack of uniformity in villous membrane thickness no doubt contributes to the discrepancy between physiological and morphometric estimates of placental diffusing capacity (Mayhew, Joy and Haas, 1984). Diffusion is likely to proceed more rapidly where the resistance of the villous membrane is lower, and less rapidly where it is higher, producing regional inhomogeneities in diffusing capacities. In conclusion, the present stereological methods offer several advantages over planar morphometric data for drawing structure-function correlations in the human placenta. Methods similar to those described here may be employed to estimate the effective diffusion distances of the serial blood plasmas which also affect the overall diffusing capacity of the placenta (Weibel, 197o; Mayhew, Joy and Haas, 1984). They may also be used to study the functional morphology of placentae during gestation and in various states associated with increases in the histological components of the villous membrane; for example, trophoblastic basement membrane thickening and proliferation of cytotrophoblast (Fox, 1964; Wang, Hamann and Hartge, I983). We are now using these methods to investigate placental morphology in pregnancies associated with reduced birthweight (Haas et al, 198o).
SUMMARY Stereological principles were used to calculate functionally significant dimensions of the human villous membrane, its arithmetic mean thickness ( T a ) and its harmonic mean thickness (Th). The former is proportional to tissue mass and oxygen consumption, the latter to diffusional resistance. For a group of 15 term placentae from uncomplicated pregnancies at high altitude, the average values w e r e T a = 4-44/am, T h = 3.56/~m a n d T a / T h = 1.26. The latter figure provides a useful quantitative expression for the efficiency of the membrane in gas and metabolite diffusion. It implies that vasculosyncytial membranes and syncytial knots decrease resistance to diffusion by 26 per cent, compared with that of a membrane with uniform thickness
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t h r o u g h o u t . T h e m e t h o d s a r e s i m p l e to a p p l y a n d p r o v i d e b e t t e r e s t i m a t e s o f t r u e t h i c k n e s s t h a n d o m e a s u r e m e n t s c o n f i n e d to t h i n sections.
ACKNOWLEDGEMENTS This work was supported by grants from the Leverhulme Trust and the National Science Foundation, BNS 76-I23 ~ 2 . We are also grateful to the Maternal-Infant Division of the Bolivian Ministry of Health, to Dr P. Sanguesa and Professor E. J. Clegg, and to others too numerous to mention. This is a research report from the US Agricultural Experiment Station, Division of Nutritional Sciences, Cornell University. REFERENCES Aherne, W. & Durmill, M. S. 0966) Morphometry of the human placenta. British Medical Bulletin, 22, 5-8. Bailey, N. T. J. (I972) Statistical Method in Biology. London: English Universities Press. Chabes, A., Peroda, J., Perez, J. et al (i968) Morphometry of human placenta at high altitude. AmericanJournal of Pathology, 50, I4a-x5a. Dubowitz, L. M. S., Dubowitz, V. & Goldberg, C. (197o) Clinical assessment of gestation age in the newborn infant. Journal of Pediatrics, 77, I-IO. Fox, H. (I964) The pattern of villous variability in the normal placenta. Journal of Obstetrics and Gynaecology of the British Commonwealth, 7I, 749-758Getzowa, A. S. & Sadowsky, A. (195o) On the structure of the human placenta with full-term and immature foetus, living or dead. Journal of Obstetrics and Gynaecology of the British Commonwealth, 57, 388-396. Gundersen, H. J. G., Jensen, T. B. &Osterby, R. (I978) Distribution of membrane thickness determined by lineal analysis. Journal of Microscopy, II3, 27-43. Gupta, M., Muyhew, T. M., Bedi, K. S. et al (t983) Inter-animal variation and its influence on the overall precision of morphometric estimates based on nested sampling designs. Journal of Microscopy, x3i , i47-154. Haas, J. D. (t 980) Maternal adaptation and fetal growth at high altitude in Bolivia. In Social and Biological Predictors of Nutritional Status, Physical Growth and Neurological Development (Ed.) Greene, L. S. & Johnston, F. S. pp. 257-290. New York: Academic Press. Haas, J. D. Frongillo, E. A., Stepick, C. D. et ai ( i98o ) Altitude, ethnic and sex differences in birth weight and length in Bolivia. Human Biology, 52, 459-477. Jensen, E. B., Gundersen, H. J. G. & Osterby, R. (1979) Determination of membrane thickness distribution from orthogonal intercepts. Journal of Microscopy, 115, t9-33. Laga, E. M., Driscoll, S. G. & Munro, H. N. (1973) Quantitative studies of human