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Skeletal growth and development of the human fetus : effect of maternal and nutritional factors. Roberts, Jill Anne 1971

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SKELETAL GROWTH AND DEVELOPMENT OF THE HUMAN FETUS: EFFECT OF MATERNAL AND NUTRITIONAL FACTORS by JILL ANNE ROBERTS B . H o E o , University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HUMAN NUTRITION in the Department of Home Economics We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f fiCrrrrj^. fis^ttrrrr s ^Li The U n i v e r s i t y o f B r i t i s h Co lumbia V a n c o u v e r 8, Canada Date a/«^ 4f% in/ i A B S T R A C T Growth i s associated with the a v a i l a b i l i t y of e s s e n t i a l nutrients and i t seems possible that these n u t r i -ents could a f f e c t the growth mechanism involved in s k e l e t a l development. To t e s t t h i s hypothesis 76 normal human fetuses aged 9 to 20 weeks were c o l l e c t e d from therapeutic abortions. Sex, weight, length, head circumference, foot length and a s k e l e t a l index were recorded; developmental age was calculated from crown-rump length, and gestational age estimated from the mother's menstrual h i s t o r y . Bones from the r i g h t arm and leg were removed and cleaned for biochemical analysis. Calcium, inorganic phos-phorus, magnesium, sodium and collagen content of 60 femora and humeri were determined, aft e r length, fresh weight, constant dry weight and f a t - f r e e weight were recorded. Length of o s s i f i c a t i o n i n the bones of the l e f t arm and leg was measured v i a s i l v e r radiography. Assuming b i l a t e r a l symmetry, biochemical and physical data could then be com-pared. A l l f e t a l data were grouped according to develop-mental age: 9-10, 11-12, 13-14, 15-16, 17-20 weeks. Analysis of variance and Duncan's New Multiple Range Test were performed to determine the s i g n i f i c a n c e of group e f f e c t . Simple l i n e a r regression was executed on the whole i i range of data to detect which variables best predicted other v a r i a b l e s . Maternal information was obtained from an interview and from medical records at Vancouver General Hos p i t a l . Age, weight, height, b i r t h weight, p a r i t y and g r a v i d i t y of the mother were recorded. A socio-economic index was c a l -culated. Adequacy of maternal d i e t during pregnancy was assessed from a d a i l y pattern r e c a l l , food frequency and preference questions. These data were used to calcu l a t e a t o t a l n u t r i t i o n score and a protein score. Maternal data were coded as pot e n t i a l independent variables and multiple regression analysis performed against f e t a l dependent v a r i a b l e s . As developmental age of the fetuses increased, the fresh length, dry weight and length of o s s i f i c a t i o n also increased i n both humerus and femur, as did the calcium and phosphorus content. In most cases long bone growth as measured by these variables advanced proportionately with f e t a l age. Thus group means of most variables were s i g n i f i c a n t l y d i f f e r e n t from each other when divided into f i v e 2 week age periods. Water content dropped propor-ti o n a t e l y with age, r e f l e c t i n g bone mineralization. Sodium content f e l l markedly i n f e t a l bones after 10 weeks. Mag-nesium and collagen remained constant. Fat extraction d i d not change the dry weight of the bones. i i i S t a t i s t i c a l c o r r e l a t i o n was found between physical and biochemical data. Generally physical variables were best predicted by other physical v a r i a b l e s . Biochemical composition of the femur could best be predicted from corresponding data i n the humerus. When gestational age was plotted against physical or biochemical variables, s t a t i s t i c a l c o r r e l a t i o n was weaker. The c o r r e l a t i o n found between f e t a l variables and maternal age, par i t y , weight and socio-economic status would indicate a d i v e r s i t y of factors influencing f e t a l growth. Whereas protein score of maternal d i e t was not s t a t i s t i c a l l y related with f e t a l parameters, general n u t r i t i o n score showed a consistent, p o s i t i v e c o r r e l a t i o n with length and dry weight of the femur and humerus. This r e l a t i o n s h i p was s t a t i s t i c a l l y s i g n i f i c a n t when develop-mental or gestational age remained constant. The res u l t s of t h i s study suggest that n u t r i t i o n of the pregnant woman i s p o s i t i v e l y correlated with some indices of s k e l e t a l growth and development of the human fetus. iv T A B L E O F C O N T E N T S PAGE ABSTRACT . . i TABLE OF CONTENTS Iv LIST OF TABLES . o v i LIST OF FIGURES v i i LIST OF PROCEDURE FORMS v i i i LIST OF PLATES • • • • • © • • • • • • • • • • ( . • • ^ • • • • • © • © © © • • © • • • o © » j_x LIST OF SCATTERGRAMS X ACKNOWLEDGMENT . x i i i REVIEW OF LITERATURE 1 A. Parameters of f e t a l growth and development 1. B i r t h weight 2. Skeletal growth and development (a) o s s i f i c a t i o n and growth (b) composition and development B. Maternal n u t r i t i o n and f e t a l growth 1. Role of n u t r i t i o n 2. E f f e c t of n u t r i t i o n on b i r t h weight 3. N u t r i t i o n and bone growth (a) animal studies (b) human studies INTRODUCTION • • o o o « » » e o e e » * o o o * * * « a * * e o « « o « o o * o o * « o e e < > 3^ MATERIALS AND METHODS 37 A. F e t a l studies 1. Long bone studies: physical 2. Long bone studies: biochemical 3 . Long bone studies: radiographical V PAGE B. Maternal studies 1. Medical h i s t o r y 2. Dietary h i s t o r y 3 . Personal h i s t o r y C. S t a t i s t i c a l analysis RESULTS . ^5 A. Fe t a l data 1. Whole fetus 2 . Long bones B. Maternal data 1. Medical growth 2. Socio-economic status 3 . Sex of fetus 4 . N u t r i t i o n a l data DISCUSSION 69 REFERENCES 80 APPENDIX 1. - METHODS . ..... 97 AP PEND X^X! 2 o "~ DATA o * o o o o o a « * * * * « * 0 « o * o * Q « o * o « » e o o o « 117 L I S T O F T A B L E S TABLE PAGE I. Composition of the whole femur of the human fetus . 10 I I . Composition of the cortex of the femur during f e t a l l i f e 12 I I I . F e t a l and long bone growth related to develop-mental age 4-7 IV. Composition of f e t a l long bones according to developmental age 48 V. Means and standard deviations of whole f e t a l variables 49 VI. Means and standard deviations of long bone variables 51 VII. Change in bone weight following f a t extraction .. 57 V I I I . Means and standard deviations of maternal variables 58 IX. E f f e c t of maternal variables on f e t a l data 60 X. E f f e c t of n u t r i t i o n a l variables on f e t a l data ... 67 XI. Estimated crown-rump length vs. developmental age of fetus 98 XII. Minimum formalin treatment for s i l v e r radio-graphy 108 XIII. Optimum s i l v e r n i t r a t e treatment for radio-graphy 109 XIV. Exposure time for f e t a l radiography 110 v i i L I S T O F F I G U R E S FIGURE PAGE Semidiagrammatic presentation of f e t a l growth of several population groups .., 2. Relation of osseous development of l i v i n g f u l l - t erm infants at b i r t h to the i r mothers' Cl 16 t Cl U 3T X I"i Cf P 3T6 CJ 9 fl G y Q O O O O O O * Q O O O O O O O O O O O O O O O O O 2 ^ 3 . Histogram of f e t a l age groups .................. k-6 4. Histogram of maternal socio-economic 5. Histogram of maternal n u t r i t i o n scores ......... 6k 6. Measurement of o s s i f i e d shaft of f e t a l bOI"16 • o o o e o o e a o e e o o o » o o o e e o * o o e o a o e o e o o o o o a o o a o a 1X1 v i i i L I S T O F P R O C E D U R E F O R M S FORM PAGE 1 o D l S t 3 i r y t l l S t O i r y O O O O » O O O O O O « J O « O O O « O O O O O O O « O Q Q O O O 112 2 e Nil t 3T i t X O n 3 1 S t S t U S o o o o o e o e o o o o o o o o o o a o o e o o o o o o o 11^ 3 o Socio-economic status o o o o o o o o o o o o © 0 o 0 o 0 o » © o o o o o 116 i x L I S T O F P L A T E S PLATE PAGE 10 Specimen in intact sac o o © © o o o © o o o © < » o & o o © © o o © o o o 99 2 , Fetus and p i aCenta o o o o o o o o e o o o o o o o o e o o o o a e o a o o o l O O 3. Eviscerated fetus? right arm and leg 3T©rnOVeCl © © o o o o o o o o o o o © o © o o © o o o o o a o o o o o o o o © o o o o o © jL 0 1 4. Six f e t a l long bones; cleaned, fresh ........... 102 5. Six f e t a l long bones; dried .................... 103 6. Fetus prepared for radiograph aft e r s i l v e r n i t r a t e treatment 104 7. Radiograph; fetus in formalin only ............. 105 8 . Radiography; fetus in s i l v e r n i t r a t e for S 15C d 3 y S o o o 0 0 o o o o o e o o o o o o o o o o o e o o o a o a o « © © o © O 0 o o 3 . O 6 9. Radiography; fetus in s i l v e r n i t r a t e for ten days » o o » o » o © * » o « » o o © © o o © o © o © © o o » o © o o © o © o © o © o © 10"7 X L I S T O F S C A T T E R G R A M S EXHIBIT PAGE 1. Developmental age vs. crown-rump length ...... 118 2. Developmental age vs. head circumference ..... 119 3. Developmental age vs. foot length ............ 120 4. Developmental age vs. f e t a l weight ........... 121 5. Developmental age vs. s k e l e t a l index ......... 122 6. Developmental age vs. gestational age ........ 123 7. Gestational age vs. foot length .............. 12^ 8. Gestational age vs. crown-rump length ........ 125 9. Gestational age vs. head circumference ....... 126 10. Gestational age vs. f e t a l weight ............. 127 11. Gestational age vs. s k e l e t a l index ........... 128 12. Developmental age vs. femoral dry weight ..... 129 13. Developmental age vs. humeral dry weight ..... 130 14. Developmental age vs. femoral water C 5^ ft. *t@ n t o o o e o o o o o o o o o o o o a o o o o o v a o o o o o o o o o o o o o o 3* 3 *^ 15. Developmental age vs. humeral water C O n t © n t o o o o o o o o e o A o o e o o o o o o o o o o o o o o o o o o o o o e o o 13^ 16. Developmental age vs. femoral length ......... I33 17. Developmental age vs. humeral length ......... 13^ 18. Developmental age vs. femoral O S S l f l C S t l O n 9 0 o e o o o o » o o o e o o o o o a e e o o e o o o o o o o o o i 3 .5 19. Developmental age vs. humeral O S S l f l C 5 l t l O n o o o o o o o o o o o o o o o o o o o e o o o o o o o o o e o o o «^ 3 ^  x i EXHIBIT PAGE 20. Developmental age vs. femoral collagen ....... 137 21. Developmental age vs. humeral collagen ....... I38 22. Developmental age vs. femoral calcium ........ 139 23. Developmental age vs. humeral calcium ........ 140 24. Developmental age vs. femoral inorganic p j h O S p h O l T U S O O O O Q O O O O O O O O O O Q O O O O O 0 O O O O O O O O O O Q O O X^"J*1 25. Developmental age vs. humeral inorganic p h O S pl"10 ITU S o o o o a o o o o o o o o e o e e o o o « o o o o Q O O O O o o o o a 1 ^4*2 26. Developmental age vs. femoral magnesium ...... 14-3 27. Developmental age vs. humeral magnesium ...... 144 28. Developmental age vs. femoral sodium ......... 145 29. Developmental age vs. humeral sodium ... ...... 146 30. Developmental age vs. femoral calcium/ O O X l a C J © IT, r a t l O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 X 31. Developmental age vs. humeral calcium/ c o X X a c j e n r a t 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * 0 0 0 0 0 0 1 4^*8 32. Developmental age vs. femoral calcium/ p h O S p l * i a t e r a t l O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 1^4*9 33. Developmental age vs. humeral calcium/ p h O S p h a t e r a t l O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 1^0 34. Maternal weight vs. maternal height .......... 151 35. Maternal age vs. maternal p a r i t y ............. 152 36. Maternal age vs. maternal g r a v i d i t y .......... 153 37. Maternal parity vs. maternal g r a v i d i t y ....... 154 38. Socio-economic score vs. socio-economic CJ r O U P 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 ^ .f? 39. Total n u t r i t i o n score vs. weighted r i U t r i t l O n S O O r e o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 1 x i i EXHIBIT PAGE 40. Total n u t r i t i o n score vs. n u t r i t i o n i r iC l65C O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Q O O O 1^ *^  41. Nu t r i t i o n index vs. weighted score ........... 158 42. Total n u t r i t i o n score vs. protein score ...... 159 43. Protein score vs. n u t r i t i o n index ............ 160 44. Protein score vs. weighted score ............. 161 A C K N O W L E D G M E N T Acknowledgment i s due to: Dr. Betty Poland, for embryological guidance and for access to both mothers and fetuses; Dr. Poland's s t a f f , for t h e i r cooperation and technical assistance; Dr. George Eaton, for s t a t i s t i c a l advice and negotiatons with the computer; and Dr. Birkbeck along with the Div i s i o n of Human N u t r i t i o n . 1 R E V I E W O F L I T E R A T U R E One of the fundamental features of development i s growth, defined as an increase in s p a t i a l dimensions and weight. Growth may be accomplished through increases i n the number of c e l l s , the s i z e of i n d i v i d u a l c e l l s , or the amount of i n t e r c e l l u l a r substance (1). F e t a l growth of a p a r t i c u l a r organ or tissue i s usually produced by a l l three components simultaneously. The observation by schultz i n 1926 (2) i n h i s comprehensive t r e a t i s e on the f e t a l growth of man and other primates, that more i s known about growth in the embryonic and postnatal periods than about the f e t a l periods, i s s t i l l v a l i d today. U n t i l recently the human fetus was considered to have a r e l a t i v e l y constant growth rate so that a small baby was necessarily a premature one. Over the l a s t 20 years obstetricians and p e d i a t r i c i a n s have become aware that the human fetus, l i k e a l l other l i v i n g things, grows at a variable r a t e . I t has also become c l e a r that the s i z e a baby has attained r e l a t i v e to the period of gestation i s important in determining the hazards i t w i l l face i n the p e r i n a t a l period (3-13). 2 A. Parameters of F e t a l Growth and Development 1. B i r t h Weight Since i t i s obviously impossible to study human f e t a l growth l o n g i t u d i n a l l y , one must r e l y on b i r t h weight curves to compare f e t a l growth. Such curves are based on the assumption that b i r t h weights a f t e r various lengths of gestation are representative of normal f e t a l weight at those times. Published charts such as those of Ba t t a g l i a (14), Lubchenco (15, 16) and Usher (17) are of li m i t e d v a l i d i t y so far as normal f e t a l development i s concerned, as information has been obtained from premature b i r t h s or spontaneous abortions. I t i s not usually known whether the mishap was due to uterine anomalies, placental defect or f e t a l abnormality, but at le a s t i t i s u n j u s t i f i e d to accept the l e v e l of f e t a l growth as r e s u l t i n g from normal gestation. Present workers i n the f i e l d of human prenatal development continue to show how d i f f i c u l t i t i s to demon-strate cause and e f f e c t i n growth. Extensive evidence from human and animal studies indicates that b i r t h weight i s primarily determined by factors r e l a t i n g to uterine envi-ronment rather than by the genetic c o n s t i t u t i o n of the fetus (18, 19). Determinants of b i r t h weight, varying i n sign i f i c a n c e and directness of t h e i r e f f e c t , have been considered by various authors (20, 21, 22). These include: r a c i a l o r i g i n (23, 24), period of gestation(25, 26), type and amount of prenatal care (27, 28), s o c i a l and economic 3 status (29, 30, 31), maternal age (32), maternal weight and height (33-38), maternal cigarette smoking (39, 40), maternal disease (41, 42), maternal prenatal n u t r i t i o n (42-46), maternal education (45), maternal occupation (47),parity and b i r t h order (48, 49), sex of infant (84), geographical location and season (143). For example, Gruenwald (50) f e e l s that the e f f i -cency with which the maternal organism s a t i s f i e s the needs of pregnancy can be judged by f e t a l growth. New values f o r b i r t h weight i n r e l a t i o n to gestational age have been proposed, and are i l l u s t r a t e d in Figure 1 (51). According to Gruenwald i t i s l i k e l y that the normal b i r t h weight curves of various population groups do not d i f f e r from one another during the f i r s t h a l f of the t h i r d trimester or longer. The l i n e a r course i s i n d i c a t i v e of unrestrained growth regulated by the growth p o t e n t i a l of the fetus in the presence of an adequate supply l i n e . A time comes when support i s no longer adequate for unrestrained growth. The lower the l e v e l of growth support received from the mother v i a the placenta, the e a r l i e r the departure from the st r a i g h t l i n e growth, and the lower i s the b i r t h weight at term (see graph). There i s a trend toward higher b i r t h weights within most population groups as a r e s u l t of improved n u t r i t i o n a l , socio-economic and medical conditions. The spectacular change in b i r t h weights in Japan during a 20 year period was caused only by better f e t a l growth and not by an increase i n duration of pregnancy (21). I t would be i n t e r e s t i n g to apply t h i s hypothesis to Meredith's world-wide comparative t r e a t i s e of b i r t h weights (52). Figure 1. Semidiagrammatic presentation of f e t a l growth (determined from b i r t h weight) of several population groups (51). 5 2. Skeletal Growth and Development a) O s s i f i c a t i o n and Growth Histogenesis of human c a r t i l a g e and bone has been well described (53, 54, 55, 56). The forerunner of the skePton i n the f e t a l body i s formed as a cartilaginous frame-work, and t h i s begins to c a l c i f y at about the eighth week of gestation. Wallgren (57) has made a det a i l e d microradio-graphical study of the process of o s s i f i c a t i o n of f e t a l bone and has shown that o s s i f i c a t i o n i n the long bones begins at the center of the cartilagenous model. A thin layer of c a l c i f i e d bone matrix i s l a i d down between the perichondrium and that portion of the shaft containing hypertrophic c a r t i l a g e c e l l s , and by extending around the shaft, forms a ring or c o l l a r . This c o l l a r i s incomplete at f i r s t , and the rate of development varies from one type of bone to another and even the long bones do not a l l develop equally r a p i d l y . The c l a s s i c review of the h i s t o -genesis of c a r t i l a g e and bone using the f e t a l humerus as an example has been presented by Streeter (58). Recently Gray has outlined the prenatal development of the human femur (59) and humerus (60). Many tables are found i n the early l i t e r a t u r e categorizing the developmental sequences of both membranous and endochondral o s s i f i c a t i o n (61-68). Using Streeter's or Boyd's (69) method of staging human fetuses, a rough approximation of age can be obtained by p l o t t i n g crown-6 rump length on a standard curve. Time of occurence of primary o s s i f i c a t i o n centers can then be related to developmental age. Several l i m i t a t i o n s of t h i s procedure must be considered. Fetuses of a given age could vary considerably in length, and two fetuses of s i m i l a r length may d i f f e r s i g n i f i c a n t l y i n degree of development. Again, the majority of studies have been performed on spontane-ously aborted or s t i l l - b o r n fetuses, development of which may not necessarily be considered normal. I n i t i a l recogni-ti o n of an o s s i f i c a t i o n center varies with the technique used, and the following methods are l i s t e d by Noback (70) in what he considered to be t h e i r order of decreasing s e n s i t i v i t y : sectioning, c l e a r i n g and a l i z a r i n staining, radiography and gross d i s s e c t i o n . With the development of heavy metal staining by Hodges (71) i n the f e t a l pig and O'Rahilly (72) i n the human fetus, s i l v e r radiography i s now considered to be as sensi t i v e as a l i z a r i n s t a i n i n g . Regardless of technique, several p r i n c i p l e s concern-ing o s s i f i c a t i o n and growth have evolved. O s s i f i c a t i o n centers may be regarded as indices of anatomical maturity, and v a r i a b i l i t y i n t h e i r appearance may r e f l e c t the v a r i -a b i l i t y of maturation of the s k e l e t a l system. One part of the body i s a c r i t e r i o n of normalcy for the other parts; whereas one of a pa i r of b i l a t e r a l o s s i f i c a t i o n centers may appear at a d i f f e r e n t time than the other center of the pair , the degree of such asymmetry i s usually s l i g h t before b i r t h . Uses of t h i s p r i n c i p l e as a diagnostic t o o l are many. 7 Radiological assessment of f e t a l maturation i n utero can predict the date of d e l i v e r y more accurately than menstrual h i s t o r y (73, 74). Epiphyseal maturation of c e r t a i n centers at b i r t h i s gaining recognition as a means of estimating the age of the infant at b i r t h (75, 76, 77) and of predict-ing neonatal respiratory d i s t r e s s syndrome (78). Use of r a d i o l o g i c a l techniques to determine bone age in children (79) i s well known. I t would seem of p r a c t i c a l s i g n i f i c a n c e % to have a s i m i l a r standard curve of f e t a l bone ages from eight weeks gestation to term; unfortunately these data cannot be found in the l i t e r a t u r e . Many researchers, have recognized the importance of heredity, race, sex, n u t r i t i o n , endocrine secretion and disease as factors which influence bone growth and the i n i t i a l appearance of o s s i f i c a t i o n centers. The weight of the skeleton i s an important fa c t o r i n the understanding of body composition and of problems i n n u t r i t i o n and disease, as found in the l i v i n g subject (80). Recently, Trotter determined the weight of the dry, f a t - f r e e osseous skeleton of 124 American, white and Negro fetuses of both sexes, ranging i n age from 16-44 weeks (81, 82, 83). A s i g n i f i c a n t c o r r e l a t i o n existed between the weight of the t o t a l osseous skeleton and body weight, as well as lengths of osseous diaphyses of humerus and femur; a l l increased with age. From the regression equations, the weights of the long limb bones were found to r e s u l t i n s l i g h t l y more r e l i a b l e estimates of s k e l e t a l weight than d i d bone lengths. E i t h e r weight o r l e n g t h o f long bones permitted more r e l i a b l e e stimates o f t o t a l s k e l e t a l weight than d i d g e s t a t i o n a l age, b i r t h weight o r length of f e t u s . S i g n i f i c a n t race, but not sex d i f f e r e n c e s were found f o r lengths of long limb bones, w i t h bones o f Negros being l o n g e r than those o f w h i t e s . The r a t i o o f the l e n g t h of femur to humerus and o f t i b i a to r a d i u s showed sex d i f f e r e n c e s , w i t h female r a t i o s h i g h e r than male, but n e i t h e r race nor sex d i f f e r e n c e s were found f o r the weight of s e l e c t e d p a r t s o f the f r e e limbs o r f o r the t o t a l s k e l e t o n . T h i s i s i n c o n t r a s t to Roche's (84) o b s e r v a t i o n t h a t d u r i n g the l a s t three months p r e n a t a l and a t b i r t h o s s i f i c a t i o n i s more advanced i n the female than the male. b) Composition and Development Knowledge of the changes i n composition o f long bones d u r i n g development probably dates from the 1925 study by Hammett (85, 89, 90) on the r a t femur and humerus. The fundamental change i n the composition o f a bone d u r i n g development i s a r e s u l t o f an i n c r e a s e i n the degree of o s s i f i c a t i o n , accompanied by a f a l l i n the percentage o f water. Hammett concluded t h a t the p r o g r e s s i v e d e p o s i t i o n o f bone ash d u r i n g growth i s the cause o f the displacement o f water, and the increment i n o r g a n i c matter p l a y s a r e l a t i v e l y i n s i g n i f i c a n t p a r t i n the dehydration which occurs w i t h age. T h i s g e n e r a l i z a t i o n seems t o h o l d f o r 9 most species, e s p e c i a l l y when the composition of the bones i s expressed on a f a t - f r e e basis* Table I from Dickerson (86) shows the composition of the whole human femur between 12 to 14 weeks gestation and term. The changes are very c l e a r - the f a l l i n percentage of water and the increase i n collagen and bone mineral, as indicated by the calcium and phosphorus content. A r i s e i n the calcium/nitrogen r a t i o indicates the increase i n degree of c a l c i f i c a t i o n of the bone. The cleaning of bone samples fo r analysis takes considerable time and c a r e f u l precautions are necessary i f the percentage of water i s to be accurately determined. I t has therefore been customary to express the composition of bone tissu e on a dry f a t - f r e e b a s i s . When t h i s i s done, the amounts of organic matrix and mineral bear an inverse r e l a t i o n to each other. No detectable f a t has been found i n the femora of fetuses up to 28 weeks gestation and at term i t amounted to 0.14% (86). These changes i n whole bone represent changes in a composite structure, for a long bone consists of bony tissue, marrow and c a r t i l a g e , and a l l of these are changing in composition and r e l a t i v e s i z e . Over t h i s period of development the weight of the epiphyses expressed as a percentage of the weight of the femur was found to f a l l from 73% to 50%, at the same time the percentage of water i n the epiphyses f e l l and the concentration of collagen and calcium Table I. Composition of the whole femur of the human f e t u s 3 F e t a l age (weeks) Constituent 12-14 15-16 20-24 25-28 30-34 Term Weight of femur (gm) 0.11 0.22 1.96 4.7 9.2 16.6 Fat in fresh bone (gm/lOOgm) 0 0 Composition 0 of fresh 0 fa t - f r e e 0.15 bone b 0.14 Water 77.8 78.4 72 .9 68.4 63 .8 63.9 Total N 1.61 1.66 2.01 2.19 2 .35 2 .71 Collagen N 0.61 0.81 1.11 1.36 1.52 1.67 Ca 2 .42 3.47 4.33 5.25 5.63 6.06 P 1.50 1.61 1.97 2 .36 2 .59 2 .84 Ca/N 1.50 2 .09 2.18 2 .40 2 .42 2 .24 a From Dickerson (86) b In g/lOOg increased by a factor of approximately three. The r a t i o of calcium to nitrogen rose and there was also a considerable increase in the r a t i o of calcium to phosphorus. Dickerson (86) suggested that the increase was due to a f a l l in the proportion of phosphate from ester phosphates, a large part of the phosphorus in the bone of the immature fetus being present in this form. Dickerson has also tabulated the main developmental changes in c o r t i c a l bone composition, expressed per lOOg of dry f a t - f r e e s o l i d s (Table I I ) . From 12 to 34 weeks the percentage of t o t a l N f e l l and that of collagen N rose, i f somewhat i r r e g u l a r i l y . This proportion of t o t a l N accounted for by collagen has been shown to increase at cer t a i n stages during development of bone in man, the pig, rat, fowl, but the stage of development at which the r i s e occurs varies from one species to another. Thus, in the human bone the main increase takes place before 22 weeks gestation and in the pig, before 65 days gestation. In the rat and fowl, on the other hand, the same increase occurs during postnatal growth (87) . In the cortex of the human femur, Dickerson (86) observed that collagen accounted for 89-96% of the t o t a l N a f t e r 9 months of age (88). The percentage of calcium in the tissue increased u n t i l the 34th week and so did the Ca/N r a t i o . The v a l i d i t y of the Ca/N r a t i o as a measure of the degree of c a l c i f i c a t i o n 12 Table I I . Composition of the cortex of the femur during the f e t a l l i f e 9 * * Constituent 12 Fet a l -14 Age (Weeks) 20-24 30 -34 Term Total N (gm/100 gm) 5 .95 5 .25 5 .03 5.06 Collagen N (gm/lOOgm) 2 .9 4.05 4 .03 4.20 Ca (gm/lOOgm) 18 .9 23.4 24 .7 24.6 P (gm/lOOgm) 9 .1 10.5 10 .9 10.8 Ratio Ca/N 3 .2 4.45 4 .9 4.9 Ratio Ca/collagen N 6 .5 5.8 6 .1 5.8 Ratio Ca/P 2 .4 2 .2 2 .3 2 .3 a Dry, fa t - f r e e bone k From Dickerson (86) 13 of bone depends upon the cleanliness of the samples of bone analysed (91). Bone begins to be l a i d down i n the c a r t i l a g e model of the human femur at about eight weeks gestation. Before t h i s the Ca/N r a t i o may be considered to be p r a c t i c -a l l y n i l (92). By 12 weeks, the r a t i o had increased to 3.0 and by 22 weeks gestation i t had increased to 4.5 (86). These changes i n the degree of c a l c i f i c a t i o n of the human bone during f e t a l development and also the r e l a t i v e degree of c a l c i f i c a t i o n of bone from f u l l term babies and that from adults agree well with the findings of Wallgren (57), based on biophysical methods. Since the c r y s t a l s of bone mineral are mainly l a i d down in association with the collagen f i b r i l s , the Ca/col-lagen r a t i o gives a measure of the degree of saturation of the collagen f i b r i l s . As seen from Table I I , t h i s r a t i o changed very l i t t l e during growth in humans. This i s i n agreement with the currently accepted view that the collagen f i b r i l s are r a p i d l y mineralized to about 80% saturation soon af t e r they are l a i d down (93). Ca/P r a t i o remained constant with age when expressed per lOOg of dry f a t - f r e e s o l i d s . This confirms the e a r l i e r observation of Swanson and lob (94, 95) who found also that the concentration of magnesium, sodium and chloride i n bone ash decreased with f e t a l development. This would imply a r i s e i n the Ca/Mg and Ca/Na r a t i o s . Various workers (85, 96) have obtained d i f f e r e n t r e s u l t s f o r Ca/Na r a t i o s depending on the species and stage of development, for the following reasons. Sodium i s found in the bone i n e x t r a c e l l u l a r f l u i d s , i n the hydrated layer of bone c r y s t a l s , and i n the bone c r y s t a l s themselves. The sodium of the bone c r y s t a l s , and also the magnesium, are thought to be absorbed on the c r y s t a l surfaces, (97, 98, 99). With development, the percentage of e x t r a c e l l u l a r f l u i d in bone drops, thus the sodium i n t h i s f r a c t i o n also f a l l s . At the same time the bone i s becoming progressively c a l c i f i e d and the sodium associated with the c r y s t a l s increases. F i n a l l y , as bone c r y s t a l s enlarge in size there i s correspondingly less sodium on t h e i r surface. The c i t r a t e of f e t a l bone increases progressively with development according to one author (100) and f a l l s according to another (101). McCance et a l (96) found a large but temporary r i s e in the concentration of c i t r i c acid 4 weeks afte r b i r t h i n the c o r t i c a l bone of pigs. The concentration of f l u o r i n e in human bone has been found to increase during prenatal (102) and postnatal (103) growth., Its rate of deposition i s more rapid i n those areas of bone where the metabolic a c t i v i t y i s g r e a t e s t 0 The value found i n adult bones i s to some extent dependent on the f l u o r i d e content of the drinking water but even where there i s none i n the water there may be an appreciable intake of the element, because tea i s an important source. Strontium in f e t a l bones has been estimated as 0.016% of the bone ash, whereas the mean value for a l l the postnatal samples was 0.022% (104). The membrane bones of the s k u l l develop rather d i f f e r e n t l y from the long bones, as indicated both by micro-radiography (105) and chemical analysis (106). In man, McDonald (106) found a small increase i n the concentration of calcium, a larger increase i n that of carbonate, and no change i n the concentration of phosphorus or collagen per unit weight of dry bone between 28 weeks gestation and term. He suggested that the apparent increase in the proportion of bone mineral present i n the form of carbonate might be part of the 'hardening' of the f e t a l head associated with maturity. B. Maternal N u t r i t i o n and F e t a l Growth and Development 1. Role of N u t r i t i o n The continued normal growth of the fetus throughout pregnancy, assuming genetic p o t e n t i a l and optimum environ-ment, depends upon the integrated development of maternal and f e t a l placental c i r c u l a t i o n , with an adequate concen-t r a t i o n of nutrients i n maternal blood and an adequate area of normal placental membrane for f e t a l t r a n s f e r . Poor f e t a l growth could i n theory r e s u l t from a) conditions a f f e c t i n g the nutrient content of the maternal blood or i t s supply to the placenta; b) poor development of. 16 damage to, or s p e c i f i c abnormalities of the placental membrane affe c t i n g transfer across the placenta, or c) disorders of the f e t a l placental c i r c u l a t i o n . Available evidence suggests that f e t a l n u t r i t i o n may be impaired at any of these s i t e s (10, 107). Evidence for the role of n u t r i t i o n i n human pregnancy i s derived from three general sources: a) records of large population groups with varying socio-economic and health status; b) data from supervised h o s p i t a l and c l i n i c groups; and c) controlled, prospective studies of patients receiving prescribed d i e t s and/or n u t r i t i o n a l supplements, frequently with laboratory observations. These and other pertinent information have been reviewed by Burke (108) and have been considered more recently i n the 1970 N.R.C. maternal n u t r i -t i o n study (109, 100). The rate of growth before b i r t h , l i k e the rate of growth afterwards, depends pr i m a r i l y upon the food supply and upon the a b i l i t y of the fetus to take i n and make use of the food. Widdowson (111) and others (112, 113, 114, 115) have reviewed how the fetus i s fed generally, body composition and placental transfer of n u t r i e n t s . Controversy s t i l l rages over n u t r i t i o n a l needs during pregnancy. For example, i n a recent l e t t e r i n the American Journal of C l i n i c a l N u t r i t i o n , Gam (116) remarks on how l i t t l e i s known about actual calcium requirements i n man, 17 eith e r for bone development, or for s k e l e t a l maintenance. During pregnancy, less than 20g calcium i s incorporated into the f e t a l skeleton, assuming the weight of the skeleton at b i r t h to be lOOg (81). Therefore, calcium retained as new bone approximates 75mg/day during pregnancy and Garn fee l s i t i s doubtful whether absorptive e f f i c i e n c y i s then so diminished as to j u s t i f y an additional allowance of 400mg/day at that time. Armstrong (117) determined blood plasma calcium of women i n t h e i r ninth month of pregnancy. His data suggest that e i t h e r a calcium 'pump' operates in the placenta supplying a higher concentration of calcium to the f e t a l blood supply than i s found in the maternal c i r c u l a t i o n , or that the calcium homeostatic mechanism operates at d i f f e r e n t l e v e l s i n maternal and f e t a l organ-isms. Widdowson and McCance (118) however, suggest the amounts of calcium, phosphorus and magnesium i n the maternal serum are not nearly enough to provide for the developing fetus near term. Cl e a r l y , the precise nature of the maternal f e t a l r e l a t i o n s h i p i s not known.. The stores of the maternal tissue act as buffers which prevent deprivation of the developing fetus as long as possible (119). I t was assumed u n t i l recently that these maternal stores e i t h e r protect the o f f s p r i n g e n t i r e l y , premitting delivery of normal young, or that i n the case of extreme dietary deficiency the fetus dies i n utero. Although there i s some truth in the ' a l l or 18 none' theory i t i s not e n t i r e l y correct since between these two extremes there e x i s t s a narrow range in which maternal n u t r i t i o n a l deficiency may r e s u l t i n arrest of f e t a l devel-opment without causing death (120, 121). In t h i s case, growth of the fetus may be retarded. 2. E f f e c t on N u t r i t i o n on B i r t h Weight I t appears c l e a r from both animal (122-125) and human data (126-129, 120), that starvation can have delete-rious e f f e c t s on f e t a l growth, r e s u l t i n g i n intra-uterine growth retardation, s t i l l b i r t h s and abortion. The magni-tudes of these e f f e c t s are greater i n those species with longer gestation periods and larger term fetuses (130). In instances of mass deprivation, as i n time of war, low b i r t h weight infants were frequently reported. Even exposures to s l i g h t l y reduced dietary intake and q u a l i t y may a f f e c t the fetus (131-141). Dokladah (142) attributed an increase in b i r t h weight i n a Czechoslovakian sample over a period of 50 years to an improvement in d i e t , although other factors may have contributed to t h i s change. Toverud (143) i n an analysis of s t a t i s t i c s gathered i n Norway, found a s i g n i f i c a n t increase i n the weight of infants born between August and October. In discussing the possible cause of t h i s difference i n weight, she mentioned the increase i n sunlight during the summer months, the greater a v a i l a b i l i t y of fresh f r u i t s and vegetables and the longer 19 time the mothers probably spent at rest during the warm months. Toverud was i n c l i n e d to attribute the seasonal differences i n weight i n her series to a combination of these f a c t o r s . Attempts to rel a t e the protein intake of a popula-t i o n of pregnant women to b i r t h weight indicated that the protein requirements are higher during pregnancy, that the requirement i s further elevated during the l a s t trimester and that an intake below 70g protein per day re s u l t s i n a small infant (128). A study of overnutrition (144) revealed that although the b i r t h weights of obese adult women f e l l within the normal range, the b i r t h weights of infants from obese mothers tended to be somewhat elevated. Thomson (145) also reported an unusually accurate c o r r e l a -t i o n between maternal intake of c a l o r i e s and f e t a l s i z e . I f the maternal intake was below 1800 Cal/day, the mean b i r t h weight was 3.09kg and the incidence of 'prematurity' was 8.5%. I f the intake was greater than 3,000 Cal/day, the mean b i r t h weight was 3.3kg and the incidence of 'prematurity' was 1.5%, (the international d e f i n i t i o n of prematurity: ^ 2,500g at b i r t h ) . I f there i s a m u l t i p l i c i t y of causes for low b i r t h weight of infants, i t would be d i f f i c u l t to explain these data unless there also happened to be an inverse r e l a t i o n s h i p between c a l o r i c intake and the incidence of toxemia, mothers who smoke, etc. 20 Other investigators have reported that there was no s i g n i f i c a n t difference i n the d i e t of women who delivered f u l l term babies and those who had premature infants, but a normal rate of intra-uterine growth can be associated with eit h e r full-term or premature infants (146-158). I f the food deprivation i s severe, the incidence of prematur-i t y may r i s e . Experiments with rats demonstrated that i f starvation was i n i t i a t e d at the midpoint of pregnancy, there was a 40% reduction in weight of the o f f s p r i n g (159) . The same experiment repeated with only moderate food deprivation did not reduce f e t a l or placental weight (160). I t has been reported that pregnant rats with only a poor dietary h i s t o r y may d e l i v e r low b i r t h weight fetuses but that food deprivation during pregnancy i s a more s i g n i f i c a n t factor (124). Although f e t a l stunting i s more severe i f food deprivation occurs during the l a t t e r part of pregnancy, f e t a l growth retardation has been reported as early as 90 days when maternal sheep were undernourished during the f i r s t h a l f of pregnancy (161). Other -experimental techniques that may inadvertently i n t e r f e r e with maternal n u t r i t i o n and influence b i r t h weight. Pregnant rats subjected to i r r a d i a t i o n of the head produced stunted young at term (162). Not only were the fetuses reduced i n s i z e but the mother also d i d not gain a normal amount of weight during her pregnancy. Poor intake and poor maternal n u t r i t i o n could have contributed d i r e c t l y to f e t a l loss and f e t a l 21 growth retardation. 