placenta. I. Morphometry. Biology of the Neonate, 23, 231-259. Mayhew, T. M. 0983) Stereology: progress in quantitative microscopical anatomy. In Progress in Anatomy (Ed.) Navaramam, V. & Harrison, R. J. Volume 3, PP. 81-I 12. Cambridge: Cambridge University Press. Mayhew, T. M., Joy, C. F. & Haas, J. D. (1984) Structure-function correlation in the human placenta: the morphometric diffusing capacity for oxygen at full term. Journal of Anatomy, t39 , 691--708. Sen, D. K., Kaufmarm, P. & Schweikhart, G. (i979) Classification of human placental villi. If. Morphometry. Cell and Tissue Research, 2oo, 425-434. Shay, J. (I975) Economy of effort in electron microscope morphometry. AmericanJournal of Pathology, 81, 5o3-512. Tominaga, T. & Page, E. W. (1966) Accommodation of the human placenta to hypoxia. AmericanJournal of Obstetrics and Gynecology, 94, 679-69I. Wang, T., Hamann, W. & Hartge, R. (i 983 ) Structural aspects of a placenta from a case of maternal acute lymphatic leukaemia. Placenta, 4, 185-196. Weibel, E. R. (I97O) Morphometric estimation of pulmonary diffusion capacity. I. Model and method. Respiration Physiology, I I, 54-75. Weibel, E. R. (t979) Stereological Methods. Volume I, Practical Methods for Biological Morphometry. London: Academic Press. Weibel, E. R. & Knight, B. W. 0964) A morphometric study on the thickness of the pulmonary air-blood barrier. Journal of Cell Biology, 2I, 367-384.
Stereological Studies on the True Thickness of the Villous m e m b r a n e in Human Term Placentae: a Study of Placentae from High-altitude Pregnancies MOIRA R. JACKSON a, CAROLINE F. JOY a, T. M. MAYHEW a,c & J. D. HAAS b a Department of Anatomy, Marischal College, University of Aberdeen, Aberdeen AB9 1AS, UK b Division of Nutritional Sciences, Martha Van Rensselaer Hall, Cornell University, Ithaca, N Y z4853, USA To whom correspondence should be addressed
INTRODUCTION In the human haemochorial placenta, maternal and fetal blood spaces are separated by the villous membrane. This comprises layers of trophoblast and fetal capillary endothelium with a variable amount of connective tissue interposed. The membrane acts as a physical barrier to the transfer of respiratory gases and other metabolites. It has a considerable mass, accounting for roughly 12 per cent of total placental weight (Aherne and Dunnill, 1966; Laga, Driscoll and Munro, I973), and a high metabolic activity. For these reasons, the thickness of the villous membrane is of physiological interest. Arithmetic mean thickness is proportional to tissue mass and so reflects tissue oxygen requirements (Weibei and Knight, 1964). Harmonic mean thickness, based on reciprocal local thicknesses, accords greater weight to thinner regions, and is a critical determinant of total placental resistance to gaseous diffusion (Laga, Driscoll and Munro, 1973; Mayhew, Joy and Haas, 1984). Using microscopically thin sections, it is possible to measure 'minimum membrane thickness' (Aherne and Dunnill, I966 ) or 'maternofetal diffusion distances' (Sen, Kaufmann and Schweikhart, 1979). Unfortunately, these estimates represent neither true arithmetic nor true harmonic mean thicknesses. The problem lies in the fact that the apparent irregularity in thickness actually arises from two main sources of variation: (x) natural variation in real thickness, partly attributable to localized thinning at vasculo-syncytial membranes (Getzowa and Sadowsky, 195o, Sen, Kaufmann and Schweikhart, i979), and (z) sectioning artefact, introduced by chance variability in the angle at which the villous membrane is sectioned during microtomy (Weibel and Knight, 1964, Gundersen, Jensen and Osterby, I978 , Jensen, Gundersen and 0sterby, 1979). Both variations must be taken into consideration before true thicknesses can be estimated and before harmonic means can be substituted into appropriate diffusion equations (Mayhew, Joy and Haas, 1984). In this investigation, artithmetic and harmonic mean thicknesses are calculated using stereological methods previously applied to the alveolar air-blood barrier (Weibel and Knight, z49
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M. R. Jackson, C. F. Joy, 7". M. Mayhew, J. D. Haas
x964) and to the glomerular filtration barrier (Gundersen, Jensen and 0sterby, 1978). Values are then used to derive a thickness uniformity index for assessing the functional significance of vasculosyncytial membranes and syncytiaI knots in determining the diffusional resistance of the villous membrane. Results are based on analyses of randomized histological sections from term placentae delivered by women living at high altitude in Bolivia.