3. N u t r i t i o n and Bone Growth a) Animal Studies The normal shape of a bone i s the r e s u l t of a balance between rate of growth in thiakness and rate of growth in length. These two processes may be affected to d i f f e r e n t degrees by changes i n the l e v e l of n u t r i t i o n . Experiments with growing animals appear to support t h i s hypothesis. In rats held at b i r t h weight for two weeks by underfeeding, the skeleton continued to grow very slowly while o s s i f i c a t i o n also proceeded slowly (163). The bones of other young animals reared on a maintenance or subsis-tance d i e t continued to grow but at a much slower rate than normal (164-168). For example, retarding growth of chickens by underfeeding was found to depress increase i n femoral thickness to a greater extent than increase i n length (87). Appleton (169) attributed v a r i a t i o n s in the si z e of young rabbits of the same age to differences i n n u t r i t i o n a l back-ground, concluding that the l e v e l of n u t r i t i o n a f f e c t s both the rate of growth and the rate of bone o s s i f i c a t i o n . The cortex of the long bones of pigs and cockerels whose growth was greatly retarded for long periods of time by underfeeding was very thin and b r i t t l e and the Ca/col-lagen r a t i o was s i g n i f i c a n t l y higher than in well nourished animals of either the same body weight or same chronological age, (170). The structure of the cortex of the bones of these animals was also abnormal and the chemical findings were possibly related to t h i s . The abnormality i n the composition of the cortex induced by underfeeding was com-p l e t e l y masked when the composition of the whole bone was considered, f o r i n both species the Ca/collagen r a t i o was the same as or lower than i n normal bones of the same age. Other animal experiments have been conducted i n which food intake was increased materially during the f i r s t few days of l i f e (171) . Rats i n t h i s group grew much fas t e r throughout t h e i r whole growth period than t h e i r littermates so that they became larger adults and remained large for the rest of t h e i r l i f e . Using a l i z a r i n staining, weight and length measurements and determination of the composition of rat femur, Dickerson (172) studied t h i s e f f e c t of accelerated growth on sk e l e t a l development. He found that f a s t e r growth rate affected maturation to d i f f e r e n t extents although body length was always proportional to body weight. E a r l i e r appearance and fusion of the epiphyses was seen. Long bones were short for body weight and length i n ra p i d l y growing animals, suggesting that the skeleton of a highly nourished animal i s i n a less advanced state of o s s i f i c a t i o n than the skeleton of a poorly nourished animal which has f i n a l l y reached the same size a f t e r a longer period of growth. Thus the morphologically immature femora of the accelerated rats may be due to a high plane of n u t r i t i o n having increased growth in thickness to a greater extent than growth in length. Hammond (173) and his associates have carried out a number of investigations on the e f f e c t of d i f f e r e n t levels of n u t r i t i o n upon the skeleton of the pig and sheep. In one of these, Wallace (174) showed that the s k e l e t a l devel-opment of lambs depends on the l e v e l of n u t r i t i o n of the mother during the l a s t s i x weeks of pregnancy and on the number of lambs carried by the ewe. The skeletons of lambs born of mothers which had been maintained on a high l e v e l of n u t r i t i o n , and more e s p e c i a l l y i f they were singletons, were in a more advanced state of development than those of twins or t r i p l e t s and of lambs born of ewes reared on a low plane of n u t r i t i o n . O s s i f i c a t i o n of bones was also found to be more advanced with the former group. This study suggested that size of skeleton was a better reference for s k e l e t a l development than was body weight. The calcium content of the d i e t is of prime importance to the growth and development of the animal skeleton. The proportion of calcium in the newborn rat seemed to be unaffected by only reducing the calcium intake of the mother during pregnancy (175) . The calcium/phosphate r a t i o of the d i e t appears to be the c r i t i c a l factor in this species. Henry and Kon (176) showed, in rats, that low concentration of phosphorus in the d i e t reduced the retention of calcium. Warkany (177) noted t h a t when r a t s r a i s e d on r a c h i t o g e n i c d i e t s were bred, a l i z a r i n s t a i n i n g of the young i n d i c a t e d t h a t 57 out o f 164 had m u l t i p l e s k e l e t a l d e f o r m i t i e s , compared to no a b n o r m a l i t i e s i n the c o n t r o l group. Many p a i r e d animal experiments have showed s k e l e t a l d i f f e r e n c e s when young were r a i s e d on v a r y i n g l e v e l s o f c a l c i u m . With i n c r e a s i n g increments o f d i e t a r y c a l c i u m , female r a t s had a longer l i f e span and were able t o r e a r more young, s t u r d -i e r o f f s p r i n g . I t was a l s o found t h a t animals which had r e c e i v e d ample food c a l c i u m but were stunted i n growth because o f o t h e r d i e t a r y d e f i c i e n c i e s ( v i t a m i n A, thiamine, p r o t e i n ) had h i g h e r c o n c e n t r a t i o n o f s k e l e t a l c a l c i u m than d i d normal r a t s o f the same age (178). M a r g i n a l p r o t e i n d e f i c i e n c y d u r i n g the r e p r o d u c t i v e c y c l e has r e s u l t e d i n lower bone growth p o t e n t i a l i n the s k u l l s o f newborn r a t s (179) and i n s e v e r l y depressed endochondral bone formation i n monkeys (180). When growing r a t s are fed d i e t s c o n t a i n i n g o n l y t r a c e amounts of magnesium or sodium the c o n c e n t r a t i o n o f t h a t m i n e r a l i n the bones f a l l s (181). Even d e f i c i e n c i e s o f t r a c e elements such as z i n c and manganese has produced s k e l e t a l r e t a r d a t i o n ( 4 ) . From s i m i l a r kinds o f s t u d i e s , t h e o r i e s have been proposed r e g a r d i n g the s p e c i f i c e f f e c t o f d i e t a r y manipula-t i o n o f endochondral o s s i f i c a t i o n (182). D e f i c i e n c i e s o f c a l c i u m , phosphorus and V i t a m i n D w i l l impair e r o s i o n o f hypertrophic c a r t i l a g e c e l l s and thus retard c a l c i f i c a t i o n . Maternal hypervitaminosis D 2 has resulted in smaller d i a -physes of f e t a l bones and has produced a l t e r a t i o n i n o s s i f -i c a t i o n with the appearance of pathological types of c a r t i l -age c e l l s i n the epiphyseal area. Uncontrolled osteoblast a c t i v i t y causes overgrowth of bone in Vitamin A deficiency, whereas in hypervitaminosis A, bone formation ceases and fractures occur from depressed osteoblastic a c i t i v i t y . In r i b o f l a v i n deficiency there i s a gradual cessation of c a l c i f i c a t i o n , the primary spongeosa disappears, the epiphyseal c a r t i l a g e narrows and i s f i n a l l y sealed o f f with bone. Pantothenate and pyridoxime d e f i c i e n c i e s , protein deficiency and i n a n i t i o n show the above e f f e c t s also, presumably due to interference with matrix formation rather than with cessation of c a l c i f i c a t i o n . Ascorbic acid d eficiency i n t e r f e r e s with the a c t i v i t y of osteoblasts, which then revert back to f i b r o b l a s t - l i k e c e l l s ; bone weak-ness and fractures r e s u l t . Vitamin E does not appear to be d i r e c t l y involved i n endochondral o s s i f i c a t i o n . b) Human Studies Although there i s a considerable amount of information available concerning the role of maternal n u t r i t i o n on the development of the fetus in experimental animals, the relevance of these data to the problem of human f e t a l deprivation, p a r t i c u l a r i l y i n the area of s k e l e t a l develop-26 ment, i s hard to assess. Usually the dietary deficiency or d e f i c i e n c i e s u t i l i z e d are gross to ensure major defects; the gestation periods are very d i f f e r e n t from that of humans; most laboratory animals are highly polytocous; and f i n a l l y the intra-uterine development i s very d i f f e r e n t from that i n man, i n that the f e t a l organism i s more highly d i f f e r e n t i a t e d at b i r t h compared to man. A l i m i t e d number of studies of newborn infants, and considerably more studies of growing children, have related n u t r i t i o n to s k e l e t a l development. Figure 2 shows the r e l a t i o n s h i p that Stuart (183) obtained between maternal d i e t and osseous development of the hand, knee and foot, based on X-Rays taken at b i r t h . In t h i s study, "poor d i e t " could be strongly correlated with retardation i n the infant's osseous development. The difference between a "good" or "excellent" maternal d i e t and a " f a i r " d i e t was not as s t r i k i n g , although there were more retarded infants i n the " f a i r " maternal d i e t group than i n the other groups. I t was obvious that few infants were advanced and many were retarded i n the "very poor" d i e t group. Stuart found an even stronger r e l a t i o n s h i p when protein content of the maternal d i e t was correlated with osseous development at b i r t h . In the "excellent" protein d i e t group, 57% of the infants were advanced and 14% were retarded, whereas i n the "poor" protein group, none were 27 65 •• 60 •• 55 J7.<o <D CD (0 U > tea jCM 0) 'O S-l ro a) T3 ro +J 0) OS ro U > <D o c ro > *6 A?. 3 4/. 9 Good or Excellent (29 Cases) F a i r (147 Cases) Poor or Very Poor (21 cases) Mean General Rating of Prenatal Diet Figure 2. Relation of osseous development of l i v i n g , f u l l -term infants at b i r t h to t h e i r mothers' diets during pregnancy (183) 28 advanced and 71% of the newborns were retarded i n osseous development. A somewhat less marked re l a t i o n s h i p was found when maternal dietary calcium was considered: "excellent" calcium d i e t s giving 32% advanced with 23% retarded, and "poor" calcium d i e t s giving 6% advanced and 64% retarded. Stuart (183) found a s i m i l a r r e l a t i o n s h i p between the c a l c i f i c a t i o n of teeth before eruption and q u a l i t y of the d i e t during pregnancy. This reinforced Berk's conclusion (184) that an adequate prenatal d i e t seemed to be an e s s e n t i a l factor i n c a l c i f i c a t i o n of a c h i l d ' s teeth during the f i r s t ten months of l i f e . Massler (185), however, in hi s discussion of prenatal c a l c i f i c a t i o n of teeth commented that almost perfect c a l c i f i c a t i o n of certain tissues before b i r t h was not surprising - the fetus or embryo i s a para-s i t e deriving a l l i t s nutrients from the mother and drawing on her calcium reserves i n the bone where necessary. Thus only severe deficiency i n the mother could a f f e c t tissues c a l c i f i e d before b i r t h . Generally, the "parasite" concept i s not accepted. The c a l c i f i c a t i o n of the t i b i a of newborn infants was found to be unaffected even when the mothers were only 14-17 years o l d and were probably c a l c i f y i n g t h e i r own bones (186). However, the si g n i f i c a n c e of t h i s finding must be questioned as maternal calcium intake was not determined. Individual bones may d i f f e r i n the susepti-29 b i l i t y to a low calcium intake by the mother. Toverud and Toverud (187) reported that the percentage of calcium in the p a r i e t a l bones and r i b s of newborn infants was lower when the d i e t of the mother contained p r a c t i c a l l y no milk and was therefore very low i n calcium. The degree of c a l c i f i c a t i o n of the infant's s k u l l at b i r t h may be influenced by prenatal f a c t o r s . Boder (188) s p e c i f i c a l l y implicated maternal exposure to sun and supplemental administration of dicalcium phosphate together with Vitamin D. A review of l i t e r a t u r e on prenatal r i c k e t s indicated that maternal health, d i e t , frequent pregnancy, and lack of exposure to sunshine may be contributing factors which exert an influence on the development of r i c k e t s in very young infants (189). Toverud (190) studied the etiology of congenital osteoporosis, finding that poor c a l c i f i c a t i o n of f e t a l bone correlated with the negative calcium and phosphorus balance common during the l a s t 2 to 3 months of pregnancy. Cockburn (191) i n exploring some biochemical aspects of intra-uterine growth retardation, reported that plasma calcium was s i n g i f i c a n t l y reduced and inorganic phosphate s i g n i f i c a n t l y increased in umbilical vein plasma of low b i r t h weight i n f a n t s . Sontag (192) attempted to c l a r i f y the r e l a t i o n s h i p between c e r t a i n maternal conditions during pregnancy and the state of well-being of the fetus at b i r t h , as measured and by length, weight and blood calcium Adevelopment of bone. 30 No c o r r e l a t i o n was shown between the following sets of factors: (a) the calcium content of the serum of the mother and the t o t a l f e t a l epiphseal area; (b) the length of infant at b i r t h and the f e t a l epiphyseal area; (c) the adequacy of the maternal d i e t and the f e t a l epiphyseal area; (d) the adequacy of the maternal d i e t and the calcium content of the serum i n the cord; (e) the amount of calcium i n the maternal d i e t and the calcium content of the serum in the cord; (f) the mother's gain i n weight and the weight at b i r t h ; (g) the mother's c a l o r i c intake and the weight at b i r t h ; (h) the mother's protein intake and the f e t a l e p i -physeal area; (j) the amount of f a t in the mother's d i e t and the f e t a l epiphyseal area; (k) the amount of calcium i n the mother's d i e t and the f e t a l epiphyseal area; (1) the amount of phosphorus in the mother's d i e t and the f e t a l epiphyseal area; (m) the menstrual age of the fetus and the t o t a l epiphyseal area; (n) the d i e t of the mother and the home conditions. From what was known about the seeming independence of the growth of new bone (or at le a s t the transformation of c a r t i l a g e into osteod tissue) and r i c k e t s , Sontag f e l t that the intake of calcium, phosphorus, and vitamin D was probably more important i n the determina-ti o n of bone density than in bone growth. Although an annotated bibliography (193) on bone density has recently been published, no l i t e r a t u r e i s available on the density of human f e t a l bones and i t s r e l a t i o n to maternal n u t r i t i o n . 31 Tompkins (194) has related epiphyseal maturation in the newborn to maternal n u t r i t i o n a l status. Hospitalized pregnant women were given varying amounts of n u t r i t i o n a l supplements (protein, vitamins, minerals) during the f i n a l 16 weeks of pregnancy. The area of o s s i f i c a t i o n was measured from radiographs of the newborns' heels and knees. Individual differences were found in the time of formation of the three centers studied. Negros developed e a r l i e r than whites, and the female, regardless of race, was more advanced than the male. Among patients who took supplements there was a s i g n i f i c a n t p r o b a b i l i t y that the t i b i a l epiphyseal center of the knee would be present in female in f a n t s . These supplements did not a l t e r the time of appearance of the epiphyseal center. Interestingly, Tompkins' population was not experiencing any serious n u t r i t i o n a l d e f i c i e n c i e s . The patients who did not receive supplements reported a d a i l y d i e t in the l a s t half of pregnancy which included, on the average, 76g protein and 86o"^:alcium. Other researchers have reported that epiphyseal development during the intra-uterine period was markedly delayed in f e t a l malnutritional syndrome. Femoral and t i b i a l epiphyses were absent in a higher percentage of the undernourished group than the controls, and even when present, the centers in the malmourished infants were smaller (195). Postnatal bone growth of infants with f e t a l growth retardation has also been investigated (196). I n f a n t s w i t h b i r t h weights lower than the tenth p e r c e n t i l e f o r g e s t a t i o n a l age had s h o r t e r f i b u l a s and r e t a r d e d d e v e l -opment o f the epiphyses a t the knee when compared to i n f a n t s with normal weight f o r g e s t a t i o n a l age. The m a j o r i t y o f i n f a n t s s m a l l a t b i r t h grew at a normal rate d u r i n g n e o n a t a l l i f e (197). U n f o r t u n a t e l y , c o n c l u s i o n s cannot be drawn from these experiments unless poor n u t r i t i o n a l s t a t u s o f the mother had been c l e a r l y d i f f e r e n t i a t e d from p l a c e n t a l d y s f u n c t i o n (198). In c h i l d r e n there i s a d e f i n i t e sequence as w e l l as date o f appearance f o r secondary c e n t e r s o f o s s i f i c a t i o n , but t h i s schedule may be i n t e r r u p t e d or r e t a r d e d by meta-b o l i c o r c o n s t i t u t i o n a l d i s t r u b a n c e s . Weight, body m a t u r i t y and even mental development may show i r r e g u l a r i t i e s i n t h e i r progress (199). E p i p h y s e a l r a t i n g i s the e a r l i e s t and f r e q u e n t l y the o n l y i n d i c a t o r o f d i s t u r b a n c e s i n growth and i s more d e l i c a t e than measures of weight or h e i g h t (200). E p i p h y s e a l r a t i n g may be i n f l u e n c e d by the a v a i l a b i l i t y o f m i nerals and V i t a m i n D i n the d i e t and the g e n e r a l l e v e l of n u t r i t i o n (201). S t u d i e s on the development o f e p i p h y s e a l o s s i f i c a t i o n i n c h i l d r e n w i t h kwashiorkor (202) and i n malnourished German c h i l d r e n (203) have shown t h a t n u t r i t i o n may a l t e r the r a t e at which a bone develops, thus masking the u s u a l e f f e c t s o f c h r o n o l o g i c a l age. Although Dickerson and John (204) p o s t u l a t e d a d e f i c i e n c y o f p r o t e i n i n the bone marrow, there were no d i f f e r e n c e s i n the composition 33 of the femur as a whole, or of the epiphyses or the cortex, that could be attributed s p e c i f i c a l l y to kwashiorkor or marasmus. 3^  I N T R O D U C T I O N The n u t r i t i o n a l process can be regarded as a continutn t h a t begins w i t h c o n c e p t i o n and i n which emergence to e x t r a - u t e r i n e l i f e i s merely a t r a n s i t i o n r a t h e r than a b e g i n n i n g . I n t u i t i v e l y , the n u t r i t i o n a l s t a t u s o f the pregnant woman co u l d a f f e c t s k e l e t a l growth and development of the f e t u s . Growth at the end of the f e t a l p e r i o d has been assessed by such c r i t e r i a as b i r t h weight and l e n g t h , f o o t l e n g t h , circumference of the head, che s t , abdomen and t h i g h , and s k i n - f o l d t h i c k n e s s e s (17). Maternal n u t r i t i o n d u r i n g pregnancy i s but one f a c t o r t h a t has been shown to a f f e c t c e r t a i n b i r t h s i z e parameters. F o r example, there i s some evidence t h a t development o f secondary o s s i f i c a t i o n c e n t e r s , as determined a t b i r t h , i s r e l a t e d to maternal d i e t (183, 194, 195) . By c o n t r a s t , the a v a i l a b l e i n f o r m a t i o n r e g a r d i n g growth d u r i n g the p r e - b i r t h p e r i o d has been obtained l a r g e l y from premature b i r t h s or spontaneous a b o r t i o n s . In each case the pregnancy was abnormal because i t f a i l e d to reach term. Dickerson and c o l l e a g u e s (86, 106) have d e s c r i b e d bone composition o f f e t u s e s 12 weeks to b i r t h . T r o t t e r (81-83) has r e c e n t l y s t u d i e d bone leng t h , weight and d e n s i t y o f the human f e t u s . Gruenwald (49-51) has r e l a t e d socio-economic f a c t o r s to f e t a l b i r t h weight, 24 35 weeks to term. Whereas the c o n t r i b u t i o n o f each work i s impr e s s i v e , no c o r r e l a t i o n has been made between these v a r i o u s s t u d i e s . I t i s not known whether the fe t u s e s s t u d i e d by Dickerson, T r o t t e r and Gruenwald were r e p r e s e n t -a t i v e o f normal growth. In a d d i t i o n , no r e s e a r c h r e l a t i n g maternal d i e t to growth o f the human f e t u s , 8-20 weeks o l d , i s a v a i l a b l e . The purpose o f t h i s p r o j e c t i s two - f o l d : (a) to d e f i n e c e r t a i n parameters of s k e l e t a l growth and develop-ment i n the normal human f e t u s , and (b) to c o r r e l a t e c e r t a i n maternal f a c t o r s w i t h these f e t a l parameters. I t i s hoped t h a t the f e t a l model developed w i l l g i v e d i r e c t i o n to f u r t h e r r e s e a r c h i n t h i s a r e a . Normal human f e t u s e s were made a v a i l a b l e f o r t h i s p r o j e c t through the c o o p e r a t i o n o f Dr. B e t t y Poland o f the Department o f O b s t e t r i c s and D i v i s i o n o f Human G e n e t i c s , U.B.C. Length, weight and e x t e r n a l measurements analogous to those taken on newborns were recorded f o r the i n t a c t f e t u s . The humerus and femur were chosen as r e p r e s e n t a t i v e models o f endochondral bone growth and, thus, o f s k e l e t a l growth. Because most of the re s e a r c h has been conducted on the femur, the humerus was i n c l u d e d i n t h i s study t o compare growth r a t e i n the f e t a l arm and l e g . Both bones were weighed, measured, and radiographed to provide p h y s i c a l i n d i c e s o f bone growth. The bones were assayed f o r c e r t a i n m i n e r als and c o l l a g e n to pr o v i d e b i o c h e m i c a l indices of bone development. Three c r i t e r i a of f e t a l growth were therefore available; whole f e t a l measurements, physical data and biochemical data of long bones. Maternal factors other than n u t r i t i o n have been related to f e t a l growth and development (20-49). To provide perspective between n u t r i t i o n a l and non-nutrit-ional factors, selected medical information, growth data, and socio-economic scores were also collected and correlated with f e t a l parameters. 37 M A T E R I A L S A N D M E T H O D S A. Fetal Studies Seventy-six human fetuses of varying ages and sex were collected immediately following therapeutic abortion v i a hysterotomy. Crown-rump length was measured and devel-opmental age calculated from a modification of Streeter's (205) graph (Table XI) . The umbilical cord was cut at the naval and the intact fetus was weighed. Head circumference, sex and limb measurements were recorded. Where possible, length of cord and weight of placenta were noted. The fetus was dissected and eviscerated using standard autopsy procedure at Vancouver General Ho s p i t a l . The right arm and leg were c a r e f u l l y removed at the c l a v i c l e and the pelvic j o i n t s , respectively, for biochemical analysis. The remainder of the fetus was placed in 10% buffered formalin, for l a t e r r a d i o l o g i c a l study. The V. G. H. Pathology Lab examined a l l placentas for lesions. Abnormal fetuses, detected either by autopsy or by placental histology, were excluded from this study. A photographic study of the following f e t a l materials and methods has been included in Appendix 1 (Plates 1-9). Appendix 1 also contains a l l tables, figures and forms rel a t i n g to methods. 38 1. Long Bone Studi e s - P h y s i c a l F l e s h and tendons were c a r e f u l l y c u t from limb bones o f the r i g h t arm and l e g . The elbow and knee j o i n t s were teased a p a r t . Humerus, r a d i u s , u l n a , femur, t i b i a , f i b u l a were washed by water p r e s s u r e , then wiped thoroughly to remove the periosteum. The l e n g t h o f the f r e s h bone i n c l u d i n g c a r t i l a g e was recorded. A M e t i e r A n a l y t i c Balance was used to weight the i n d i v i d u a l bones to 0.01 mg. A l l the bones were d r i e d to c o n s t a n t weight at 105°C. Mean percent dry matter per long bones per f e t u s was c a l -c u l a t e d to provide an index o f s k e l e t a l weight. The water content (% f r e s h bone) o f femur and humerus was found by s u b t r a c t i o n . F a t was e x t r a c t e d from each f e t a l bone using the G o l d f i s c h F a t E x t r a c t i o n Apparatus (206). The bones were then d r i e d to constant f a t - f r e e weight. The d i f f e r e n c e between d r y weight and f a t - f r e e weight was c a l c u l a t e d and expressed as a percentage. 39 2. Long Bone Studi e s - B i o c h e m i c a l S i x t e e n f e t u s e s were p l a c e d i n f o r m a l i n b e f o r e d i s s e c t i o n . Because f o r m a l i n treatment impairs b i o c h e m i c a l a n a l y s i s , bone composition o f these specimens was not c o n s i d e r e d . A t o t a l of s i x t y femora and humeri were analysed i n d i v i d u a l l y . Samples o f bone powders weighing 50mg or whole bone fragments i n the case o f those not l a r g e enough f o r powdering, were heated with 1ml 6N h y d r o c h l o r i c a c i d i n s e a l e d tubes i n a 100°C b l o c k h e a t e r f o r 48 hours. A f t e r a c i d h y d r o l y s i s , the mixture was n e u t r a l i z e d w i t h l m l 6N potassium hydroxide. S u f f i c i e n t HCl was then p i p e t t e d t o d i s s o l v e the c a l c i u m phosphate p r e c i p i t a t e . A f t e r making up to volume wi t h d e i o n i z e d - d i s t i l l e d water, samples o f the s o l u t i o n were taken f o r e s t i m a t i o n o f ca l c i u m and magnesium by atomic a b s o r p t i o n (207), sodium by flame emission (208), and i n o r g a n i c phosphorus by a c o l o r i m e t r i c method (209). The percentage o f hydroxypro-l i n e estimated by Neuman and Logans procedure (210) w i t h the m o d i f i c a t i o n suggested by Leach (211) was converted i n t o percentage o f c o l l a g e n on the assumption t h a t human c o l l a g e n c o n t a i n s 14.1% h y d r o x y p r o l i n e (212). A l l v a l u e s were expressed as g/lOOg d r y f a t - f r e e bone. 3. Long Bone Studi e s - R a d i o l o g i c a l The e v i s c e r a t e d f e t u s w i t h r i g h t arm and l e g removed was f i x e d i n f o r m a l i n f o r 2 to 7 days (Table X I I ) . F o l l o w i n g O ' R a h i l l y ' s method (72) of s i l v e r radiography, the f e t u s was immersed i n a 0.5% aqueous s o l u t i o n o f s i l v e r n i t r a t e f o r a p e r i o d o f 2 to 11 days depending on the l e n g t h o f the specimen (Table XIII) . The f e t u s was then r i n s e d , d r i e d thoroughly and pinned f l a t , d o r s a l s i d e down, us i n g wooden t o o t h p i c k s , on a styrofoam s l a b . A l l 76 specimens underwent t h i s treatment. A lOma p o r t a b l e roentgen u n i t was employed. The f e t u s e s were radiographed on non-screen f i l m at 58kv, 10-40mas (Table XIV) and a t a r g e t - f i l m d i s t a n c e of 24". The X-Rays were processed manually. The radiographs were i l l u m i n a t e d on a screen and, u s i n g a m i l l i m e t e r eyepiece w i t h e i g h t - f o l d m a g n i f i c a t i o n , (Flubacher & Co., Horgen, Switzerland) l e n g t h and width of o s s i f i e d bone s h a f t s were measured ac c o r d i n g t o F i g u r e 6. Jf-1 B o Maternal S t u d i e s l o M e d i c a l H i s t o r y The p a t i e n t ' s h i s t o r y was taken by Dr. Poland e i t h e r preceding the t h e r a p e u t i c a b o r t i o n o r w i t h i n 24 hours t h e r e a f t e r . Information i n c l u d e d age of the mother and f a t h e r , number o f c h i l d r e n ( p a r i t y ) , number o f preg-nancies i n c l u d i n g the present one ( g r a v i d i t y ) , p revious a b o r t i o n s o r s t i l l b i r t h s , e t h n i c o r i g i n , past m e d i c a l , o b s t e t r i c and f a m i l y h i s t o r y , method o f b i r t h c o n t r o l used, and p r e n a t a l f a c t o r s . G e s t a t i o n a l age o r d u r a t i o n o f pregnancy, was c a l c u l a t e d by adding 14 days to the date o f the l a s t menstrual p e r i o d and s u b t r a c t i n g t h i s estima-t i o n o f c o n c e p t i o n from the date o f a b o r t i o n . The reason f o r the present a b o r t i o n and the method o f a b o r t i o n were re c o r d e d . The m a j o r i t y o f i n d i c a t i o n s i n v o l v e d p s y c h i a t r i c reasons but three normal f e t u s e s from spontaneous a b o r t i o n s were i n c l u d e d . 2. D i e t a r y H i s t o r y D i e t a r y i n f o r m a t i o n was obtained from an i n t e r v i e w a t the p a t i e n t ' s bedside three t o f i v e days a f t e r the a b o r t i o n . A standard procedure was followed (Form 1), based on s h o r t form d i e t a r y h i s t o r i e s presented i n the l i t e r a t u r e (213-215) . F i r s t a d a i l y d i e t a r y p a t t e r n was obtained by asking the p a t i e n t what she u s u a l l y ate d u r i n g the course o f a day throughout her pregnancy. A more p r e c i s e i n d i c a t i o n o f maternal d i e t was obtained from a food frequency q u e s t i o n . The type o r form and amount of food consumed was recorded, and the p a t i e n t chose the time p e r i o d ( i . e . day, week, month). The p a t i e n t was then asked s p e c i f i c food l i k e s and d i s l i k e s t o v a l i d a t e the preceding q u e s t i o n s . Any other r e l e v a n t i n f o r m a t i o n from the 15 minute i n t e r v i e w was recorded under g e n e r a l comments. This c o u l d i n v o l v e f a m i l y p r e f e r e n c e s , p r e v i o u s n u t r i t i o n a l s t a t u s , d i f f e r e n c e between pregnant and non-pregnant n u t r i t i o n , any h i s t o r y o f n u t r i t i o n a l a i l m e n t s , f o l l o w i n g o f fad d i e t o r v e r y e r r a t i c e a t i n g h a b i t s , and comments concerning the g e n e r a l a u t h e n t i c i t y o f the h i s t o r y . M a t e r n a l n u t r i t i o n was assessed u s i n g Crump's r a t i n g (45) which was based on previous s t u d i e s (115) and on Recommended D a i l y Allowances (216, 217) . Number o f s e r v i n g s per week f o r each o f 6 food groups was c a l c u l a t e d from i n t e r v i e w data and expressed i n fo u r p o s s i b l e ways; (Form 2) (a) T o t a l N u t r i t i o n Score (0-133); sum o f number of s e r v i n g s per week across a l l food groups. (b) Weighted N u t r i t i o n Score (0-30); each food group was assigned a maximum value o f 5 and response s c a l e d a c c o r d i n g l y (c) N u t r i t i o n Index (0-5); based on Weighted Score d i v i d e d by number of food groups i n v o l v e d (d) P r o t e i n Score (0-40); sum of number o f s e r v i n g s per week i n milk and eggs, meat, f i s h , cheese food groups. Comments concerning the g e n e r a l adequacy of the maternal d i e t i n V i t a m i n D, c a l c i u m , p r o t e i n and i r o n , based on types and amounts o f food eaten, were noted on t h i s form. 3. P e r s o n a l H i s t o r y F o l l o w i n g the d i e t a r y h i s t o r y the p a t i e n t was u s u a l l y r e l a x e d enough to answer more p e r s o n a l q u e s t i o n s . Age, weight, h e i g h t , e t h n i c o r i g i n , o c c u p a t i o n and grade o f s c h o o l completed were recorded f o r both the mother and f a t h e r o f the f e t u s . In a d d i t i o n , b i r t h weight of mother was sought. Information which c o u l d not or would not be g i v e n by the p a t i e n t was obtained from h o s p i t a l records where p o s s i b l e . A s h o r t form socio-economic index was c a l c u l a t e d u s i n g Crump's r a t i n g (23), i n which the o c c u p a t i o n o f f a t h e r , e d u c a t i o n o f mother and f a t h e r and m a r i t a l s t a t u s o f mother were c o n s i d e r e d . Information was coded as t o t a l s c o r e (0-72) and as Socio-economic Group (1-4) a c c o r d i n g to the o u t l i n e (Form 3 ) . 44 C. S t a t i s t i c a l A n a l y s i s Whole f e t a l measurements, o s s i f i c a t i o n and b i o -chemical data of femora and humeri, n u t r i t i o n a l , m e d i c a l , growth and socio-economic i n f o r m a t i o n from the mother were coded f o r a n a l y s i s on U.B.C.'s IBM 360/67 computer. Means, standard d e v i a t i o n s , degrees o f freedom and simple c o r -r e l a t i o n m a t r i c e s were generated. Simple and m u l t i p l e r e g r e s s i o n a n a l y s i s was performed on the whole range of data f o r l i n e s o f l e a s t squares, c o e f f i c i e n t s of d e t e r -mination and F p r o b a b i l i t i e s . T h i s procedure was under-taken to d i s c o v e r which v a r i a b l e s b e s t p r e d i c t e d o t h e r v a r i a b l e s . The C o e f f i c i e n t o f C o r r e l a t i o n (r) squared i s the C o e f f i c i e n t of Determination (R^). Because R^ g i v e s the p r o p o r t i o n o r percentage o f the v a r i a n c e shared by the two v a r i a b l e s , t h i s value was co n s i d e r e d a more u s e f u l means o f e x p r e s s i o n . The c l o s e r v? i s to 1.0, the b e t t e r the f i t i s on the r e g r e s s i o n l i n e . Each v a r i a b l e was then c l a s s i f i e d a c c o r d i n g to age group o f f e t u s (9-10, 11-12, 13-14, 15-16, over 16 weeks developmental age) . Two week age groups were chosen to allow comparison w i t h Dickerson's data (86). A n a l y s i s o f v a r i a n c e was executed along w i t h Duncan's New M u l t i p l e Range T e s t a t the 5% l e v e l to t e s t the s i g n i f i c a n c e o f each group mean. 45 R E S U L T S A. F e t a l Data F e t a l data were grouped according to developmental age into f i v e age periods of two weeks each. The number of specimens in each group ( t o t a l 76 specimens) i s presented in Figure 3. Experimental error, and the fact that only 60 femora and humeri were analysed, reduces the t o t a l sample s i z e . The smallest variable size i s 57. Results of analysis of variance are expressed in Tables III and IV. Total number of observations, means of each age group, and units of expression are given. F p r o b a b i l i t y indicates the l i k l i h o o d of obtaining an age group e f f e c t for that variable by chance alone. I f a s i g n i f i c a n t F was found at the 5% l e v e l , Duncan's New Multiple Range Test (218) was executed to determine which means were s i g n i f i c a n t l y d i f f e r e n t from each other. Duncan's Test adjusts the "least s i g n i f i c a n t difference" t - t e s t so that the number of means in comparison are included in the c a l c u l a t i o n . Means sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t from each other; means assigned a d i f f e r e n t l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l . 46 Figure 3 . Histogram of f e t a l age groups 26 -• 24 • — 22 ro -P o E-i c t r •P ro rH 20 • 18 -16 • 14 2 12 fa 10 •• 8 6 -4 .. 2 •• 25 15 18 11 9-/o /3-/v is-if, n-aa Developmental age (weeks) Table I I I . Fetal and Long Bone Growth related to Developmental Age Variables n Unit 9 -10 11 Age -12 of Fetus 13-14 (weeks) 15-16 16 F Prob. Crown-rump length 76 mm 61 .00a 87 • 04b 115 ,44c 139.91d 171 .71e 0.0 Developmental age 76 days 69 .87a 83 .76b 97 .94c 110.91d 131 • 71e 0.0 Gestational age 74 days 69 .60a 86 .50b 102 .06c 109.09c 132 .86d 0.0 Fe t a l weight 67 g 14 .20a 41 .03b 94 ,55c 169.77d 340 .96e 0.0 Skeletal index 58 % 16 .58a 20 .65b 25 .11c :2 9.35d 31 .13d 0.0 F-dry weight 60 mg 3 ,40a 17 .2 0a 75 .09b 181.21c 384 .06d 0.0 H-dry weight 60 mg 4 .20a 18 .18b 64 .20c 12 7.34d 2 52 ,19e 0.0 F-water content 58 % 86 .16a 83 .61b 78 .78c 75.73d 73 .49d 0.0 H-water content 58 % 83 .57a 79 .83b 75 .62c 71.80d 69 .88d 0.0 F-fresh length 57 mm 13 .32a 21 .93b 31 .29c 39.43d 48 .50e 0.0 H-fresh length 57 mm 13 ,11a 20 .88b 28 .92c 3.621d 43 .50e 0.0 F - o s s i f i c a t i o n 76 mm 5 .51a 11 .52b 18 .99c 25.39d 32 .50e 0.0 H-o s s i f i c a t i o n 76 mm 6 .23a 12 .06b 19 .12c 2 5 .2 8d 32 .01e 0.0 F = femur, H = humerus 0.0 indicates F probability < 10~ 8 I f F p r o b a b i l i t y ^0.05 Duncan's New Multiple Range Test was performed at the 5% l e v e l . Means sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t from each other; means assigned a d i f f e r e n t l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l . Table IV. Composition of Fetal Long Bones According to Developmental Age Age of Fetus (weeks) F Variable Unit 9-10 11--12 13-14 15-16 16 prob. F-collagen 18.23 21 .18 20.86 21.32 21 .36 0.1285 H-collagen 16.72a 20 .95b 21.55b 21.60b 20 ,86b 0.0013 F-calcium c 0 10.26a 13 . l i b 16.60c 17.62cd 19 .90d 0.0 H-calcium -Q 9.72a 14 .80b 17.57c 17.84c 19 .81c 0.0 F-phosphorus 6.09a 6 . 51a 7.77b 8.80b 8 .87b 0.0000 H-phosphorus o • 5.75a 7 .23b 7.88bc 8.18bc 8 .80c 0.0000 F-magnesium 0.57 0 .52 0.52 0.47 0 .51 0.2584 H-magnesium 0 o 5 8 0 .55 0.55 0.48 0 .50 0.3811 F- sodium <• 4.72a 1 .35b 0.98b 1.06b 0 .98b 0.0000 H-sodium "0 4.12a 1 ,06b 0.91b 1.09b 0 .86b 0.0 F-calcium/collagen ra t i o 0.56a 0 .64a 0.79b 0.83b 0 .93b 0.0000 H-calcium/collagen ra t i o 0.57a 0 .72b 0.82bc 0.83bc 0 .95c 0.0000 F-Oa/P ra t i o 1.68a 2 .02b 2 .15b 2 .18b 2 .25b 0.0000 H-Ca/P ra t i o 1.68a 2 .05b 2 .22c 2.18bc 2 .25c 0.0 n = 59 for each variable F = femur, H = humerus 0.0 s i g n i f i e s F prob. 10~ 8 0.0000 s i g n i f i e s 10 _ 8<: F prob.<10~ 5 I f F probability 4:0.05 Duncan's New Multiple Range Test was performed at the 5% l e v e l . Means sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t from each other; means assigned a d i f f e r e n t l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l . 49 1. Whole fetus Means, standard deviations and sample sizes of the whole range of variables computed are presented in Table V. Sixty-eight percent (mean t 1 standard deviation) of the specimens col l e c t e d were 70-138mm in length from crown to rump and therefore had a developmental age of 74-112 days or about 11-16 weeks. Table V. Means and standard deviations of whole f e t a l variables Unit Sample Size Mean Standard Deviation C. R. Length mm 76 104.1 34.15 Developmental Age days 76 92 .72 18.55 Gestational Age days 74 94.39 22 .57 Fetal Weight g 67 89.40 91.32 Head Circumference mm 67 105.4 36.32 Foot Length mm 76 18.34 8.057 Skeletal Index % 58 22 .29 5 .423 Sex 1=S 76 1.553 0.5005 2=* From Table I I I , as developmental age of the fetuses increased, CR length, gestational age, weight, head circum-ference, foot length and s k e l e t a l index increased proport-ionately. Developmental age was best predicted by f e t a l length, as would be expected from the method of determination 2 (R = 0.995). Because of extrapolation from the graph v i a a table, this value was not 1.00. Head circumference (R 2= 0.96), foot length (R 2 = 0.95), weight (R 2 = 0.85), and s k e l e t a l index (R 2 = 0.77) a l l predicted developmental age with an F p r o b a b i l i t y of <10~ a. As seen in Table I I I , each of the above variables separated cleanly into the 5 age periods, except s k e l e t a l index which tapered o f f a f t e r 16 weeks. Whereas gestational age was s t i l l a s i g n i f i c a n t predictor of developmental age, scatter reduced the Co e f f i c i e n t of Determination to 0.67, and only 4 d i s t i n c t groups were found in Table I I I . Scattergrams of these variables are presented in Appendix 2 (Exhibits 1-6)„ The complete regression data have been deposited with the School of Home Economics and are available upon request. Gestational age i t s e l f i s predicted by foot length (R 2 = 0.70), C.R. length of fetus (R 2 = 0.67), head circum-ference (R 2 = 0.67), weight (R 2 = 0.58) and sk e l e t a l index (R 2 = 0.49). A l l the above relationships were s i g n i f i c a n t at p =<10~4 but greater scatter was evident compared to si m i l a r regression against developmental age (Exhibits 7-11) . More male specimens than female specimens were co l l e c t e d ( r a t i o 42:34). Because sample size was small and sex was unevenly d i s t r i b u t e d throughout the age range, performing separate regression analysis on each group would not have been meaningful. 