MATERIALS AND M E T H O D S Material was collected as part of a much larger prospective study of maternal adaptation to altitude during pregnancy and its consequences for subsequent growth and development of the neonate. Details of the larger project may be consulted in Haas (I98O). Briefly, Amerindian women and non-Indian women of European ancestry were selected on the basis of uncomplicated, full-term gestation and the availability of records on age, socioeconomic status, migratory and reproductive history, dietary intake, health, blood biochemistry and haematology, anthropometry for nutritional status, genetic markers and smoking habits. Present results are confined to those from placentae belonging to a randomized subsample of I5 European mothers drawn by lottery from the larger population samples (Haas, i98o ). This number of placentae provided standard errors of less than 6 per cent of each group mean for the three thickness variables under scrutiny (see below). This level of precision is usually considered adequate for statistical comparisons between groups. All mothers had been born and raised at an average altitude of 36oo m in the Bolivian capital of La Paz. In order to verify gestational ages estimated from the last menstruation, a clinical examination (Dubowitz, Dubowitz and Goldberg, 197o) was administered to all infants within 24 hours of delivery. Infants were between 266 and 282 completed days of gestation. All were weighted by an attending neonatologist within minutes of delivery. It is important when making measurements involving fetal capillaries within villi to standardize handling of the placenta, especially the timing of clamping of the umbilical cord. In this study, the cord remained unclamped and uncut for up to one minute from delivery of the infant. Clamps were applied at two sites, the nearer being about io cm from the umbilicus, and the cord was cut between them. The clamp on the maternal side was subsequently released for roughly 15 sec for the purpose of sampling cord blood. It was thenre-applied until expulsion of the placenta. Following expulsion, the cord was immediately clamped and trimmed to within 5 mm of the fetal aspect of the placenta. Placentae were weighed after removing attached membranes and blood clots. Gestational ages, birthweights and trimmed placental weights are provided in Table i. Tissue s a m p l i n g Systematic samples of tissue from two quadrants per placenta were removed, fixed in isotonic formal saline and embedded in paraffin wax. From each of four to five blocks of tissue per placenta, a single arbitrary section (thickness c. 3/~m) was cut and stained by the Masson trichrome method. Light microscopical fields of view were obtained from the sections by systematic random sampling procedures (Weibel, I979; Mayhew, i983) and prepared as colour transparencies. Sets of 2o transparencies per organ were projected onto a flat, white cardboard screen for stereological evaluation. The final magnification ( x 2ooo) was standardized with a stage micrometer calibration scale.
Human Villous Membrane Thickness
25I
Table I. Gestational age, newborn birthweight and trimmed placental weight for a group z5 subjects from 36oo m altitude in Bolivia
Subject number IO17 IOI8 X037 xo5o Io54 1o65 I077 HI7 Ill 9 I132 3oi0 4oi2 4o47 407~ 4o97
Gestational age (days)
Weight of newborn (kg)
Weight of placenta (g)
268 266 274 274 274 269 282 273 272 277 267 278 272 274 278
2.93 2"50 2.82 3.22 2.80 2.93 3.29 2A3 2.8O 3.30 3.03 3.22 3.00 2.50 3.oo
466 335 368 49I 383 479 588 377 393 494 586 597 435 429 383
Stereological m e t h o d s All projected images were analysed by the same person, who had no prior knowledge about the placentae or their origins. In order to estimate arithmetic mean thickness (Ta) and harmonic mean thickness (Th), the projection screen was ruled with a set of equidistant, parallel, straight lines. Where these test lines randomly intercepted profiles of villous membranes, intercept lengths from the trophoblastic surface to the luminal surface of capillary endothelium were measured by ruler (see Figure i). Altogether, 47-57 intercept lengths per placenta were sampled. As far as it was possible to identify them, stem villi were excluded from these measurements. Their histological and morphometric characteristics suggest that stem villi play a
/5 Figure i. Section through villus illustrating chance encounters between test lines and profiles of villous membranes.
The positions and lengths of five random intercepts are indicated (L1-L~). cl = capillary lumen; fc = fetal capillary endothelium; st = stromal tissue; tr = trophoblast.