51 2. Long Bones Means, standard deviations and sample sizes for the variables computed are presented in Table VI. Generally, data from the femora and humeri were comparable, with the suggestion that the humerus i s s l i g h t l y more developed fo r i t s dry weight than i s the femur. Table VI. Means and standard deviations of long bone variables femur humerus Stand. Stand. Variables Unit n Mean Deviat. Mean Deviat. Dry weight mg 60 69.15 104.4 52 .53 68.03 Water content % 58 81.66 4.55 78.28 4 .99 Fresh length mm 57 25.80 10.97 24.13 9.54 O s s i f i c a t i o n mm 76 16.04 8.65 16.33 8.24 Collagen CP O CD 59 20.44 3 .60 20.14 3 .68 calcium 59 14.14 3 .68 14.86 4 .03 phosphorus o >, c r-t U 0 59 7.03 1.29 7.23 1.45 magnesium 59 0.52 0.10 0.55 0.11 sodium 59 2 .02 1.90 1.75 1.67 Ca/collagen r a t i o 59 0.70 0.16 0.73 0.16 Ca/P r a t i o 59 2.00 0.27 2 .03 0.26 i The constant dry weight of both femora and humeri increased with developmental age (Exhibits 12-13). The non-linear rel a t i o n s h i p may explain why the femoral data separated into only 4 s i g n i f i c a n t age groups, suggesting a lag period followed by a proportionately larger deposition of mineral and organic matter aft e r 12 weeks. However each of the 5 group means for humeral dry weight were s i g n i f i c a n t . The weight of the intact fetus was the best predictor of the dry weight of both bones (femur = 0.98, humerus R^  = 0.99). Dry weight of the corresponding bone, devel-opmental age of fetus, o s s i f i c a t i o n of the bone, f e t a l length and length of the fresh bone were also good predictors of a bone's dry weight (R^ =0.80). Whereas gestational age predicted bone weight with p<10 -^, scatter around the regression li n e was greatly increased (R^ = 0.54). Bio-chemical variables were even poorer predictors of the dry weight of both femura and humeri. An inverse r e l a t i o n s h i p was found between water content of femora and humeri, developmental age and a l l other variables computed (Exhibits 14-15) . Four s i g n i f -icant age groups were found suggesting that as the fetus ages, organic and mineral material replaces the water in f e t a l bones, reaching a plateau at 15 weeks. The water content of the corresponding bone, length and o s s i f i c a t i o n of bone, f e t a l length and developmental age best predicted the percentage of water in both bones (R^ =0.90) . The calcium content of the bone resulted in less scatter when plotted against water content than did gestational age against water content. The lengths of the fresh long bones increased proportionately with developmental age and could be separated into 5 d i s t i n c t age groups. The lengths of the femora and humeri were best predicted by the o s s i f i c a t i o n of the bone (R 2 = 0.99) and also by the wet length of the cor-responding bone. F e t a l length, developmental age, weight of fetus, dry weight and water content of the bone were also good predictors, in that order. Again, calcium content of the femora and humeri was the only biochemical variable with l i t t l e scatter and was more useful in predicting bone length than was gestational age. As developmental age increased so did length of o s s i f i c a t i o n in both bones; this e f f e c t was s i g n i f i c a n t in each of the 5 age groups (Exhibits 18-19). Indeed, th i s variable was the best long bone predictor of f e t a l age (R 2 = 0.96). Length of o s s i f i c a t i o n of the humerus best predicted o s s i f i c a t i o n length of the femur and vice versa. Fresh bone lengths predicted o s s i f i c a t i o n nearly as well (R 2 = 0.99), followed by C.R. length, developmental age, weight of fetus, dry weight and water content of the bone. Gestational age and calcium content showed a good c o r r e l a -tion with bone o s s i f i c a t i o n (R 2 = 0.68). A non-linear e f f e c t was seen when collagen content of femora and humeri was plotted against developmental age (Exhibits 20-21) . As re s u l t when f e t a l age was grouped into two-week periods l i t t l e s i g n i f i c a n t difference was found. Group means for femoral collagen ranged between 18.23 - 21.36 g/lOOg dry bone, with a 12.85% p r o b a b i l i t y of 5h a s i g n i f i c a n t difference between age groups. However, c o l l a -gen in the humeri of fetuses 9-10 weeks old was s i g n i f i c a n t l y less than that found in fetuses 11-20 weeks o l d . This suggests that the t o t a l amount of collagen increases more rapidly with age in younger fetuses and deposition slows afte r ten weeks. There were no highly s i g n i f i c a n t predict-ors of collagen in f e t a l bone. Femoral collagen was best predicted by humeral collagen (R 2 = 0.50) whereas humeral phosphate best predicted humeral collagen (R 2 = 0.56). There was a l i n e a r r e l a t i o n s h i p (Exhibits 22-23) between developmental age of fetus and femoral calcium 2 2 (R = 0.65) and humeral calcium (R = 0.55). When analysed according to f e t a l age group the amount of calcium in the humerus was s i g n i f i c a n t l y d i f f e r e n t in 9-10 week, 11-12 week and 13-20 week fetuses. Femoral calcium showed a s i g n i f i c a n t group e f f e c t into 4 ages, with some overlap. Group means suggest a greater increase in calcium depos-i t i o n in the humerus during the 11-12 week period than in the femur, followed by a plateau from 13-20 weeks. Depos-i t i o n appears more gradual in the femur; approximately the same f i n a l value per lOOg dry bone was seen in both femora and humeri. Individual v a r i a t i o n and experimental error cannot be ruled out in differences of this magnitude. The phosphorus content of the bone best predicted i t s calcium content (femur R2 = 0.76, humerus R2 = 0.85). In each bone, calcium was the biochemical variable that best predicted physical variables in that bone. 55 A non-linear e f f e c t was suggested by the scatter-gram of phosphorus content plotted against developmental age (Exhibits 24-25) . There was a s i g n i f i c a n t grouping e f f e c t into 2 f e t a l age periods for femoral phosphorus and into 3 for the humeral values, although some overlap was seen. Humeral P was s i g n i f i c a n t l y lower than femoral P in the 9-10 week age group but was s i g n i f i c a n t l y higher at 11-12 weeks and about the same per lOOg dry bone in the remaining age groups. Correlation between femoral and humeral P values produced R2 = 0.69. Humeral P had only one good predictor; humeral calcium with R2 = 0.92. Many other variables showed p <10"^ indicating a non-zero slope, but much scatter was evident. S i m i l a r i l y , scatter was high with femoral phosphate and i t s best predictor was femoral calcium (R 2 = 0.87). With advancing age of the fetuses, magnesium content of both bones decreased (Exhibits 26-27) . The relat i o n s h i p was so s l i g h t that the means were not s i g n i f i c a n t l y d i f f e r -ent when grouped into 5 f e t a l age periods. The only pre-d i c t o r of the magnesium content of femora and humeri was the sodium content of the same bone (R^ = 0.30). This was also the only variable s i g n i f i c a n t at p-^lO""^. Correlation between femoral and humeral magnesium was 0.51, suggesting e i t h e r great variations between the two bones or poor method s e n s i t i v i t y . An inverse, non-linear r e l a t i o n s h i p was seen when sodium content of f e t a l bones was plotted against developmental age (Exhibits 28-29). As re s u l t , in both humeri and femora the sodium content was s i g n i f i c a n t l y higher in the bones of 9-10 week old fetuses than the remaining period of 11-20 weeks. Group means were s i m i l a r but there was less sodium per lOOg dry humerus than per lOOg dry femur. The best predictor of the sodium content of one bone of a fetus was the sodium content of the cor-responding bone of the same fetus (R 2 = 0.96) but 7-9 variables showed p <10~^ indicating that the slope of the regression line was not 0. The Ca/collagen increased in a non-linear fashion when plotted against developmental age (Exhibits 30-31). The Ca/collagen r a t i o in the femur of 9-12 week old fetuses was s i g n i f i c a n t l y lower than that in the 13-20 week period. In the humerus, some overlapping into 3 s i g n i f i c a n t age periods was seen. The r a t i o had the same s t a r t and end value in both bones; humeral Ca/collagen bowed more in the middle range. The best predictor was the Ca/collagen r a t i o of the corresponding bone at R2 = 0.80, but 6-8 variables clustered below t h i s with p <10"^. These included length, dry weight, and water content of the bone, length of fetus, weight, developmental and gestational age. A non-linear r e l a t i o n s h i p was seen when the calcium/phosphate r a t i o of both bones was plotted against developmental age of fetus (Exhibits (32-33) . In fetuses 9-10 weeks old, t h i s r a t i o in the femur was s i g n i f i c a n t l y less than 2.0. In the remaining 13-20 weeks, the r a t i o was greater than 2.0 in t h i s bone. The Ca/P r a t i o in the humerus was s i m i l a r but separated into 3 s i g n i f i c a n t age groups with some overlap. The best predictor of t h i s r a t i o was the Ca/P r a t i o in the corresponding bone, although i t was seen from the regression data that a l l other variables except one were s i g n i f i c a n t at p<10~^. The percent change in weight of each long bone following f at extraction was calculated. I f the results expressed in Table VII can be explained by experimental error, no f a t was found in the bones of fetuses aged 9-20 weeks. Table VII. Change in bone weight following fat extraction Long Bone (%) Mean Standard Deviation femur -1.5256 2 .62 t i b i a -0.5943 1.98 f i b u l a 0.4847 3 .69 humerus -0.6093 3 .29 radius -0.4572 3 .66 ulna -0.0900 2 .89 58 B. Maternal Data Selected variables were coded and compared against each other for simple l i n e a r regression data. These potential independent variables were then correlated with the f e t a l data as dependent variables using stepwise regression a n a l y s i s . Independent variables which correlated s i g n i f i c a n t l y with each other (r = 0.3) were isolated from each other in successive runs. 1. Medical-Growth Information Means, standard deviations and number of observa-tions for the s i x variables are shown in Table VIII. Table V I I I . Means and standard deviation of maternal variables Variable Sample Size Unit Mean Standard Deviation Age 76 years 29 8 Height 67 cm 164 6 Weight 66 kg 57.6 8.2 B i r t h Weight 45 kg 2.9 0.8 P a r i t y 75 no. c h i l d . 1.8 2.0 Gravidity 75 no. pregn. 3.3 2 .3 A s i g n i f i c a n t relationship was seen between maternal weight and height (R2= 0.24, p = 0.0001), whereas no rela t i o n s h i p was detected between maternal b i r t h weight and present weight ( p = 0.6253) . Parity and g r a v i d i t y 59 correlated s i g n i f i c a n t l y with maternal age; the older woman had a greater chance of having more children and more preg-nancies than a younger woman. A highly s i g n i f i c a n t r e l a t i o n -ship was found between p a r i t y and gr a v i d i t y for obvious reasons. Scattergrams for the above are presented in Appendix 2 (Exhibits 34-37) . No other relationships between a l l combinations of the above variables were detected. When these potential independent variables were analysed in stepwise regression, certain s i g n i f i c a n t cor-relations were seen. Results are presented in Table IX. F i r s t developmental age was held constant; then gestational age was chosen as the s i g n i f i c a n t independent v a r i a b l e . The data suggest that younger women produced fetuses with longer, more o s s i f i e d bones at each age of development, with a larger head circumference and a higher Ca/P r a t i o in the humeri. None of the above relationships were seen when gestational age was held constant. Maternal weight appeared to be inversely correlated with biochemical indices of the f e t a l humerus. This suggests that l i g h t e r women produced fetuses with more phosphorus, magnesium, calcium and a higher Ca/collagen r a t i o in the humeri than fetuses of the same developmental age from heavier women. When gestational age was held constant the same relat i o n s h i p held only for humerus phosphorus and magnesium. Interpretation of t h i s finding i s d i f f i c u l t ; 60 Table IX. E f f e c t of maternal variables on f e t a l data Developmental Age Gestational Age constant constant Independ . Depend. F Depend. F Var. Var. prob . Rel. Var. prob. Rel. Age F-Len 0.0000 H-Len 0.0000 -HeadC 0.0152 -H-Ca/P 0.0177 -F-oss 0.02 76 -H-Oss 0.0415 -We ight H-Pho 0.0007 — H-Pho 0.0013 — H-Mag 0.0079 - H-Mag 0.0116 -H-Cal 0.12 9 -H-Ca/C 0.0311 -Height H-Pho 0.0381 -B i r t h F-Len 0.0000 _ H-Col 0.0085 + Weight H-Len 0.0000 - HeadC 0.0134 + H-Ca/C 0.0054 - DeAge 0.0210 + FootL 0.0071 -F-Oss 0.0142 -GeAge 0.0159 -H-Dry 0.0244 -H-Col 0.0238 + Parity F-Len 0.0000 — H-Ca/P 0.0058 — H-Len 0.0000 — H-Ca/P 0.0009 -FootL 0.0098 -Gravidity F-Len 0.0000 _ H-Ca/P 0.0135 — H-Len 0.0000 -H-Ca/P 0.0000 -HeadC 0.0072 -FootL 0.0338 -F-Oss 0.0401 -We ig ht 0.0445 -Socio-econ We igh t 0.0116 + Score Socio-econ Weight 0.0356 Group Weight 0.0066 + + F e t a l Sex HeadC 0.0020 _ H-Pho 0.0207 + We ight 0.0422 + F = femur H = humerus femora and humeri data were strongly correlated with each other yet maternal weight only affected humeral varia b l e s . Also, maternal height, which correlated with maternal weight, showed no s i g n i f i c a n t c o r r e l a t i o n w i t h any of the f e t a l v a r i a b l e s . Maternal b i r t h weight was inversely correlated with certain physical f e t a l data. For example, when developmental age was held constant, mothers who weighed more at b i r t h appeared to produce fetuses with shorter bones, shorter feet, less o s s i f i e d femora and l i g h t e r humeri. However there appeared to be a d i r e c t c o r r e l a t i o n between maternal b i r t h weight and humeri collagen. Certain d i r e c t relationships were discovered when gestational age was held constant. There is the suggestion that mothers with fewer children produced fetuses which had longer feet and longer bones than women with a larger family, i f developmental age was held constant. There appeared to be l i t t l e r e lationship between parity and these dependent variables when expressed as a function of gestational age. Similar responses were found when number of preg-nancies was considered. In addition, when developmental age was kept constant, as g r a v i d i t y increased, f e t a l head circumference, weight and femoral o s s i f i c a t i o n decreased. As with parity, no conclusive results were seen when 62 g r a v i d i t y was expressed as a function of gestational age. 2. Socio-economic Status Mean socio-economic score out of a possible 72 was 33.4 t 12.9; mean group was 2.7 t 0.74. There were 52 observations in each case as the remaining 24 women were unwilling to supply the information. Relative frequencies of the socio-economic groups i s shown in Figure 4. The scattergram of socio-economic score versus socio-economic group (Exhibit 38) i s presented in Appendix 2. As seen in Table IX, socio-economic data were p o s i t i v e l y correlated with f e t a l weight, whether expressed as a score or group, as a function of developmental or gestational age. 3. Sex of Fetus Results of stepwise regression analysis suggest that females of the same developmental age had a greater head circumference than males, whereas when gestational age was considered, females had lower humeral phosphate values and weighed less than males. 4. N u t r i t i o n a l Data Relative frequencies, means and standard deviations for each n u t r i t i o n variable are presented in Figure 5. Sample size was 70 since 6 women were discharged from the 63 Figure 4 . Histogram of Socio-economic Groups CN in ro o U c 3 tr CD U fa > -H -P ro r-i CD a; 2 6 2 4 2 2 2 0 1 8 1 6 1 4 1 2 1 0 8 6 4 2 low. .high 2 4 2 0 Mean S.D. 2 o 7 0 „ 7 I II III 3V Socio-economic group o-/v IS-J9 so-74. Socio-economic score Socio-economic Status Figure 5a. Histograms of general maternal n u t r i t i o n score 14 12 10 • 8 -6 4 2 + low. ^. high 16 13 d 3 3 1 13 r . f . 40 Nean S.D. 36 • Wean S.D. 90 no 34 Ai 4 32 28 24 20 16 12 8 4 low high 22 11 r . f . 40 36 32 28 24 20 16' 12 • 8 • 4 low v high Neon S.O. 3.5 22 _3 21 15 3a- *»- S0-i0-7fl- SCr fO- ItXt 136; 39 49 S9 it 79 i i 19 ICI in 1*9 133 TOTAL NUTRITION SCORE jo- is- Jt>- *s-/* 19 *H A9 WEIGHTED NUTRITION SCORE A&-*J-3-0-3£-to- MS-J.1 Jt.1 At 3.9 *V V.9 NUTRITION INDEX 65 Figure 5b. Histogram of maternal protein score low. high > •P re CO 22 20 18 16 --o c CD 3 C 12 14 -10 • 8 •• 6 --21 Me an S .D. 28 12 17 16 1 2 . 4 2 0-9 /0-/9 Z0T*9 30-39 TOTAL PROTEIN SCORE 66 h o s p i t a l before the dietary hi s t o r y could be taken. A l l scores were arranged on a continuum from low to high and grouped a r b i t r a r i l y . A normal d i s t r i b u t i o n was obtained for t o t a l n u t r i t i o n score, weighted score and n u t r i t i o n index; probably only 4 women could be described as having an inadequate d i e t according to the c r i t e r i a used. Protein scores clumped at the upper range of the d i s t r i b u t i o n ; again only a small number could be c l a s s i f i e d as having a low animal protein intake. Total n u t r i t i o n , weighted score and index were manipulations of the same data and as such correlated well with each other (R 2 = 0.85) . A d i r e c t relationship was also seen between protein score and t o t a l n u t r i t i o n (R 2 = 0.44), weighted score (R 2 = 0.32) and index (R 2 = 0.33). In a l l of the above correlations, F p r o b a b i l i t y was s i g n i f -icant at p <10~4. Scattergrams are included in the Appendix (Exhibits 39-44). No further relationships were detected between n u t r i t i o n a l , maternal or socio-economic data at the 5% l e v e l of s i g n i f i c a n c e . Table X presents the s i g n i f i c a n t relationships that resulted when n u t r i t i o n a l factors were tested as pote n t i a l independent variables in multiple regression analysis. Protein score of the maternal d i e t did not a f f e c t any f e t a l variables, e i t h e r when developmental or gesta-67 Table X. E f f e c t of n u t r i t i o n a l variables of f e t a l data Developmental Age Gestational Age constant constant Independent Depend F Depend F Variable Var. prob. Rel. Var. prob. Rel. Total F-Len 0.0000 + H-Dry 0.0165 + n u t r i t i o n H-Len 0.0000 + F-Dry 0.0199 + H-Dry 0.0001 + F-Pho 0.0317 + F-Dry 0.0015 + F-Len 0.0404 + F-Pho 0.0365 + H-Len 0.0435 + Weighted F-Len 0.0000 + H-Dry 0.0010 + score H-Len 0.0000 + F-Dry: 0.0012 + F-Dry 0.0000 + F-Len, 0.0087 + H-Dry 0.0000 + H-Len 0.0117 + F-Pho 0.0496 + F-Pho 0.0262 + Nu t r i t i o n F-Len 0.0000 + H-Dry 0.0010 + index H-Len 0.0000 + F-Dry 0.0012 + F-Dry 0.0000 + F-Len 0.0079 + H-Dry 0.0000 + H-Len 0.0105 + F-Pho 0.0284 + Protein no s i g n i f i c a n t r e l . no s i g n i f i c a n t r e l score F = femur, H = humerus 68 t i o n a l ages were held constant. Total n u t r i t i o n , weighted score and index were comparable in e f f e c t , with t o t a l n u t r i t i o n score predicting the greatest number of variables with the lowest p r o b a b i l i t y . Results suggest that general n u t r i t i o n of the mother was d i r e c t l y related to the length and dry weight of both long bones studied. Although this r elationship held when gestational age was considered, p r o b a b i l i t y of chance rela t i o n s h i p was greater and more scatter was seen. 69 D I S C U S S I O N I t can be shown that there are drawbacks to basing the age of the fetus on i t s crown-rump length. Aside from experimental error in measurement fetuses of the same length are not necessarily the same age from conception, and vice versa. Just as the estimated gestational age of a newborn is an important c l i n i c a l datum which must not be disregarded whatever the infant's b i r t h weight or length, estimation of f e t a l age from maternal dates must be con-sidered. Large scatter in regression data from gestational age against a l l other variables can be explained in three ways: (a) fetuses the same age in utero grow at highly variable rates; (b) for p s y c h i a t r i c reasons, i r r e g u l a r menstruation or poor memory, the mother was unable to give an accurate date of her l a s t menstrual period; (c) i f conception did not occur 14 days a f t e r the s t a r t of her l a s t menstrual period, estimated gestational age would be inaccurate. Any or a l l of these reasons could contribute to errors in the gestational age assigned to each specimen. Batt a g l i a (219) has suggested that r e l i a b l e menstrual h i s t o r i e s be selected. Since such selection could eliminate 10-40% of the sample, does th i s remainder constitute a normal reference group (220)? A number of researchers 70 ignore t h i s s i t u a t i o n by proposing a va r i e t y of other specimens as models for the study of f e t a l biology, e.g. rhesus monkey. Because normal fetuses of exactly known gestational age are r a r e l y available for analysis, human studies l i k e the present one must be content with expressing results according to developmental age and thus f e t a l length. With increasing developmental age of the fetuses studied, the length, dry weight and extent of o s s i f i c a t i o n increased in both the humerus and femur. These factors showed a strong positive c o r r e l a t i o n with each other. The weights, rather than the lengths of the limb bones were found to r e s u l t in a more r e l i a b l e estimate of f e t a l weight. Trotter, i n his research on older fetuses (81), described a s i g n i f i c a n t c o r r e l a t i o n between weights of the t o t a l osseous skeleton to b i r t h weight and to lengths of the osseous diaphyses of the humerus and femur; each increased with age. The many tables in the l i t e r a t u r e (61-68) des-cr i b i n g the developmental sequence of both membraneous and endochondral o s s i f i c a t i o n have been concerned with the time of appearance not the extent or length of o s s i f i e d diaphyses. The present work has shown conclusively that long bone o s s i f i c a t i o n as detected from s i l v e r radiography i s a simple and accurate parameter of f e t a l age. This could be substantiated by performing the technique on a large backlog 71 of therapeutically aborted fetuses. The res u l t i n g bone age table, based on the lengths of the o s s i f i e d diaphyses of femora and humeri, could then be used to date spontan-eously aborted specimens. Bone composition results are in agreement with Dickerson's research (86) on the human femur although the present study was concerned with both a younger, more narrow age range, and a larger t o t a l sample s i z e . The fundamental change in the composition of a bone during development i s an increase in i t s degree of o s s i f i c a t i o n . This is accompanied by a decrease in the percentage of water (85). Hammett's observation was substantiated in t h i s study. I t was found that bone length and o s s i f i c a t i o n best predicted water content. However, because cleaning of bone for analysis takes considerable time and controlled con-d i t i o n s to determine accurately the percentage of water, i t i s customary to express composition of bone tissue on a dry fat-fre e b a s i s . The results of t h i s study seem to confirm Dickerson's statement (86) that no fat i s present in the f e t a l femur during the 12-28 week age range. However, the effectiveness of petroleum ether to penetrate the bone and to break the lipoprotein complexes in the marrow could be questioned. A micro-soxhlet apparatus would have been a more sensitive technique although problems in drying and weighing a bone of such size (l-400mg) would s t i l l have to be solved. Collagen did not increase s i g n i f i c a n t l y i n the femurs of fetuses 9-20 weeks developmental age. Although s i m i l a r results were found by Dickerson (86) in femoral c o r t i c a l bone, i t i s surprising that collagen would not increase when expressed as g/lOOg whole bone. Femoral collagen was the only variable examined that did not predict developmental age at the 5% l e v e l of s i g n i f i c a n c e . Humeral collagen was s i g n i f i c a n t l y higher in 11-20 week fetuses than in 9 - 10 week specimens. Differences in humeral and femoral collagen content are not r e a d i l y explainable. Whereas calcium increased l i n e a r l y in both femora and humeri of 9-20 week old fetuses, inorganic phosphorus increased in a non-linear manner. Therefore the Ca/P r a t i o (indicator of bone mineralization) was s i g n i f i c a n t l y less than 2.0 at 9-10 weeks, and constant at 2.0 in the remaining age range. Dickerson (86) and Swanson (94, 95) reported r e l a t i v e l y constant Ca/P r a t i o s when expressed per lOOg dry f a t - f r e e s o l i d s . However, fetuses less than 12 weeks were not studied by e i t h e r researcher. The results of the present work suggest that e i t h e r the r a t i o of calcium to inorganic phosphate deposition i s not constant with bone growth, or younger specimens have proportionately larger amounts of organic phosphorus resulting in contamination. Perhaps the r a t i o increase a f t e r 10 weeks was due to a decrease in the proportion of phosphate from ester phos-73 phates - a large part of the phosphorus in the bones of immature fetuses being present in ester form. Because c r y s t a l s of bone mineral are p r i n c i p a l l y l a i d down in association with collagen f i b r i l s , the Ca/collagen r a t i o gives a measure of the degree of satura-tion of collagen f i b r i l s . The r a t i o was found by Dickerson (86) to change very l i t t l e during growth in humans. This i s in agreement with the currently accepted view that collagen f i b r i l s are rapidly mineralized to about 80% saturation soon afte r they are l a i d down. The results of t h i s study indicate a s i g n i f i c a n t l y lower r a t i o in younger fetuses. Again, bone composition of 9-10 week old fetuses has not been reported. Perhaps the c a l c i f i c a t i o n mechanism is not f u l l y developed to mineralize a surplus of collagen f i b r i l s in the cartilaginous model. This explanation i s reasonable because c a l c i f i c a t i o n of long bones does not begin u n t i l the fetus i s 8 weeks old (57) . Sodium content of f e t a l bones, 9-10 weeks old was s i g n i f i c a n t l y higher than in bones from fetuses aged 11-20 weeks. Sodium i s found in the bone i n the extra c e l l u l a r f l u i d , in the hydrated layer of bone cr y s t a l s and in the bone c r y s t a l s themselves. Although a dynamic process i s occuring, the trend seen here confirms the findings of Swanson and lob (94, 95). Magnesium i s also thought to be inversely related to f e t a l age (94, 95). The constant value of bone magnesium found in this study, and the poor c o r r e l a t i o n between humeral and femoral magnesium would suggest poor method s e n s i t i v i t y . Some comments can be made concerning the f e t a l model evolved in this project. Generally, physical v a r i -ables best predicted other physical v a r i a b l e s . S i m i l a r l y , biochemical variables best predicted other biochemical va r i a b l e s . With the exception of magnesium and collagen, which remained constant, a l l biochemical variables cor-related s i g n i f i c a n t l y with physical data at better than the 5% l e v e l . This is a reasonable, but u n t i l now undocumented, find ing. In t h i s study, femoral and humeral data were found to be comparable. U n t i l now, studies of s k e l e t a l growth and development in the human fetus have been limited to the femur. Data presented herein show that the rate of growth between the humerus and the femur, as assessed by physical and biochemical variables, i s s i m i l a r . C o e f f i c -ients of Determination associated with femur variables were generally higher than those associated with corresponding humerus variables when plotted against developmental age (less scatter about the regression l i n e ) . However, humerus results generally had a more s i g n i f i c a n t F p r o b a b i l i t y than did femur re s u l t s , and therefore a greater separation into 75 age groups was seen in this bone. The reason for t h i s pattern i s not r e a d i l y explainable. The study did not detect a consistent sex d i f f e r -ence among the f e t a l variables analysed. Roche (84) and others have observed that o s s i f i c a t i o n i s more advanced in the female than in the male during the l a s t three months prenatal and at b i r t h , whereas the b i r t h weights of males are generally higher. . Perhaps these e f f e c t s are not detectable u n t i l a f t e r 20 weeks developmental age. Caution must be exercised i n drawing conclusions from the e f f e c t of most maternal variables on f e t a l v a r i a b l e s . A s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n does not e s t a b l i s h a causal r e l a t i o n s h i p . A d i r e c t c o r r e l a t i o n found between maternal variables (e.g. age, parity, gravidity) and new-born variables (e.g. b i r t h weight and length) has been observed (32, 48, 49). Apparently this r e l a t i o n s h i p extends into e a r l i e r f e t a l l i f e . Previous studies have shown that maternal height, weight and b i r t h weight (33-38) are d i r e c t l y correlated with certain newborn growth parameters. The negative c o r r e l a t i o n found in t h i s study contradicts previous findings. The reason for t h i s contradiction i s unclear. I t i s not inconceivable that maternal age, weight, b i r t h weight, p a r i t y and g r a v i d i t y could be correlated in some way with f e t a l s k e l e t a l growth, but factors such as ethnic o r i g i n (23, 24), maternal anxiety (27, 28), smoking (39, 40), season (143) and paternal variables must also be considered in the analysis. One of these additional v a r i -ables could be mediating the observed e f f e c t of socio-economic status on f e t a l weight (49-51). The potential for undertaking studies on the mothers of the experimental fetuses was rather limited because of the emotional factors associated with the performance of a therapeutic abortion. Technical problems associated with conducting research in a hospital manifested themselves through attitudes of nursing personnel and f a c i l i t i e s a v a i l a b l e . Accordingly, i t was important to l i m i t the amount of information obtained from the mother without prejudicing the needs of the study. This l i m i t a t i o n c u r t a i l e d the scope and accuracy of the maternal dietary h i s t o r y . The study concerned a unique group of women. Seventy-three out of seventy-six had been granted t h e i r abortion for psychiatric reasons. They were e a s i l y upset, very g u i l t - r i d d e n , and generally reluctant volunteers. I t was not feasible to validate the questionnaire with blood or urine samples, or with 7-day dietary h i s t o r i e s following discharge. Whereas the questionnaire i t s e l f could have been validated on normal volunteers, i t would have been of doubtful significance to extrapolate from a normal s i t u a t i o n to the abortion patients. The i n t e r v i e w placed emphasis on the quantity and v a r i e t y of the die t in a r e l a t i v e sense. Scores were arranged on a continuum from low to high. Crump (45) devised t h i s scale from a study of 483 pregnant women in Nashville, Tennessee. He validated the o r a l dietary h i s t o r y using 7-day food records from the same patients. Diets were c l a s s i f i e d as "poor" (scores 24-33), " f a i r " (34-52), "good" (53-70) and "excellent" (71-133). From his c alculations a score of 60 was found to represent an intake of two-thirds the Recommended Daily Allowance during pregnancy. Ninety-five percent (mean plus/minus two stand-ard deviations) of the n u t r i t i o n scores in the present study f e l l into Crump's "good" or "excellent" c l a s s i f i c a t i o n s . Only 4 women could be considered to have " f a i r " diets during pregnancy. This would indicate that scores were taken from the upper range of the normal population d i s t r i b u t i o n ; not surprising considering that the procedure and cost of a therapeutic abortion e f f e c t i v e l y l i m i t s the operation to those in the middle-to-upper socio-economic range. Because the scores represented a continuum from f a i r to excellent, i t was considered v a l i d to regress n u t r i t i o n a l data against f e t a l data. Perhaps egg, cheese, meat and milk content of the maternal d i e t gave an inaccurate protein score, for t h i s variable showed no relat i o n s h i p with any of the f e t a l parameters examined. On the other hand, n u t r i t i o n score (a measure of protein, vitamins, minerals and calories) d i r e c t l y correlated with length and dry weight of both humeri and femora. In turn, dry weight of bones was the best predictor of f e t a l weight, and length of bone best predicted bone o s s i f i c a t i o n . Both bone length and weight were s i g n i f i c a n t predictors of f e t a l length and developmental age, and good indicators of bone calcium. Skeletal growth and maturation are obviously under control of a fundamental b i o l o g i c a l growth mechanism (1). These data suggest that n u t r i t i o n a l factors may a f f e c t the e f f i c i e n c y with which this mechanism functions. In conclusion, normal s k e l e t a l growth and devel-opment of the human fetus can be described in terms of a c o r r e l a t i o n between whole f e t a l measurements, physical growth and biochemical composition of e i t h e r the femur or the humerus. Using this model, further n u t r i t i o n a l research could be conducted to explore the r e l a t i o n s h i p between f e t a l bone growth and diet during pregnancy. Length and weight of e i t h e r bone, as well as being s i g n i f -i c a n t l y correlated with maternal n u t r i t i o n in this study, are r e l a t i v e l y simple and accurate parameters to analyse, and would allow considerable increase in sample size during a s i m i l a r time period. Bone calcium would be the best predictor of bone composition and could be compared with cord blood calcium and maternal blood calcium. Biochemical findings could then be related to maternal consumption of milk and other calcium-rich foods during pregnancy. Pos-s i b i l i t i e s are as numerous as the number of dimensions presented; study i n this area offers an i n t e r d i s c i p l i n a r y blend of n u t r i t i o n , embryology, biochemistry and psychology. 80 R E F E R E C N E S 1. Langman, J . : Medical Embryology. 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C : Maternal undernutrition and retarded f e t a l development in merino sheep. Nature. 201:1341-1342 (1964). 162. Brent, R. L., McLaughlin, M. M.: The i n d i r e c t e f f e c t of i r r a d i a t i o n on embryonic development. Amer. J . Dis. C h i l d . 100:94-102 (1960). 163. Jackson, C. M.: Structural changes when growth i s suppressed by undernutrition in the albino r a t . Amer. J . Anat. 51:347-379 (1932). 164. Waters, H. J . : The capcity of animals to grow under adverse conditions. Proc. Soc. Promotion Agric. S c i . 29:71-79 (1908). 165. Waters, H. J . : The influence of n u t r i t i o n upon the animal form. Proc. Soc. Promotion A g r i c . S c i . 30:70-75 (1909). 166. Aron, H.: N u t r i t i o n and growth. P h i l l i p p . J . S c i . 6:1-9 (1911) . 167. Drummond, J . C : Observations upon the growth of young chickens. Biochem. J . 10:77-88 (1916). 168. McCance, R. A.: Severe undernutrition in growing and adult animals. 1. Production and general e f f e c t s . B r i t . J . Nutr. 14:59-73 (1960). 169. Appleton, A. B.: The r e l a t i o n between rate of growth and rate of o s s i f i c a t i o n in the fetuses of rabbits. C. R. Assoc. Anat. (24th meeting, Bordeau) 1929, pg. 3. 93 170. Dickerson, J , W. T., McCance, R. A . : Severe under-n u t r i t i o n in growing and adult animals; dimensions and chemistry of long bones. B r i t . J . Nutr. 15:567-576 (1961). 171. Widdowson, E. M., McCance, R. A.: Some effects of accelerating growth. Proc. Roy. Soc. B. 152:188-206 (1960). 172. Dickerson, J . W. T., Widdowson, E. M.: Some ef f e c t s of accelerating growth. Proc. Roy. Soc. B. 152:207-217 (1960). 173. Hammond, J . : Physiological factors a f f e c t i n g b i r t h weight. Proc. Nutr. Soc. 2:8-12 (1944). 174. Wallace, R. R.: The growth of lambs before and aft e r b i r t h in r e l a t i o n to the l e v e l of n u t r i t i o n . J . Agric. S c i . 38:367-376 (1948). 175. Sherman, H. C , Booher, L. E.: The calcium content of the body in r e l a t i o n to that of the f o o l . J . B i o l . Chem. 93:93-103 (1931). 176. Henry, K. M., Kon, S. K.: The e f f e c t of age and the supply of phosphate on the assimilation of calcium by the r a t . Biochem. J . 41:169-176 (1947). 177. Warkany, J . : Appearance of s k e l e t a l abnormalities in o f f s p r i n g of rats reared on d e f i c i e n t d i e t s . Anat. Rec. 79:83-100 (1941). 178. Sherman, H. C : Calcium in Foods and N u t r i t i o n . Columbia Univ. Press, N. Y., 1948. 179. Shaw, J . H.: Marginal protein deficiency during the reproductive cycle in rats: Influence on body weight and development of s k u l l s and teeth of of f s p r i n g . J . Dental Res. 49:350-358 (1970). 180. Jha, G. J . : Bone growth in protein deficiency. Amer. J . Path. 53:1111-1121 (1968). 181. watchorn, E., McCance, R. A.: Subacute magnesium deficiency in r a t s . Biochem. J . 31:1379-1390 (1937) . 182. Irving, J . T.: Calcium Metabolism (Wiley & Sons, N. Y., 1957, pg. 98) . 94 183. Stuart, H„ C : Findings on the examination of new-born infants and infants during the neonatal period which appear to have a relationship to the diets of t h e i r mothers during pregnancy. Fed. Proc. 4:271-281 (1945). 184. Berk, H.: Some factors concerning the incidence of dental caries in c h i l d r e n . Amer. J . Dent. Assn. 30:1749-1754 (1943). 185. Massler, M., Schour, I., Poncher, H. G.: Developmental patterns of the c h i l d as reflected in the c a l c i f i -cation pattern of the teeth. Amer. J . Dis. C h i l d . 62:33-67 (1941). 186. Booher, L. E., Hansmann, G. H.: Studies on the chemical composition of the human skeleton. 1. C a l c i f i c a t i o n of the t i b i a of the normal newborn infant. J . B i o l . Chem. (4:195-205 (1931) . 187. Toverud, K. U.: Norsk. Mag. Laegevidenskap. 95:688-691 (1934). 188. Boder, E.: Further studies in congenital c r a n i a l osteoporosis. J . Pediat. 41:305-313 (1952). 189„ Rector, J . M.: Prenatal influences in r i c k e t s . J . Pediat. 6:161-166 (1935). 190. Toverud, K. U.: Etiology of congenital osteopososis. Acta. Paediat. 12:267-273 (1932). 