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M. R. Jackson, C. F. Joy, T. M. Mayhew, J. D. Haas
minimal role in maternofetal gas diffusion (Laga, Driscoll and Munro, I973; Sen, Kaufmann and Schweikhart, i979; Mayhew, Joy and Haas, I984). Given that the encounters between test lines and villous membrane profiles are random in position and orientation, it has been shown (Weibel and Knight, i964; Gundersen, Jensen and 0sterby, i978 ) that the mean intercept length ( L ) is related to T by the simple equation Ta = LJ2 and, taking the mean of reciprocal intercept lengths, that the harmonic mean intercept length (Lh) is related to T h by T h -- 2Lh/3 The dimension T is a measure of the volume (and hence the mass) of villous membrane associated with a unit area of trophoblastic surface (Weibel and Knight, i964). This dimension therefore influences the relative amount of oxygen that is consumed in the villous membrane as the gas is diffusing from the maternal to the fetal erythrocytes. Being based on reciprocal intercept lengths, T h places greater emphasis on thinner regions of the villous membrane: that is, on precisely those regions (vasculosyncytial membranes) considered to be best suited to facilitating passive transfer by diffusion. From these considerations it is clear that T equals T h and that the ratio ( T J T h ) equals I when the membranes are uniform in thickness. However, for membranes that, like the villous membrane, are irregular in thickness, ( T J T h ) > I. Therefore, the ratio T J T h affords a convenient index of thickness uniformity which we denote by the symbol Itu. D a t a handling Values of Ta, T h and Itu were calculated for each placenta after averaging random intercept lengths and reciprocal intercept lengths measured on all transparencies from all histological sections. The estimates were corrected for tissue shrinkage, the linear correction factors for these preparations being between 1.25 and 1.5o. Subsequently, intercept length size-frequency distributions were constructed for each placenta and employed to determine the overall percentage frequency distribution. Knowing the group mean T a for this observed distribution allowed us to calculate the expected distribution for a villous membrane of uniform thickness (see Gundersen, Jensen and 0sterby, 1078). Observed and expected distributions of intercept lengths were compared using a chi-squared test (Bailey, I972). Individual placental values of T , T h and Itu were later used to calculate group means and standard deviations (s.d.). As measures of the observed variability between placentae, and of intercept-to-intercept variability within placentae, coefficients of variation (c.v. = ioo x s.d./mean) were also calculated. All these calculations were handled using pre-recorded BASIC programs run on a Hewlett-Packard HP85 personal computer.
RESULTS The results are presented in Tables 2 and 3 and in Figure 2. Our qualitative observations support previous impressions. The thickness of the villous membrane is not equal throughout in microscopically thin sections. It is clear that part of the lack of uniformity reflects the fluctuating geometrical relationships between the trophoblast, connective tissue stroma and fetal capillaries. The latter tend to have a sinuous course and sometimes appear dilated, particularly those near the periphery of villi where they are separated from the trophoblast by minimal amounts of
253
Human Villous Membrane Thickness Table 2. Estimates of arithmetic mean, reciprocal mean and harmonic mean intercept lengths for villous membrane profiles
Placenta number IOI 7 IOI8 IO37 ]05 ~ lO54 lO65 1~ I I 17 I119 1132 3oio 4o12 4o47 4o7~ 4o97
Intercept length (#m) L
Reciprocal intercept length (/~m 1) L, 1
Harmonic mean (,um) Lh
IO.O1 + 7.25 (72.6) a 12.55+8.4o(67.o ) 7-34 + 5-76 (78-4) 8.05 + 4.82 (59.9) 9.6o+6.55(68.3) 8.73 + 5-45 (62.3) 5.65 + 4.74 (84.0) 8.1o + 5.68 (7o.I) 7"5~ ) 7.47+4.93(66.1) 9.25 + 8.78 (95.o) lO.55+7.7o(73.o ) 9.14 + 7 .26 (79.5) 9.55 + 5.66 (59.5) 9.68 + 5.86 (6o.5)
o.163 +o.i35 (82.6) a o.126+o.o94(74.2) o.224 + o. I72 (76.5) 0.2o9 + 0.188 (9o.2) o. 182 + o.I6O (87.9) o.167 + o.t23 (73.8) 0-313 "4-o.225 (71.7) o.225 +0.188 (83.4) ~ o.2o6"I-o.144(69.7) o.2oo + o. 15o (75.4) o.17o+o.158(93.1 ) o.220 + o.2o3 (92.2) o. 156 ___o. I Io (7o.3) o. 152 + o. lO2 (67.0)