191. Cockburn, F.: Some biochemical aspects of i n t r a -uterine growth retardation. Arch. Die. C h i l d . 44:136- (1969). 192. Sontag, L. W., Pyle, S. I., Cape, J . : Prenatal condition and the status of infants at b i r t h . Amer. J . Dis. C h i l d . 50:337-342 (1935). 193. Garn, S. M.: An annotated bibliography on bone densitometry. Amer. J . C l i n . Nutr. 10:59-67 (1962) . 194. Tompkins, W. T. Wiehl, D. G.: Epiphyseal maturation in the newborn as related to maternal n u t r i t i o n a l status. Amer. J . Obstet. Gynecol. 68:1366-1377 (1954). 195. Scott, K. E.: Epiphyseal development i n f e t a l mal-n u t r i t i o n syndrom. New. Eng. J . Med. 270:822-824 (1964). 95 196. Wedgewood, M., Holt, K. S.: A longitudinal study of the dental and physical development of 2-3 year old children who were underweight at b i r t h . B i o l . Neonatorum. 12:214-232 (1967). 197. Wilson, M., Meyers, H., Peters, A.: Postnatal bone growth of infants with f e t a l growth retardation. P e d i a t r i c s . 40:213-223 (1967). 198. Peck, W.: Malnutrition of newborn secondary to placental abnormality. New. Eng. J . Med. 250:905-907 (1954) . 199. Frances C : Factors influencing appearance of centers of o s s i f i c a t i o n during e a r l y c h i l d h o o l . Amer. J . Dis. C h i l d . 57:817-830 (1939). 200. Frances, C : Factors influencing the appearance of centers of o s s i f i c a t i o n during early childhood. Amer. J . Dis. C h i l d . 59:1006-1012 (1940). 201. Driezen, S., Stone, R. E., Spies, T„: The influence of chronic undernutrition on bone growth in ch i l d r e n . Postgrad. Med. 29:182-193 (1961). 202. Jones, P. R. M., Dean, R. F. A.: The e f f e c t of kwashiorkor on the development of the bones of the knee. J . Pediat. 54:176-184 (1959). 203. Berridge, F. R., Prior, K, M.: The s k e l e t a l develop-ment of children at the beginning and end of periods of supplemental feeding. Spec. Rep. Ser. Med. Res. Council, London, no. 287, (1940). 204. Dickerson, J . W. T., John, P. M. V.: The e f f e c t of protein c a l o r i e malnutrition on the composition of the human femur. B r i t . J . Nutr. 23:917-924 (1969). 205. Streeter, G. L.: Developmental Horizons of Human Embryos, Washington, D. C , Carnegie I n s t i t u t e , Embryology Reprints, v o l s . I & I I , (1951). 206. Labconco: Information on Operation. Goldfisch Fat Extraction Apparatus, pg. 1-4. 207. "Calcium and Mangesium in blood serum". Atomic absorption method for Unicam SP90 Atomic Absorption Spectrophotometer. 2 08. "Sodium in serum and urine". Flame emmission method for Unicam SP90 Atomic Absorption Spectrophotometer. 96 209. Taussky, H. H., Shorr, E.: A microcolourimetric method fo r the determination of inorganic phos-phorus. J . B i o l . Chem. 202:675-685 (1953). 210. Neutnan, R. E„, Logan, M. A.: The determination of collagen and e l a s t i n in t i s s u e s . J . B i o l . Chem. 186:549-556 (1950). 211. Leach, A. A.: Notes on a modification of the Neuman and Logan method for the determination of the hydroxyproline. Biochem. J . 74:70-71 (I960). 212. Eastoe, J . E.: The amino acid composition of mammalian collagen and g e l a t i n . Biochem. J . 61:589-600 (1955). 213. Burke, B,: The dietary history as a tool in research. J . Amer. Diet. Assn. 23:1041-1046 (1947). 214. Abramson, J„ H., Slome, C , Kosovsky, C : Food frequency interview as an epidemiological t o o l . Amer. J . Pub. Health. 53:1093-1101 (1963). 215. Epstein, L„ M., Reshef, A., Abramson, J . H., Bealik, 0.: V a l i d i t y of a short dietary questionnaire. I s r a e l J . Med. S c i . 6:589-597 (1970). 216. Recommended Daily Allowances. N. R. C. Publication 1694, National Academy of Science, Washington, D. C , (1968) . 217. Dietary Standard for Canada. Canadian B u l l e t i n on N u t r i t i o n 6:1, Canadian Council on N u t r i t i o n , March, (1964) . 218. Duncan, D. B.: Multiple range and multiple F t e s t s . Biometrics. 11:1-42 (1955). 219. Battaglia, F. C : Intra-uterine growth. J . Pediat. 1015 (1969) o 220. Murphy, E. A., Abby, H.: The normal range - a common misuse. J . Chron. Dis. 20:79-88 (1967). 97 APPENDIX 1 9 8 Table XI. Crown-rump length versus developmental age of fetus CR days CR days CR days length a f t e r length after length a f t e r (mm) ovu1'n (mm) ovul'n (mm) ovul'n 1.0 18 40-41 58 113-114 97 1.5 20 42 59 115-116 98 1.8 22 43-44 60 117-118 99 2.0 22 45-46 61 119-120 100 2 .8 24 47-48 62 121-122 101 3.0 25 49 63 123-124 102 3 .5 26 50-51 64 125-126 103 4.0 27 52-53 65 127-128 104 4.5 28 54 66 129 105 5.0 28 55-56 67 130-131 106 6.0 29 57-58 68 132-134 107 6.5 29 59-60 69 135-136 108 7.0 30 61-62 70 137-138 109 7 .5 31.5 63-64 71 139 110 8.0 32 65 72 140-141 111 9.5 33 66-67 73 142-143 112 10.0 33 68-69 74 144-145 113 11.0 34 70-71 75 146-147 114 12 .0 35 72-73 76 148-149 115 13.0 35 74 77 150 116 14.0 36 75 78 151-152 117 15.0 37 76-78 79 153 118 16.0 37 79-80 80 154-155 119 17.0 38 81 81 156-157 120 18.5 39 82 82 158 121 20.0 40 83-86 83 159-160 121 21.0 41 87-89 84 161-162 122 22.0 41 90-91 85 163-164 124 23 .0 43 92-93 86 165 125 24 .0 43 94 87 166 128 25.0 44 95-97 88 167 130 26.0 45 98-99 89 168 131 2 7.0 51 100-101 90 169 132 28-29 52 102-103 91 170-172 133 30-31 53 104 92 173-174 134 32-34 54 105-106 93 175-177 135 35-36 55 107-108 94 178-179 136 37 56 109-110 95 180 137 38-39 57 111-112 96 99 Plate 1. Specimen in i n t a c t s*c 100 2. Placenta and fetns Female: Crown-rumD l e n g t h - 114mm D e v e l o p m e n t a l aoe - 97 rlavs P l a t e 3 . E v i s c e r a t e d f e t u s w i t h r i a n t arm and leer removed 102 P l a t e 4. S i x f e t a l lone? bones; c l e a n e d (femur, t i b i a , f i b u l a , humerus. rad i u s , u l n a ) 103 Plate 6. Fetus nreoared ^or radioaraphy a f t e r s i l v e r n i t r a t e treatment P l a t e 7. Radnoaraphr f e t u s i n f o r m a l i n f o r 1 day P l a t e 8. R a d i o q r a p h r f e t u s i n s i l v e r n i t r a t e f o r 6 days 107 P i s t e 9. R a d i o a r a o h ; f e t u s i n s i l v e r n i t r a t e f o r 10 d a y s 108 Tabl e X I I . Minimum f o r m a l i n treatment f o r s i l v e r r a d i o -graphy Crown-rump l e n g t h mm For m a l i n Treatment days 4 0 - 6 0 2 60- 7 5 3 7 5 - 90 4 90-110 5 110-130 6 over 130 7 Table XIII. Optimum s i l v e r radiography n i t r a t e treatment for f e t a l Crown-rump length mm S i l v e r n i t r a t e treatment days 40- 60 2 60- 70 3 70- 80 4 80- 90 5 90-100 6 100-110 7 110-120 8 120-130 9 1.30-140 10 over 140 11 110 Table XIV. Exposure time for f e t a l radiographs on G. E. Model F unit (58kv., lOma) Crown-rump length Exposure time mm sec 40- 60 1 60- 90 2 90-130 3 130 and over 4 I l l Figure (o. Measurement of o s s i f i e d shaft of f e t a l bone a. Length of o s s i f i c a t i o n of long bone (femur, t i b i a , f i b u l a , humerus, radius, ulna) b. Width of o s s i f i c a t i o n at proximal metaphysis of each long bone c. Width of o s s i f i c a t i o n at d i s t a l metaphysis of each long bone I f necessary, a bone was divided into two or three parts by pencil lines perpendicular to the plane of the bone; length of each section was measured and t o t a l length found by add i t i o n . Measurements were recorded in millimeters. 112 Form 1. Dietary History Specimen Number 1 0 J ' A. Daily pattern r e c a l l : time food amount Breakfast Mid-Morning Lunch Mid-Afternoon Dinner Mid-Evening 113 B. Food Frequency Questionnaire How often and how much do you eat of the following types of foods? M i l k J 1 .^J^!L^r7 JeJ\ Cheese s-J"^-/-V-^^>. ^^vyi**^ a. — ^/ j^e^-^r^T^^j j^/>cj Je, F i s h — T *e^*^*n^*prT*l.<rrTt*tKrzXJi. Poultry / j j s L . Bread — ^ ^ / ^ J J ^ * * ^ e . s ^ > * * J * ^ - t J s t / ^ > j ^ > Cereal products Jr^»'**<*^^aJ^^ ^^r^/JJ-^ Vegetables: green j/hs<<ajr, „<L-e*asr7 J. ,j d?«m*«a~£i*, . Si*»J*a*<£n y^^je^u-JoLci^. ye 11 ow .^<ast*4*&?Jk<i, *(wn^r2f,. J. t <t-t t w >t ? potatoes — < d * x J < « 4 * i ^ . F r u i t : c i t r u s *m*tarrr^s*L, .^rt^x^i^. y%^u^tjrj non-citrus .£><Jhfaf£4, Arrzrr?*aisr?s*A+, /H*«<t '4 f« Swe e t s 4.s-f^ y *rsi* •fJjp. Fats J>-rs77*^, ^r~x> Jarr^^sL^J^ v C y ^ ^ Beverages ^, jluj^Pj N u t r i t i o n a l Supplements <n( Cigarettes -r^*-^-*- ^*^t^t»Jpa^ A**^*/^. 114 Likes and D i s l i k e s D. General Comments 115 Form 2. N u t r i t i o n a l Status Specimen Number_ /03 7 Dietary Intake bv S n e c i f i c Food Groups (Crump et a l . Am . J . Obstet. Gynec. 77:562, 1959) Food Group (number serving s per week) Meat Milk Eggs Cereal Veg. F r u i t Butter Cheese 5 Excellent 2 8+ 21+ 2 8+ 14+ 14+ 2 8+ 4 Good 21-2 7 16-20 21-27 . 11-13 11-1.3 21.-2 7 3 F a i r 14-20 11-15 14-2 0 8-10 8-10 14-20 2 Poor 7-13 6-10 7-13 5- 7 5- 7 7-13 1 Very Poor 3- 6 3- 5 3- 6 3- 4 3- 4 3- 6 Food group no. servings per week rating Milk Meat, eggs, cheese Cereal Vegetables Fru i t Butter /6 Protein Score ( 4 9 ) >JP9 >w -* >/v J ? >j* s Total N u t r i t i o n Score (133) Weighted Nu t r i t i o n Score (3 0) N u t r i t i o n Index (5) Comments: Form 3. Socio-economic Status Specimen Nnmhgr 1031 Short Form Socio-economic Index (Crump et a l . J . Pediat. 51:678, 1957) Score Occupation of Father Education of Education of Ma r i t a l (or mother) Mo the r Father Status 9 professional, semi-professiona1 college 4 college 4 give average 8 o f f i c i a l , p r o p r i e t . of known manager, c o l . student college 3 college 3 values for 7 c l e r i c a l college 2 college 2 "married" 6 s k i l l e d college 1 college 1 (4 - 8) 5 semi-skilled grade 12 grade 12 4 protective service high school student grade 11 grade 11 divorce, sep. 3 service (except desert., protect or domestic) grade 10 grade 10 widow 2 domestic service grade 9 grade 9 (average 1-3) 1 farm labourer grade 8 grade 8 0 unskilled labourer less than 8 less than 8 single Socio-economic Group Score X 2 Range Group I 0 - 1 4 low Group II 1 5 - 2 9 | Group III 30 - 49 1 Group IV 50 - 72 high Index Answer Given Score Occupation of Father Education , of Mother Education of Father Ma r i t a l Status 7 S 7 7 TOTAL SCORE (X2) SOCIO-ECONOMIC GROUP s z 117 APPENDIX 2 OEP INO CONST C O E F F F R A T 10 F P R O B STD ERR STD E R R STD ERR RSQ VAR VAR A B ( B ) ( B ) (A.) I B ) < Y) //? DE AGE C R L E N 3 6 . 3 4 0 . 5 4 1 8 0 . 1 1 8 5 0 05 0 . 0 0 . 5 4 4 6 0 . 4 9 7 6 0 - 0 2 1 . 2 8 3 0 . 9 9 5 3 THE AND H * " ARE U S E D TO PLOT T H E R E G R E S S I O N L I M E ; THE » * " I S USED WHEN A P L O T P O I N T C O V E R S DATA P O I N T S 1 4 0 . 0 / I / / / / / / / 1 2 4 . 0 / / 11 / / / . 5 / / 1 0 8 .0 / . 1 2 / 21 / . 1 1 / / / • / 1 * 2 / 1 / * 9 2 . 0 0 - 11 / 2 . 1 / 1 / 1 . / 2 1 2 / 4 * 7 6 . 0 0 / 1 2 2 / 1 1 1 . / j_2 / "" ~ " " 1 « / 1 2 / 1*1 / /_ * / " 1 / *1 / 1 6 0 . 00 / / ! / / / / 7 7 7 / / / / / / / / / / / / I A / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / 1 / / / / / / / / / / / / / / 7 / / / / T 4 5 . 0 0 7 3 . 0 0 101 . 0 Crnrn) 1 2 9 . 0 1 5 7 . 0 1 8 5 . 0 ExHib'iT \. T)e we to ( 3 « e n to \ cuje (V) u e r s u 5 crown-rump DEP I NO CONST COEF F FR AT 10 F P R O B STD ERR S T D ERR STD ERR RSO VAR VAR A 8 ( B ) { B) ( A ) ( B ) (Y ) //? H E A D C D E A G E -72.47 1.919 1355. 0.0 4.927 0. 52120-01 7.299 0.9603 THE AND « • * " ARE USED TO P L O T THE R E G R E S S I O N L I N E ; T H E » * " IS U S E D WHEN A P L O T P O I N T C O V E R S D A T A P O I N T S 2 00. 0 t / / / 170.0 / / / / / / / / / 140 .0 / / 3 1 / 1 / 11 (mm) / 1. / 1 / 1 1 . _/ 1 / - " * / 1 / 110.0 11 1 / 1 1 . / 1 1 1 / 1 / 1 / 1 . 1 / 1 / 1 * 1 1 / 41 / . 1 80.00 - 11 111 7 I / 1 . / 1 1 / 1 . 1 / 12 1 1 / * 50. 00 / / ! / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / 1 / / / / / / / / / / / / / / / / / / / 1 / / / 7 / / / / / / / / / / / / / / / 1 60. 00 76.00 92.00 (dai4s) 108.0 124.0 140.0 Exh'ibirrf. T)e.\je.\c>pmef\-tcL\ £>_e (X) versus head. c\rcurr&e.re.nee. (V) DEP i NO CONST C Q E F F FRAT 10 F P R 0 8 STD ERR STO ERR S T D ERR RSQ VAR VAR A 8 ( B ) < B ) ( A ) ( B ) ( Y) FOOTL O E A G E - 2 0 . 9 3 0 .4 -236 1 0 8 0 . 0 . 0 1 . 2 1 8 0 . 1 2 8 9 0 - 0 1 1 . 8 0 5 0 . 9 5 0 7 THE " . « ' AND ARE U S E D TO PLOT T H E R E G R E S S I O N L I N E ; THE ••*" I S USED WHEN A P L O T P O I N T C O V E R S D A T A P O I N T S J 3 7 . 0 0 / 1 1 1 / . 1 / 1 / • / 1 / * / / 1 / 3 1 . 0 0 / * / • / 1 / * / 1 1 / 1 • / 1 1 1 / 1 . 1 / 2.5. 0 0 _ - 1 • 7 / 1 . 1 ( m m ] / 1 1 1 V J / * / 1 1 / * / 1 1 1 1 / 1 . 1 / 1 9 . 0 0 / / • 1 .1 1 1 1 / / • / 1 11 1 11 / •V / 1 22 2 / # 1 1 / 1 3 . 0 0 / * 12 1 / . i 1 / 1 1 / .1 / 1 11 1 / * / 11 1 / 1 ^ / 1 7 . 0 0 0 - 1 . 11 //I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / i (da us) 6 0 . 0 0 7 6 . 0 0 9 2 . 0 0 3 y 1 0 8 . 0 1 2 4 . 0 1 4 0 . 0 J £xhibit 3. l)e\/e.lcpmemTa.\ aqe. versus -fooT leogTh (Y) DEP INO C O N S T C O E F F F R A T I O F P R O B STD ERR S T D ERR STD ERR RSQ VAR VAR A B <B) ( B ) ( A ) <B) 1Y } /Al WEIGH D E A G E - 3 3 8 . 4 4 . 6 1 3 4 0 2 . 6 0 . 0 2 1 . 7 4 0 . 2 2 9 9 3 2 . 2 0 0 . 8 7 7 9 THE " . " AND « * " ARE USED TO P L O T THE R E G R E S S I O N L I N E ; T H E " * " IS U S E D WHEN A P L O T P O I N T C O V E R S DATA P O I N T S 4 4 0 . 0 / / / / / 3 5 0 .0 / / / / / / / / 2 6 0 .0 4 ^ / / / / / / / / 1 7 0 . 0 ~ 3 / 1 . / 1 / / / . 1 1 1 / 1 / / . 1 1 1 / 1 1 8 0 . 0 0 - . 1 / 1 1 1 / .1 1 11 / 1 1 / . 1 1 1 1 1 / 2 1 1 3 3 1 1 / . 1 / 12 121 1 1 - 1 0 . 0 0 / 11 2 1 / //I / / ? / / / / / / ? / / / / / / / / / ! / - / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / J 6 0 . 0 0 7 6 . 0 0 9 2 . 0 0 ^a<6^ 1 0 8 . 0 124.0 L40.0 Exh'ibiT -V. De.\zciaf>ona<\ta.\ age (x) versus, -petal we'ighr _V) DEP TND CONST CD E P F F R A T I 0 F PROB STD ERR STD ERR STD ERR RSQ VAR VAR A B (B) ( B ) { A ) ( B ) {Y) DRUWP D E A G E -1.278 0.2541 173.0 0.0000 1.826 0.19320-01 2.706 0.7555 T H E " . « AND ARE USED TO P L O T THE R E G R E S S I O N L I N E ; THE »«*» I S USED WHEN A PLOT POINT COVERS DATA POINTS /HA 32.00 / _/ / / / 1 1 1 T 27 .00 / / / T / / /_ / / / l l l l l . i 22.00 / / 7 / / l l i n / / / / / / i i 17.00 / / / / / 1 1 1 / / / 12.00 / / ± / / / / / 7. 000 / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I (days) 6 0. 00 76.00 92.00 108.0 124. 0 140.0 DEP IND CONST C O E F F F R A T I O F P R O B S T D ERR S T D ERR STD ERR R SQ VAR VAR A B ( 8 ) ( B ) (A) (B) <Y) fJt3 G E A G E OEAGE 1 . 9 4 0 0 . 9 9 7 1 1 1 4 . 3 0 . 0 0 0 0 8 . 8 1 5 0.9325D-01 13.06 0.6712 THE AND « * " ARE USED TO PL 3 T THE R E G R E S S I O N L I N E ; THE " * •• I S USED WHEN A PLOT POINT COVERS DATA POINTS 1 4 3 . 0 1 2 5 .0 1 0 7 .0 t /_ / / J_ / / L / / / /_ / / / T / / J_ / / _l / / / 89.00 1 .1 1 . 1 1 . 1 . 1 1 1 * 1 / l_ l / / i i l . 12 / / f 71 . 0 0 1 1 1 1 / / / 7 / / .1 2 1 / 5 3 . 0 0 60.00 76 .00 / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I (day*) 92. 00 108 .0 124.0 140.0 Exhibit 6. Leo&\oprr>cntaL\ aqc versus ^esta.ti'or>ek.l duje DEP IND CONST COEF F F R A T I O F P R O B STD ERR S T D ERR S T D ERR R SQ VAR VAR A B (B) ( 8 ) ( A ) ( B ) ( Y ) /jW F O O T L THE G E A G E - 9 . 7 8 0 " . " AND ARE USED 0 . 2 9 7 9 128 . 6 0 . 0 0 0 0 2 . 5 4 8 0 . 2 6 2 7 D - 0 1 4 . 4 7 7 0 . 6 9 6 7 TO P L O T T H E - R E G R E S S I O N L I N E ; THE « * « IS U S E D WHEN A PLOT P O I N T C O V E R S DATA P O I N T S 3 7 . 0 0 / / 1 1 1 1 / / / 1 1 / / / • 1 3 1 . 0 0 / / • / / / • 1 • / / / 1 1 1 2 1 2 5 . 0 0 / / 1 1 1 / / 1 * 1 2 / / / • 1 1 • / / / 1 1 2 1 19 . 0 0 / / • 1 1 . 1 1 1 / / / • 1 1 1 2 1 / / / . 1 1 1 1 11 1 1 - 11 1 3 . 0 0 / 1 / 1 1 1 1 1 / / / 1 1 2 * / / / 1 1 1 1 11 1 7 . 0 0 0 / . 1 / 1 2 1 1 / / | / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / ] / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 5 3 . 0 0 7 1 . 0 0 8 9 . 0 0 1 0 7 . 0 1 2 5 . 0 1 4 3 . 0 J Exhibit y. Gestatic/)a[ age (X) versus foot length [ Y) OEP INO CONST C O E F F F R A T I O F PROB STD ERR STO ERR S T D ERR RSQ VAR VAR A B (B) (B) ( A ) <B) (Y) G E A G E CRLFN 38.04 0.5414 114.3 0 . 0 0 0 0 5.544 0 . 5 0 6 5 D - 0 1 1 3 . 0 6 0 . 6 7 1 1 THE AND " *•• ARE U S E D TO PLOT THE R E G R E S S I O N L I N E ; THE I S USED WHEN A PLOT P O I N T C O V E R S DATA P O I N T S 143 .0 / / / / / 1 1 125 .0 / / / 1 . / / / / / / T / .107 .0 / / / . 1 / / / / / 1 1 1 . 1 1 89.00 / / / / / 1 1 12 1. 1 1 71 .00 / / - f 1 1 1 1 / / _/ / / / / / 5 3 . 0 0 /?)///////////////////1 ///////////////////!///////////////////I///////////////////I7//7///A/////////// I (m/n) 45. 00 7 3. 00 101 .0 129 .0 157.0 1 8 5 . 0 E/hibir 9. Gestational age (,V) versus cro*J*-ru/np lertc^Pi (x) OEP IND CONST COEFF FRATIO FPR08 STO ERR STO ERR STO ERR RSO VAR VAR A B (B) (8) <A) (B) (Y) HEADC GEAGE -18.48 1.313 111.7 0.0000 12. 05 0. 1243 21.17 0.6660 THE ".» AND "*« ARE USED TO PLOT THE REGRESSION LINE; THE «*" IS USED WHEN A PLOT POINT COVERS DATA POINTS 200. 0 / / / / / / / 170.0 / / / / / 1 / 1 140.0 / I / - , , 1 1 / 1 / 1 1 / 1 • . / / . 1 1 / 1 / / / 110.0 - 1 11 / . 1 / 1 1 1 / 1 / 1 / 1 / 1 / 1 . 1 1 / 1 1 1 8 0 . 0 0 - 1 1 1 / / . 1 / 1_1 / . 1 1 / 1 211 / . 1 / . 1 / . 1 50.00 //| / ///////A/////////71 // 77/7/////////////!///////////////////1//////////////////?1///////////////////1 53 .00 71.00 89.00 (datis^ 107.0 125.0 143.0 Exhibit <?. &e&tcktiona.l a<ye versus head c'ircu/*rfere>nce- £V_) DEP IND C O N S T COEE F F R A T I O F P R O B S T D ERR S T D ERR STD ERR R SQ VAR VAR A B ( 8 ) J B J ( A ) ( B ) <Y ) WE IGH GE AGE -200 .7 3 .074 76.44 0.0000 34.10 0. 3 5 1 6 5 9 . 9 1 0 . 5 7 7 2 THE AND ***** ARE USED TO PLOT THE R E G R E S S I O N L I N E ; T H E » * " IS U S E D WHEN A PLOT P O I N T . C O V E R S DATA P O I N T S 440.0 / / / / / / / 3 5 0.0 / / / / / / / 260 .0 4 > -I t f T / / 7 / / 170.0 / L / / / / / / / 80.00 T / / / / / 11 1 2 21 1 21311 -10 .00 2 1 .1 1 / / j / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / ! 5 3.00 71. 00 89. 00 107.0 1 2 5 . 0 143 . 0 gxh'bir 10. G-ettational age (x) versus fetal weight ( Yj OEP I NO C O N S T C O E F F FRATIO F P R O B STD ERR STD ERR STD ERR RSQ VAR VAR A B ( B ) - ( B ) { A ) CB) ( Y ) ORUWP G E A G E 6.439 0.1679 53.45 0.0000 2.228 0.2296D-Q1 3.913 0.4884 T H E AND " * » • ARE USED TO P L O T THE R E G R E S S I O N L I N E ; THE IS U S E D WHEN A PLOT POINT COVERS DATA POINTS 3 2.00 / / / / / / 27 .00 / / / 1 / / / 11 22 .00 / / T / / i i i . / / j_ / / / i I I 1 7. 00 1 . / (_ / / / 111 11 / / / / 12.00 / / / / / / 7. 000 / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 53.00 71.00 89.00 107.0 12 5. 0 143.0 ExHtb'ir II. Gestational ac^c, (_x) versus ske\e.ta.\ iocL*x £v) OFP I NO C O N S T C O E F F F R A T I O F P R O B STO FRR STD ERR STD ERR RSO V A P VAR A B (B) ( B) ( A ) ( B ) <Y ) F - D P Y OF AG F - 4 1 2 .2 5 . 1 9 1 3 1 9 .9 0 . 0 2 7 . 44 0 . 2 9 0 2 4 0 . 6 4 0 . 8 5 1 0 THE AND ARE USED TO P L O T THE R E G R E S S I O N L I N E ; THE " * " IS USED WHEN A PLOT P O I N T C O V E R S DATA P O I N T S 4 5 0 . 0 / / / / 3 6 0 . 0 / / / / / / / / / / / 2 7 0 .0 / / / I / / / / / / I 1 8 0 . 0 / / / / 9 0 . 0 0 / / / 1 / . 1 / 1 / / . 1 1 1 / . 1 / . 1 / 1 1 / 1 1  / 1 / 1 1 1 * 4 2 2 1 t ... 1 1 2 1 1 1 1 1 . . . . . . . - 0 . 0 - 11 2 1 2 1 1 1 / / ! / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I (riau*,) 6 0 . 0 0 7 6 . 0 0 9 2 . 0 0 ^ 1 0 8 . 0 1 2 4 . 0 1 4 0 . 0 E*h)b\r ML. Dcve-lepsyiental ctqe. _>0 versus femora-i drq weiqMr _V_ DEP IND CONST C O E F F F R A U D FPROB STD ERR STD E R R S T D ERR RSQ VAR VAR A B ( B ) ( B ) ( A ) <B) <Y) )30 H~ DRY D E A G E -269.4 3.472 482.4 0.0 14.94 0.1581 22.14 0.8960 THE • ' . « ' AND ARE U S E D TO PLOT T H E R E G R E S S I O N L I N E ; THE " * « I S USED WHEN A P L O T P O I N T C O V E R S DATA P O I N T S 270. 0 / / / / / / / / 210.0 / / / / / 150.0 / / T / / l l / i / * / 2 / / / 90. 00 1 1 1 / / 1  / / 1 / 1 1 / 1. / / / . 1 30.00 - 1 / 1*11 1 7 : I~T 322T / 12. 12 / 1 11 121 2 1 / 11 111 / / / / -30.00 / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I 60.00 7 6.00 9 2. 00 108.0 124.0 140.0 f x A i b i f /_. Dcv<si'of>mer)-t<a.l ex^e. 0 0 versus. hume.ra.\ ctrtj we.ia^ht iv) DEP IND CONST COEF F FRATIO F PROB STD ERR STD ERR STD ERR RSQ VAR VAR A 8 ( B l ( B) (A) (B) (Y) F-H20 DEAGE 102.3 "0.2224 256.2 0.0 1.314 0.1390D-01 1.946 0.8206 THE «.»