6.i 4 7.92 4.46 4.79 5.5~ 5.99 3-19 4.44 4.43 4-85 5-oi 5.89 4.54 6.42 6.57
a Mean + s.d. (c.v. per cent). Table 3. Estimates of true arithmetic mean thickness, harmonic mean thickness and uniformity index for the villous membrane
Placenta number IOI 7 IOI8 IO37 105~ 1054 IO65 I O77 I 117 I I t9 1132 3OlO 4o12 4o47 4o7o 4o97 Group mean s.d. c.v. (per cent)
Arithmetic mean thickness (pm) T
Harmonic mean thickness (/lm) Th
Thickness uniformity index Ta/T h
5.00 6.27 3.67 4.O2 4.80 4.36 2.82 4.05 3.75 3.74 4.62 5.28 4.57 4.77 4.84
4.00 5.28 2.97 3.t9 3.67 4.OO 2.13 2.96 2.96 3.23 3.34 3.93 3.o3 4.28 4.38
1.22 I. 19 1.23 !.26 X.3X I .O9 1.33 1.37 1.26 1.15 1.39 1.34 t.5I 1.12 t.tl
4.44 o.817 i8. 4
3.56 0.777 21.8
1.26 o.118 9-4
s t r o m a l tissue o r o n l y b y basal l a m i n a . A t s u c h sites, t h e t r o p h o b l a s t is u s u a l l y a t t e n u a t e d a n d f o r m s r e c o g n i s a b l e v a s c u l o s y n c y t i a l m e m b r a n e s . A t o t h e r sites, t h e t r o p h o b l a s t t h i c k e n s i n t o s y n c y t i a l k n o t s , t h e capillaries are n a r r o w e r a n d m o r e c e n t r a l l y located, a n d i n c r e a s e d a m o u n t s o f s t r o m a l tissue i n t e r v e n e b e t w e e n t h e t r o p h o b l a s t a n d capillary e n d o t h e l i u m . T h e s e o b s e r v a t i o n s a p p l y to all r a m i f i c a t i o n s o f t h e villous tree, t h e d i f f e r e n c e s b e t w e e n v a r i o u s classes o f villi b e i n g q u a n t i t a t i v e r a t h e r t h a n qualitative. N o a t t e m p t was m a d e to
=54
M. R. Jackson, C. F. Joy, T. M. Mayhew, J. D. Haas
30-
Cr
20"
O~ 0= e= 0 r n
10-
Ta; L aI 0
'~ 0
TI 10
210
30
410
I 50
Intercept length, jam Figure 2. Observed frequencydistribution of random intercept lengths pooledfor 15 placentae. Mean intercept length
(L,) and the arithmetic meanthicknessof the actual membrane(Ta) are indicated. The distribution differssignificantly from that expectedfor a membraneof uniformthicknessthroughoutfor whichno randomintercept can be less than T a in length. characterize terminal villi and intermediate villi separately. Therefore, the following quantitative findings refer to the villous membrane on these ramifications collectively. Individual random test line intercepts varied in length from less than 2/am to more than 5o/am. Despite this large intra-placental variation (c.v. 6o to 95 per cent), mean intercept lengths per placenta varied only between 5.6/am and 12.6/am, the corresponding range for harmonic mean intercept lengths being 3.2 to 7-9/am (Table 2). The overall size-frequency distribution of observed intercept lengths was calculated and plotted from the percentage frequency distributions of the 15 placentae. This histogram (Figure 2) comprises ten size classes each representing an equal increment of intercept length based on the largest distance recorded (i.e., 53.2/am). The observed distribution (c.v.c. 73 per cent) differed significantly from the expected distribut/on for a uniformly thick villous membrane (chi-squared value, 6o.4; degrees of freedom, 9; P < o.ooi). Estimated group means (c.v.) for the three membrane thickness variables were 4.44/am 0 8 per cent) for T , 3.56/am (22 per cent) for T h and 1.26 (9 per cent) for Itu (Table 3). In spite of the differences in T a and T h between placentae, and the su bstantial differences in intercept and reciprocal intercept lengths within placentae, the observed c.v. for Itu was comparatively small. In other words, the uniformity index was relatively constant from one placenta to the next.
DISCUSSION This investigation has sought to provide more reliable information on the true thickness of the villous membrane in human term placentae by deriving estimates of its arithmetic and harmonic
Human Villous Membrane Thickness
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mean thicknesses. Before comparing present estimates with previous findings, or discussing their functional significance, it is worth considering the errors influencing them.
Sources o f error Generally speaking, these are of two types, viz. random (or accidental) error and systematic error or bias (Mayhew, i983). Random errors are introduced by the natural 'biological variation' between placentae and by variation within organs imposed by the experimental sampling design. Within-organ variation therefore includes differences between tissue blocks within placentae, between fields of view within blocks, between villi within fields and between intercepts within villi. Some of this variation is also attributable to the precision of the method of measuring intercept lengths. Fortunately, it has been demonstrated repeatedly that within-organ errors often have little influence on the overall precision ofmorphometric estimates (see Shay, 1975; Gupta et al, 1983; Mayhew, 1983). By means of pilot studies, we confirmed for ourselves that analysing a reasonably large number of organs was the most economical way of minimizing these errors. The fact that we elected to sample fewer blocks of tissue per placenta than in some earlier morphometric studies (Aherne and Dunnill, 1966; Lags, Driscoll and Munro, 1973) is of little significance from the viewpoint of determining the precision of our final estimates of thickness. Indeed, a good indication of the precision of final estimates is afforded by calculating their coefficients of error ( = standard error/group mean). The values were 4.8 per cent (Ta), 5.6 per cent (Th) and z.4 per cent (Ira) when expressed as percentages. Systematic errors are introduced by the sampling design, by various technical limitations and by the constraints of the geometric model on which the pertinent stereological relationships are based (Weibel and Knight, 1964; Gundersen, Jensen and0sterby, 1978; Weibel, 1979; Mayhew, 1983). The methods we have used rely on the fact that the length of a random test line intercept across a membrane is determined solely by local membrane thickness and by the angle between the test line and the normal to the membrane (Weibel and Knight, 1964; Gundersen, Jensen and 0sterby, 1978). The main condition to be fulfilled is that of random orientation between membranes and test lines. Since this is exclusively a sampling problem, we have tried to meet this condition by randomized sampling at all stages of the selection process (tissue blocks, sections, fields of view, intercepts). The methods are robust enough to be only minimally affected by systematic errors due to variations in the surface curvature and thickness distribution of the villous membrane. However, technical biases from shrinkage and section thickness effects do influence the validity of present estimates. We have tried to compensate for shrinkage in order to compare our results with those obtained in other laboratories. Estimates have not been corrected for section thickness. Approximate corrections are available (e.g., Weibel, 197o), but it should be borne in mind that in thicker parts of the villous membrane the effects of section thickness are diminished in comparison to thinner regions. Suitable corrections therefore require knowledge of the distributions of membrane thickness (Gundersen, Jensen and 0sterby, 1978) which are, in any case, best reconstructed from semi-thin sections for light microscopy or ultra-thin sections for electron microscopy. Vasculosyncytial membranes, the thinnest regions, are estimated to account for only IO to z6 per cent of the total surface area of terminal and intermediate villi in such sections (Aherne and Dunnill, 1966; Sen, Kaufmann and Schweikhart, I979). In view of the sectioning artefact due to chance encounters between membranes and section planes, these percentages are probably underestimates.
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C o m p a r i s o n with previous findings Direct comparisons are not possible because of the lack of adequate information on the measuring convention. The term 'minimum membrane thickness' (Aherne and Dunnill, I966 ) probably refers to the apparent thickness of vasculosyncytial membranes measured at right angles to the trophoblastic surface. Such distances may overestimate true arithmetic mean thickness at these sites by about 27 per cent (see Jensen, Gundersen and 0sterby, t979, P- 22, formula 9)- Therefore, the value of 3. 5 pm for minimum membrane thickness could represent an arithmetic mean thickness of 2.7/~m for vasculosyncytial membranes. The term 'maternofetal diffusion distance', as used by Sen, Kaufmann and Schweikhart (I979) , is misleading. It, too, seems to be an orthogonal mean intercept length, calculated for the entire villous membrane including stem villi. Since it is not derived from reciprocal intercept lengths, the dimension is not a harmonic mean thickness and so cannot be interpreted as an effective diffusion distance. If their value of 5.94 pm is an orthogonal intercept length, it would correspond (Jensen, Gundersen and 0sterby, 1979) to a true arithmetic mean thickness of 4.67 pm. This figure is reasonably close to our estimate of 4.44 pm which is based on thicker sections and excludes stem villi. Our estimate of 3.56 ~m for the harmonic mean thickness of highland placentae compares with one of 4.08 pm for a group of low altitude placentae from Bolivia (Mayhew, Joy and Haas, 1984). These findings suggest that the harmonic mean thickness of io/~m adopted by Laga, Driscoll and Munro (i973) is far too high for substituting into diffusion equations. Indeed, their estimates appear to be informed guesses relying on mean villous diameter, central rather than peripheral fetal capillaries, and values of Itu more appropriate to the lung than to the placenta (see below).
Biological implications Our interest in harmonic mean thickness stems from a desire to calculate placental diffusing capacities for oxygen by combining the structural dimensions and physicochemical properties of the maternofetal diffusion pathway (Laga, Driscoll and Munro, I973; Mayhew, Joy and Haas, i984). Such studies demonstrate that the resistance of the villous membrane is the principal contributor to total diffusional resistance. Since the resistance of this membrane depends on its harmonic thickness, reliable estimates of this dimension are necessary. Substituting 'minimum membrane thickness' into the pertinent equations would tend to overestimate, and 'maternofetal diffusion distance' to underestimate, oxygen diffusing capacity, the former leading to even larger discrepancies between physiological and morphometric diffusing capacities than exist already (Mayhew, Joy and Haas, 1984). The present study suggests that there may be a negative correlation between newborn birthweight and harmonic mean thickness of the villous membrane (refer to Tables I and 3). Though the correlation is not statistically significant (perhaps due to inadequate numbers, or to too narrow a range of birthweights), a negative correlation might be anticipated in view of the positive and significant correlation between morphometric diffusing capacity and birthweight which exists during the last trimester of pregnancy (Mayhew, Joy and Hass, I984). Our use of the uniformity index I~u warrants further comment. The value of 1.26 by itself conveys little information because it is difficult to imagine what this means in a physical sense. However, comparisons with values for other tissue barriers help. Thus the basement membrane of the human renal glomerulus--considered to be relatively uniform in thickness--has an Itu of about I. i in non-diabetics and in patients with diabetes mellitus between zero and nine years' duration (Gundersen, Jensen and0sterby, I978, p. ,to, table 2). Similar values are found in other mammals. In contrast, the alveolar-capillary membrane of the lung has an average Itu of 2.2 in
Human Villous Membrane Thickness
~57
the rat (Weibel and Knight, 1964). In this particular barrier, the thickness may vary from only o.~/am in extremely attenuated regions to about 8/am in the thickest regions. A better way of interpreting Itu is tO consider its functional significance. The ratio T a / T h provides an indication of the efficacy of the villous membrane in permitting gas transfer by diffusion. Thus the figure 1.26 can be regarded as indicating that the diffusing capacity of the villous membrane is 26 per cent more than it would be if the membrane was uniformly thick throughout. In the rat lung the corresponding increase would be i zo per cent, in accordance with the fact that the lung is a more effective gas exchange organ. In the case of the placenta, it is clear that the improvement is achieved by concentrating much of the tissue mass away from vasculosyncytial membranes and in thicker regions, particularly at syncytial knots. It is therefore of considerable interest that the placenta should respond to hypoxic stress by increased syncytial knotting (e.g., Fox, i964; Tominaga and Page, 1966; Chabes et al, i968 ). The low inter-placental variation in Itu estimates, despite c.v. values of 18 per cent and 22 per cent for T a and T h respectively, and despite all of the random errors mentioned above, may be a further sign of the functional importance of this index. We may envisage Itu as reflecting a balance which has to be struck between the metabolic activity of the trophoblast (which requires a certain tissue mass and arithmetic mean thickness) and the need of a thin barrier for diffusion of gases and metabolites (which requires a certain harmonic mean thickness). Though T and T h may vary according to the needs of the individual fetoplacental unit, present results suggest that the structural compromise is quantitatively similar in different individuals. The real lack of uniformity in villous membrane thickness no doubt contributes to the discrepancy between physiological and morphometric estimates of placental diffusing capacity (Mayhew, Joy and Haas, 1984). Diffusion is likely to proceed more rapidly where the resistance of the villous membrane is lower, and less rapidly where it is higher, producing regional inhomogeneities in diffusing capacities. In conclusion, the present stereological methods offer several advantages over planar morphometric data for drawing structure-function correlations in the human placenta. Methods similar to those described here may be employed to estimate the effective diffusion distances of the serial blood plasmas which also affect the overall diffusing capacity of the placenta (Weibel, 197o; Mayhew, Joy and Haas, 1984). They may also be used to study the functional morphology of placentae during gestation and in various states associated with increases in the histological components of the villous membrane; for example, trophoblastic basement membrane thickening and proliferation of cytotrophoblast (Fox, 1964; Wang, Hamann and Hartge, I983). We are now using these methods to investigate placental morphology in pregnancies associated with reduced birthweight (Haas et al, 198o).
SUMMARY Stereological principles were used to calculate functionally significant dimensions of the human villous membrane, its arithmetic mean thickness ( T a ) and its harmonic mean thickness (Th). The former is proportional to tissue mass and oxygen consumption, the latter to diffusional resistance. For a group of 15 term placentae from uncomplicated pregnancies at high altitude, the average values w e r e T a = 4-44/am, T h = 3.56/~m a n d T a / T h = 1.26. The latter figure provides a useful quantitative expression for the efficiency of the membrane in gas and metabolite diffusion. It implies that vasculosyncytial membranes and syncytial knots decrease resistance to diffusion by 26 per cent, compared with that of a membrane with uniform thickness
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M. R. Jackson, C. F. Joy, T. M. Mayhew, J. D. Haas
t h r o u g h o u t . T h e m e t h o d s a r e s i m p l e to a p p l y a n d p r o v i d e b e t t e r e s t i m a t e s o f t r u e t h i c k n e s s t h a n d o m e a s u r e m e n t s c o n f i n e d to t h i n sections.
ACKNOWLEDGEMENTS This work was supported by grants from the Leverhulme Trust and the National Science Foundation, BNS 76-I23 ~ 2 . We are also grateful to the Maternal-Infant Division of the Bolivian Ministry of Health, to Dr P. Sanguesa and Professor E. J. Clegg, and to others too numerous to mention. This is a research report from the US Agricultural Experiment Station, Division of Nutritional Sciences, Cornell University. REFERENCES Aherne, W. & Durmill, M. S. 0966) Morphometry of the human placenta. British Medical Bulletin, 22, 5-8. Bailey, N. T. J. (I972) Statistical Method in Biology. London: English Universities Press. Chabes, A., Peroda, J., Perez, J. et al (i968) Morphometry of human placenta at high altitude. AmericanJournal of Pathology, 50, I4a-x5a. Dubowitz, L. M. S., Dubowitz, V. & Goldberg, C. (197o) Clinical assessment of gestation age in the newborn infant. Journal of Pediatrics, 77, I-IO. Fox, H. (I964) The pattern of villous variability in the normal placenta. Journal of Obstetrics and Gynaecology of the British Commonwealth, 7I, 749-758Getzowa, A. S. & Sadowsky, A. (195o) On the structure of the human placenta with full-term and immature foetus, living or dead. Journal of Obstetrics and Gynaecology of the British Commonwealth, 57, 388-396. Gundersen, H. J. G., Jensen, T. B. &Osterby, R. (I978) Distribution of membrane thickness determined by lineal analysis. Journal of Microscopy, II3, 27-43. Gupta, M., Muyhew, T. M., Bedi, K. S. et al (t983) Inter-animal variation and its influence on the overall precision of morphometric estimates based on nested sampling designs. Journal of Microscopy, x3i , i47-154. Haas, J. D. (t 980) Maternal adaptation and fetal growth at high altitude in Bolivia. In Social and Biological Predictors of Nutritional Status, Physical Growth and Neurological Development (Ed.) Greene, L. S. & Johnston, F. S. pp. 257-290. New York: Academic Press. Haas, J. D. Frongillo, E. A., Stepick, C. D. et ai ( i98o ) Altitude, ethnic and sex differences in birth weight and length in Bolivia. Human Biology, 52, 459-477. Jensen, E. B., Gundersen, H. J. G. & Osterby, R. (1979) Determination of membrane thickness distribution from orthogonal intercepts. Journal of Microscopy, 115, t9-33. Laga, E. M., Driscoll, S. G. & Munro, H. N. (1973) Quantitative studies of human placenta. I. Morphometry. Biology of the Neonate, 23, 231-259. Mayhew, T. M. 0983) Stereology: progress in quantitative microscopical anatomy. In Progress in Anatomy (Ed.) Navaramam, V. & Harrison, R. J. Volume 3, PP. 81-I 12. Cambridge: Cambridge University Press. Mayhew, T. M., Joy, C. F. & Haas, J. D. (1984) Structure-function correlation in the human placenta: the morphometric diffusing capacity for oxygen at full term. Journal of Anatomy, t39 , 691--708. Sen, D. K., Kaufmarm, P. & Schweikhart, G. (i979) Classification of human placental villi. If. Morphometry. Cell and Tissue Research, 2oo, 425-434. Shay, J. (I975) Economy of effort in electron microscope morphometry. AmericanJournal of Pathology, 81, 5o3-512. Tominaga, T. & Page, E. W. (1966) Accommodation of the human placenta to hypoxia. AmericanJournal of Obstetrics and Gynecology, 94, 679-69I. Wang, T., Hamann, W. & Hartge, R. (i 983 ) Structural aspects of a placenta from a case of maternal acute lymphatic leukaemia. Placenta, 4, 185-196. Weibel, E. R. (I97O) Morphometric estimation of pulmonary diffusion capacity. I. Model and method. Respiration Physiology, I I, 54-75. Weibel, E. R. (t979) Stereological Methods. Volume I, Practical Methods for Biological Morphometry. London: Academic Press. Weibel, E. R. & Knight, B. W. 0964) A morphometric study on the thickness of the pulmonary air-blood barrier. Journal of Cell Biology, 2I, 367-384.