* AND ARE USED TO PLOT THE REGRESSION LINE; THE «*•• IS USED WHEN A PLOT POINT COVERS DATA POINTS /SI 89.80 / / / / 1 1 / / / / 86 .40 11 / / /_ 7 / / i _ i . / 83 .00 / / / . 1 1 12 1 11 / / ± / / / 11 1 1 79.60 / / / / / 1 . 1 1 / / / 76. 20 1 1 *1 / / / / / / 1 / 72. 80 . 1 //I/ / / / / / / / / / / / / / / / / / / I 60. 00 76 .00 92.00 (day*) 108.0 124. 0 140.0 £xhtb»r /<y. Developmental a^e versus femora.! wafer content (.YJ DEP VAR H -H2 0 THE IND C O N S T VAR A DEAGE 1 0 0 . 1 « . » AND " * » ARE USED C O E F F B - 0 . 2 3 5 9 TO PLOT THE F R A T I O F P R O B STD ERR STD ERR STD ERR RSO (B ) ( B ) (A) <B) <Y) /SJt 186.4 0 . 0 0 0 0 1.633 0. 1728D-01 2.419 0.7690 R E G R E S S I O N L I N E ; THE " * « IS U S E D WHEN A P L O T P O I N T C O V E R S DATA P O I N T S j 8 8 . 50 / / 1 1 <, / / / / 1 / 1 1 / . . 1 84 . 6 0 / • / 1 1 1 / .1 / / • 1 / / / 1 • 1 1 8 0 . 70. / / 1 . 1 1 1 1 . 1 / 1 / / 1 1 « 1 \ J / / / 1 1 1 1 1 1 / / / 1 1 1 1 2 lb. 8 0 / / 1 1 1 . 1 1 1 / / / 1 / / / 1 1 1 7 2 . 9 0 / / 1 . 1 / / / 1 . 1 2 / / / 1 1 . 1 6 9 . 0 0 / / 2 i //I ///////////////////I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | 6 0 . 0 0 7 6 . 0 0 9 2.00 ^daiiS^ 108.0 124.0 140.0 J Exh'ib'ir s (if!) Versus humeral Mater contenT (VJ DEP I NO CONST CO EPF FRATIO F PROB STD ERR STD ERR STD ERR RS Q VAR VAR A B (B) (B) {A) IB) IY) F-LEN DEAGE -27.65 0.57 64 1044. 0.0 1.687 0.1784D-01 2.498 0.9491 THE '«." AND "*•» ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS /33 51 .00 / / / / / 1 / / / 42.00 / / / 2 1 3 3.00 (mm) / / 7 / / l i i l l l / / / / / l l l 24. 00 / / / / 2 1 1 11 2 * 1 1 / / _/ 7 15. 00 2 . 1 1 . 1 1 / / / 11 / / 6. 000 / / I / / / / / / / / / / / / / / / / / / / I 60.00 76.00 92.00 (daus) 108.0 ///////////////////I 124.0 140.0 Exhib'if lk. Dsv&lopmenTcti aqe. £*) versus femoral len^Th (Y) DEP IND CONST CO EE F F R A T I O F PROB STO ERR STD ERR STD ERR RSQ VAR VAR A B <BI ( B ) (A) (BJ {Y ) /SV H - L E N DE AGE -22. 25 0. 5OO2 966.6 0.0 1 .521 0.1609D-01 2.253 0.9452 THE AND " *•» ARE USED TO P L O T THE R E G R E S S I O N L I N E ; THE •»*" IS USED VI HE N A PLOT POINT COVERS DATA POINTS 46 .00 / / 7" / / / / / i i 38.00 1 / / / / / / 30. 00 / / T / I / / / 1 / 1 1 1 . / / 1 22.00 - I / 1 1 1 . / 1 2 1 / 1 / 1 1 1 / / / 1 / 1 * / 11 14.00 - .1 / 1 1 7 n*" / / / 6. 000 7/I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / ] / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / 1 / / / / / / / / / / / / / / / / / / / 1 60.00 76.00 92.00 108.0 124.0 140.0 Bjfh'ib'iT 17, De\ze)opn?eryT<zl ct^e. (x) versus humeral /enajh (Y) DEP IND CONST C O E F F F R A T I O F P R O B STO ERR STD ERR S T O ERR RSO „ ^ VAR VAR A 8 <B) <B) ( A ) iB) <Y) F - O S S DEAGE - 2 6 . 3 7 0 . 4 5 7 4 1 3 9 1 . 0 . 0 1 . 1 6 0 0.1227D-01 1.718 0.9613 THE « . " AND " * « ARE U S E D TO P L O T THE R E G R E S S I O N L I N E ; THE I S USED WHEN A PLOT P O I N T C O V E R S DATA POINTS 3 8 .00 / / 1 • / / / • 1 / / / • 1 1 31.00 / / 1 / / / 1 . 1 1 . / / / 1 1 1 1 2 2 4 . 00 / / • I I 1 1 • / / 1 1 » / V / / / / 1 1 . 1 1 / / / 1 1 1 1 .1 • 1 7 . 00 / / 1 1 1 . 1 1 1 / / / 1 1 • I 1 1 / / / l 1 1 .2 2 21 1 10 . 00 / / i i . . l 111 1 1 / / / I i . I n i i / / / u . i . l I 3 . 000 / / 11 . 1 2 //]///////////////////1///////////////////1///////////////////I///////////////////|///////////////////| 6 0.00 76.00 9 2 . 0 0 ^ d a _ s ^ 1 0 8 . 0 124.0 140.0 Dei/<s lopmenTcx\ aqe. (x) versus •femorexi ossification £v) OEP I NO CONST COEFF FRATIO F PROB STD ERR STD ERR STD ERR RSQ VAR VAR A B (B) (B) (A) (B) (Y) /^ -» H-OSS DE AGE -24.15 0.4366 1579. 0.0 1.039 0.1099D-01 1.539 0.9658 THE ".»' AND ARE USED TO PLOT THE REGRESSION LINE; THE " i * " IS USED WHEN A PLOT POINT COVERS DATA POINTS ) 37 .00 - • \ / 1 / / • / • / 2 / • / / 1 / 30 .00 - .1 1 / 1 / 1 / / 1. / 1 / 1 1 . / 1 1 3 / 1 / • 2 3.00 - 1 / 11 . / 1 1 ( rrtm\ / * \. J / / 11 / 1 1 / * / 1 1 1 / 2 . 1 16.00 - 1 1 / 1 / 1 . 1 1 / 1 / 1 .1 / 1 1211 / . 11 / 111 / • 1 1 1 / 1 9. 000 - 1. / 1 / 1 * 1 I / / 1 1 . 1 / 1 / . 2 / 1 * 1 / • / 2 . 000 -/ / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | 60 .00 76.00 92.00 108.0 124.0 140.0 J Exnibir /9. Dc-ue\oprnenTa.{ age. (X.) versus hurnestxt ossification DEP IND CONST COEFF FRATIO FPROB STO ERR STO ERR STD ERR RSQ VAR VAR A B (B> (B) iA) (B> (Y, *Sr F-COL DEAGE 17.08 0.36230-01 2.019 0.1571 2.410 0.25490-01 3.570 0.0348 THE «.» AND «*« ARE USED TO PLOT THE REGRESSION LINE; THE '•*» IS USED WHEN A PLOT POINT COVERS DATA POINTS 30 .70 / / / / / / / 26.90 / / / / 1 / 1 1 7 I I T / 7 " ~ ~ / 23.10 ($/jOO$ bone) t 1 1 2 1 1 / 1 1 1 / 1 1 / . 1 1 / 1 1 1 1 / 1 l 19 .30 / _/_.„ / l l / l n / l 15.50 / 1 1 1 / / 1 1 / " i l l / / / 1 1 1 / / / / 11 .70 / / I / / / / / / / / / / / / / / / / / / / I ///////////////////I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I (dtaus}-60.00 76.00 92.00 3 108.0 124.0 140.0 Exh'ib'iT #C. Develop merfta.1. Q.a&- (XJ versus Peroral collagen DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A 8 (B) ( B) (A* <B) <Y) J3S H-COL DEAGE 14.20 0.6403D-Q1 6.499 0.0130 2.374 0.2512D-01 3.517 0.1040 THE " AND "•*» ARE USED TO PLOT THE REGRESSION LINE; THE »•*" IS USED WHEN A PLOT POINT COVERS DATA POINTS 32 .00 / / / / / / L / 27. 00 / / / , _ / / 11 / / 1 1 / 1 111 1 1 22. 00 - 1 1 11 1 / 1 1 1 . 1 / 2 1 1 / 1 1 1 . 1 / / . 1 / 1 1 / 1 . 1 / / 1 1 1 1_ 17.00 - I I / 2 1 1 / 1 _ / 1 / / 1 / / / 12.00 T / / / / / 7.000 t . . / / i / / / / / / / / / / / / / / / / / / / ! / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / ] / / / / / / / / / / / / / / / / / / / I 60.00 76. 00 92.00 108.0 124.0 140.0 £jth'ibir £). 3>e\jelopnnenT<xl a^e _X) versus humer<x\ collagen £Vj DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ > VAR VAR A B {B) (8) {A) (B) C Y } /S? F-CAL DEAGE -0.6499 0.1595 102.8 0.0000 1.487 0.15730-01 2.202 0.6474 THE "." AND »*" ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS 21. 40 / /_ / / / / / 1 / 1 18. 40 / 1 / J ' / / 1 21 / 1 1 . 1 5.40 / / T / / / u / i / I l / u / l / l 12.40 - 1 . 1 / . 1 / 1 1 / 1 / / 1 11 1 "7 1 1 1 / / 9.400 - . 1 1 I / / / / / / 6 .400 //I///////////////////1///////////////////1///////////////////I///////////////////17//7///////////////1 60 .00 76.00 92.00 ^ d a ^ 108.0 124.0 140.0 £xh'ibiran. Developmental age versus fen^oro.! calcium i?) DEP IND CONST COEF F FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B ( B) (B) (A) (B) (Y> H-CAL DEAGE -0.1232 0.16 16 69.53 0.0000 1. 832 0. 19380-01 2.713 0.5539 THE AND ARE USED TO PLOT THE REGRESSION LINE; THE «*»• IS USED WHEN A PLOT POINT COVERS DATA POINTS /to 22. 20 / / / / / / /_ / ,1 18. 50 1 1 / / / / / / 1 1 1 / / 1. 14. 80 / / / 2 1 1 1 * / / jL I / 1 1 1. 1 1 11. 10 / /_ / / / . 1 1 11 / / / / 7.400 / / / 7 / / 3 .700 7/17/////////// ///////T/7//7/////////7/7//I 7/77//////////////71///////////// />//// f 77/77////////////// I (days) 60 .00 76. 00 92. 00 1 08.0 124.0 140.0 EixhibiT H3. Dcvelopryj<sA-t<a.l ctge [*) versus humero.1 calcium DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B (B) (B) (A) IB) <Y) F-PHO DEAGE 2.708 0.46420-01 44.77 0.0000 0.6558 0.6937D-02 0.9714 0.4443 THE " AND ••*«' ARE USED TO PLOT THE REGRESSION LINE; THE «»*« IS USED WHEN A PLOT POINT COVERS DATA POINTS 9.600 / / / / / 8 .300 *1 / / / / / 7.000 (q/JOu'gbQne) I / / 2* / / / / / / 1 2 1 . 1 jul_ _. J _ 1 1 5 .700 / L / / / / / L. 4 .400 / / / / / 3. 100 //1 / // // // // // /A///7//1//////77///////////1 60. 00 76. 00 92.00 Ldaus) 108 .0 124.0 140. 0 £xhib)TDcvc\opmenTa.\ age versus femora\ inorganic phosphorus DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B ( B) ( B) (A) (B) (Y) H-PHO DEAGE 2.902 0.4666D-01 31.17 0.0000 0.7900 0.8358D-02 1.170 0.3576 THE ».•• AND •'*» ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS -< 9. 800 / / / / / / / 7 8. 200 1 1 1 1 / / / / 1 1 1 1 1 11 111 . 11 6 .600 (QJIOOQ bone) f / / I / / 5.000 / /_ / / / / / / 3. 400 / / T / / / / / / / 1 .800 / / i / / / / / / / / / / / / / / / / / / / ! / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / J / / / / / / / / / / / / / / / / / / / i (days) 6 0 .00 76.00 92. 00 108. 0 124.0 140.0 Exhibit jf£. Developmenta-l aae (x)uersus humeral morqaiiic. phosphorus (y) DEP I NO CONST COEFF, FRATIO FPROB STD ERR STD ERR STO ERR RSQ VAR VAR A 8 {B) (B) (A) <8) { Y* F-MAG DEAGE 0.6585 -0. 14460-02 3.965 0.0487 0. 68640-01 0.72620-03 0.1017 0.0661 THE »«.» AND "*« ARE USED TO PLOT THE REGRESSION LINE; THE «*" IS USED WHEN A PLOT POINT COVERS DATA POINTS 0. 9 200 / / / / / 0.8000 / / L 1 / / _ _ / / / 0. 6800 / 0.b600 / / / L / / / l u i i l l l 1 l u l l i l i 1 2 1 1 1 1 1 1 1 1 . 0.4400 / T / / / / 0.3200 / / 60. 00 i / / / / / / / / / / / / / / / / / / / j / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / M i l l n n / / / / / / / / / / / i 76. 00 92.00 108 .0 124.0 140. 0 £xh'ibir T>e\J<°lo amenta! aae, C%) versus, -femora,! /naqnesi um DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR V AR A 8 < B ) ( B) (A) (B) (Y J H- MAG DEAGE 0.6890 -0.1542D-02 4.217 0.0424 0.7097D-01 0. 7508D-03 0.1051 0.0700 THE " .»' AND ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS -< 0.9100 / / / / / / / 0. 7700 / / / _/ / / / / 0. 6300 / 1 . 1 1 1 1 1. 1 1 / / / 0. 4900 / / / / / 1 1 1 1 1 / / / 0.3500 / / L / / / 0.2100 / / i / / / / / / / / / / / / / / / / / / / i 7 7 / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / \ / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 60 .00 76.00 92. CO (days) 108. 0 124.0 140 .0 Exhibit J?7. DevelopnneiTa.1 a-^e £x*_) versus, humeral magnediuryn (* V) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B (8) IB) IA) IB) (Y) F-SOD DEAGE 7.603 -0. 6025D-01 29. 72 0.0000 1.045 0.1105D-01 1.548 0.3467 THE AND »•*•» A R E U S E D TO PLOT THE REGRESSION LINE; THE «*" IS USED WHEN A PLOT POINT COVERS DATA POINTS ) 8. 300 - 1 1 / 1 / / / / / / / / 6 .700 -/ / / / / / 1 1 / / / 1 5_.iQ0_ -/ / taste ) f 1 \ j J / 1 1 / . / • / 1 3 .500 - 1 / • / / » / / 1 / 1 / 1 / 1 / « 1 .900 -/ 1 2 1 . / 1 / 21 21 1 / 1 11 1 . 1 1 / 22 1 1 1 2 1 1 1 / 1 1 1 1 1 . 1 1 / 1 1 1 1 1 / 1 1 1 / 0.3000 -/ / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / \ / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / 1 60 .00 76.00 92.00 (•d-aUsJ 108 .0 124.0 140.0 ) Exh'ibiT AS. Jtevel&pmeitcil aae (x) versus femoral sodiiino if) DEP IND CONST COEFF VAR VAR A B H-SOD DEAGE 6.424 -0.5045D-01 THE AND ARE USED TO PLOT THE FRATIO FPROB STD ERR (B ) (3) (A) 25.48 0.0000 0.9448 REGRESSION LINE; THE IS USED WHEN STD ERR STO ERR R SQ (B) (Y) 0.99940-02 1.399 0.3127 A PLOT POINT COVERS DATA POINTS 7.400 / _ / / / / / / (_ t 6.000 / /_ / / / T / 4 _600 -_ / / / /_ / / / 3.200 1.800 / / / " / _/_ / / / f 1 1 "7 2 1 / 1 . 1 / 1 12 11 1_ / 1 1 21 1 1 / 1 11 1 1 / 1 21 1 1 .1 7 i i i i r •/ i i 0.4000 / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / //////////////////I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 60 .00 76.00 92. 00 ^ a ° ^ ^ 108.0 124.0 140.0 Exhibit 49. Developmental <zoe _*) versus. humeroS sodiiunn _ V) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B ( B) (B) (A) (B) (Y) FCACO DEAGE 0.9567E-01 0.64790-02 68.47 0. 0000 0. 7402D-01 0.7830D-03 0.1096 0.5501 THE AND «*•» ARE USED TO PLOT THE REGRESSION LINE; THE «*" IS USED WHEN A PLOT POINT COVERS DATA POINTS 1. 000 / I 7 / / / / j 7 0. 8700 / 1 1 / U / 1 / / 1 / 0.7400 (ratio) I / / / 1 / 1 1 11 / 1 / / 1 . 1 / 0.6100 / 1 . 1 1 / / . 1 / 1 / . 1 1 / / . l l 1 0.4800 / / / / / 0. 3500 - L . . / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I I III i l l HI III11 III l\ 60.00 76.00 92.00 ^ d < a _ r 3 108.0 124.0 140.0 Exhibit 30. Developmental age (*) versus femoral calcium jcotlaqen ratio C^) DEP I NO CONST COEFF FRATIO FPR08 STD ERR ' STD ERR STD ERR RSQ VAR VAR A 8 (8) i B , (A) IB) (Y) HCACO DEAGE 0.1894 0.5872D-02 52.88 0.0000 0.76330-01 0.8075D-03 0.1131 0.4857 THE » .« AND ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS 1 .140 / / / / 0.9900 / / / / / 0.8400 f f / / / 11 11 / / / 1 1 1 1 1 1 0.6900 / / / / / 11 / / / 0.5400 / / J. f / / 0. 3900 1 //! 77777777///////////!///////////////////1 60.00 76.00 92.00 108.0 124. 0 140.0 £xh'ih>'rf SI. T)e\/elopnte.nt<x\ cxge (_X) versus humera.\ ca\c\umlcoHcxqer\ ract'io DEP I NO CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RS Q VAR VAR A 8 (B) (B) (A) (8) {Y) /*? FCAPH OE AGE 1. 129 0.93840-02 38.14 0.0000 0.1.436 0.1519D-02 0.2128 0.4052 THE ". " AND "*«• ARE USED TO PLOT THE REGRESSION LINE; THE ••*« IS USED WHEN A PLOT POINT COVERS DATA POINTS 2.410 1 1 / / _ . / / 1 . 1 / 1 1 1 / . . 1 / 1 1 1 / 1 11_ _ 1 1_ 1 . _ _ 1 / 1 2.170 - 1 1 . / 1 1  / 1 1 .1 / 1 1 _/_ __ _ _ 1 1 • / ' " 1 ~"" 1 / 1 1 . / / 111 1 / 1 1.930. _ _ _ _ _ _ 1 1 / 1 / . 2 / f a t i a ) I  V ' I . 1 / _/ . ; i . i / * 1.690 - . 1 1 / 1 / 1 I / / / 1 1 / 1 1 / _ __ / 1.450 - 1 / 1 / / /___ . . / / / / 1 1.210 - , _ _ _ _ / / i / // // / / / / / / / / / / / / / / i ////77 7/ I'/i i II II i in ii II II/in 11 II II 11 I\II a II it II ii ii7iii/\ i fin II HI/HI in II i 60.00 76.00 92.00 (dalds) 108.0 124.0 140.0 Exhibit 3&. Develop me*nta.l exqe C*) versus femoral calcium / phos/ohajte: ratio _V) DEP IND CONST .CO EPF FRATIO F PROB STD ERR STD ERR STD ERR RS Q VAR VAR A B < B) < B ) { A) IB) (Y) JSt> HCAPH DEAGE 1. 131 0.96 770-02' 51.62 0.0000 0 .1273 0.1347D-Q2 0.1886 0.4796 THE «.« AND »*« ARE USED TO PLOT THE REGRESSION LINE; THE »*« IS USED WHEN A PLOT POINT COVERS DATA POINTS 2 .400 ~~- I I ~. / 1 / 1 1 1 1 / / / / 1 1 1 / 1 11 / 2.180 / / / 1 . 2 / 1 1 1 / 1 1 / 11 / / 1__ 7 : i " T T / i i 1.960 - 1 gratia) / / 1 / . 1 / / 1 1 . /_ _ 7 " ~ ~ . / / . 1 1 .740 / • 1 / F ~ i n / / / 1 . 520 / / / / / / / 1 .300 //I///////////////////!///////////////7/7/177777//////////////I 7/777 /77///////////I//7/////7////////771~~ 60.00 76.00 92.00 t***^) 108.0 124.0 140.0 &th~ib'.T 33. Develop ry>enta\l age £>0 versus humeral CO Icium j phasphatez rectio _V) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B (B) (B) (A) (B) (Y) M-WEI M-HEI -233.0 3.127 17.45 0.0001 122.6 0.7486 35.01 0.2376 THE AND M*« ARE USED TO PLOT THE REGRESSION LINE; THE «*'• IS USED WHEN A PLOT POINT COVERS OATA POINTS 392 .0 / L / t I 355. 0 / / / / / / / / / / / 318.0 4*9)-/ / 7 / / / t / / / 2 81.0 / / / / 1 244.0 I / / / / 1. 11 / / / / / / 207.0 / / / I / / / / / / / / / / / / ///////I / / / / / / / / / / / / / / / / / / / j - / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I 146.0 153.0 160.0 167.0 174.0 181.0 Exhibit 2*1. Nare.fn<x\. height (v) u_r__s rnaYema.\ u/eighT _Y) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B «B) (B) (A) (B) (Y) M-PAR M-AGE -2.860 0.1641 50.04 0.0000 0.6952 0.2320D-01 1.463 0.4719 THE AND ARE USED TO PLOT THE REGRESSION LINE; THE »*« IS USED WHEN A PLOT POINT COVERS DATA POINTS 9. 000 / / / / / / / / 7 .200 / / A / / / 5. 400 / / (r>r> ch'tleLten) / / / / / / 3.600 / / / / / / / / _ / " '" 1 2 1. 800 f I / 1 1 1 . 1 / / / / / 0. 19 07E-05 - 1 1 2 3 . 4 4 2 1 2 2 / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / ) / / / / / / / / / / / / / / / / / / / ! / / / / / / / / / / / / / / / / / / / I / ciears.) 14.00 21.00 28.00 3 35.00 42.00 49.00 JExh'ib'iT 3S- Na.ferr>aA aae. C*} versus. matema.\ par.Tu _Y^ ) DEP IND CONST COEFF FRATIO FPROB STO ERR STD ERR STD ERR R SQ VAR VAR A B (B) (B) (A) <B. (Y) ^ s M-GRA M-AGE -2.213 0.1912 55.40 0.0000 0.7698 0.2569D-01 1.620 0.4973 THE " ." AND ARE USED TO PLOT THE REGRESSION LINE; THE »*" IS USED WHEN A PLOT POINT COVERS DATA POINTS 10. 00 / / / / / / / 8. 200 / / / / / 6 .400 / / 7 l l J_ , : / " / 1 1 1 . 2 / 4.600 / 1 1 2 1 1 1 2 1 1 2 / / / / / . _____ 7 1 2 1 1 1 1 2 2 .800 / / _/ / / / / / 1.000 - 1 1 2 .2 4 4 2 1 1 2 2 1 1 / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 14.00 2 1. 00 2 8.00 ^ 35.00 42.00 49.00 Exh'ih'iT 34>. /iat-ema.\ aqe (Lfecws) versus mexterrto.i Cfr&uidiTcf _V_) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSO VAR VAR A B (B) {B) (A) (BJ IY) 'SV M-GRA M-PAR 1.406 1.011 215.4 0.0000 0.1874 0.68890-01 1.038 0.7937 THE AND «*« ARE USED TO PLOT THE REGRESSION LINE; THE «*" IS USED WHEN A PLOT POINT COVERS DATA POINTS "A" REPRESENTS 10 OR MORE DATA POINTS 10.00 / / / / / / _/_ / 8. 200 / / /_ / / / 6. 400 / / / / t 4. 6 00 / / / 2 / / / / _/ / 2. 800 / / / 3 / / / / . / 1 .000 - A / / I / / / / / / / / / / / / / / / / / / / j / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I (no. children) -0.0 1.800 3.600 5.400 7.200 9.000 Exhibit £7. Na.tetna.1 par'tfy (*) Versus rna+erna.1 arai/ictitij ( V) DEP I NO V AR V AR SESGP SESSC THE AND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ A. B ( B) (B) (A) (BJ <YI 0.9613 0.5070D-O1 202.2 0.0000 0. 1274 0. 3566D-02 0.3467 0.7831 »*" ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS ~z r 4.000 / / / / / / J_ I 3 .300 / / /_ / / / 1 6 1 3 2 2 4. 3 1 1 2.600 / / / / / / J_ / / / 1 1 * 3 2 8 4 1.900 / / / / / 1. 200 / / / > / / 0.5000 / / //!/////////////7///7/1 /'////////1 ///////// \ ///////////////////!///////////////////!////////////////// / I 5.000 18.00 31.00 44.00 57.00 70.00 Exhih'iT JS. Socio-economic* Score (x) Versus socio-econoryiicj group _V) DEP IND CONST COEFF FRATIO FPR08 STD ERR STD ERR STD ERR RSQ VAR VAR A B (8) <B) (A) <B) ( Y) N-WSC N-TOT 6.160 0.1670 342.0 0.0 0.8297 0.9027D-02 1.364 0.8593 THE AND "*•» ARE USED TO PLOT THE REGRESSION LINE," THE IS USED WHEN A PLOT POINT COVERS DATA POINTS 29. 00 / / / / 1 1 1 25 .60 / / / / / 11 / 1 / / _ j_ j j 1* 1 1 / — * / / _ j ^2 i n i / I . i n I 22 .20  / / 1 1 1 1111 1 1 18 .80 / _/ *_2 11_ / / / 1 1 15.40 / / / / / / / / / 12.00 / //I / ////////////7//"///1 nn ftu//////nun ///////f///////////1///////////////////I/ //////////////////1 30. 00 5 0. 00 70.00 90 .00 110. 0 130.0 ExhibiT 39. Total nutrir,on score, i*) versus weighted nutrition score C V) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A B IB} (B) (A) IB) (Y) /S? N-IDX N-TOT 1.050 0.2762D-01 328.5 0. 0 0.1401 0.1524D-02 0.2303 0.8544 THE AND »*" ARE USED TO PLOT THE REGRESSION LINE; THE «*» IS USED WHEN A PLOT POINT COVERS DATA POINTS 4. 800 4.200 / /_ / / _/_ / / t_ / 1 1 1 1 1 3 .600 3.000 2. 400 / / / / / J_ / / T i j_ / / j / / j_ / / / / ]_ / / _/_ / / / _/_ / / j_ / / i i i 2 2 1 1 2 . 2 1 * 1 1 1 . 2 1 1 1 111 1 1 1 1111 1 1 .2 2 1 1 T T 1 . 1 1 .800 30 .00 50 .00 70.00 90. 00 110.0 130.0 £:xniJb/T */0 • ToTa.1 nut'rit-/on score _V) uersus nutrition 'index. _W DEP IND CONST COEFF FRATIO FPROB STD E*R VAR VAR A B ( B) (B) (A) N-IDX N-WSC 0.2448E-01 0 .1657 0. 2469D 05 0. 0 0.2262D-01 THE ANO ARE USED TO PLOT THE REGRESSION LINE; THE »* M, IS USED WHEN "A" REPRESENTS 10 OR MORE DATA POINTS STD ERR STO ERR RSQ (B) ( Y) 0.1055 0-02 0.28710-01 0.9977 A PLOT POINT COVERS DATA POINTS 4.800 4.200 3.600 3. 000 2. 400 / / 7 / J_ / / j_ / / / f_ / / _/_ / / T / j_ / i j_ / t ]__ / j_ / / j_ t / _/ 7 f f j 7 j_ / / T T 3. . 7 . 7 2. 1 .800 12 .00 15.40 1 8. 80 22. 20 25.60 29.00 Exhibit 47. Nutrition index. ( V*) ver&us ivemhtecL nutrition score _ X) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STD ERR R SQ VAR VAR A B < B) < B) ( A ) I B ) ( Y , N-PRO N- TOT -7. 749 0.3947 43.59 0.0000 5.495 0. 59780-01 9.033 0.4377 THE ".« AND ARE USED TO PLOT THE REGRESSION L I N E ; THE " * « • I S USED WHEN A PLOT P O I N T COVERS DATA P O I N T S /S? 48 .00 L t I T IT / / J_ / 39.00 / / / / / / 11 1 30 .00 / / 7 / / l l / / / / / l l i l l l l i r 21 .00 / __ / / / u / / / 7 I 2; oo i I I / / / / / / 3.000 //| ////'////////////// / I / / / / / / / / / / / / / / / / / / / j / ////////////////// I////////////////// / j / / / / / / / / / / / / / / / / / / / " I 30.00 50.00 70.00 90.00 110.0 130.0 Ejth\b\r . ToTal nutrition score (X} versus protein score DEP I NO CONST COEF F FRATIO FPROB STD ERR STD ERR STD ERR RSQ VAR VAR A 8 (B) (B) (A) (B) if) N-WSC N-PRO 16.39 0 .17.18 26.83 0.0000 0.9983 0. 3317D-01 2.990 0.3239 THE AND •»*" ARE USED TO PLOT THE REGRESSION LINE; THE "*« IS USED WHEN A PLOT POINT COVERS DATA POINTS /60 29.00 / J f / I / / 2 5. 60 / / / / / / 1 1 " i r 22.20 / / / 1 1 1 / / / / / / 1 1 1 1 1 . 18. 80 / / / / / T I JL / 1 1 1 1 15. AO / / / r / f / / 12.00 - 1 . / / j / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / | / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I / / / / / / / / / / / / / / / / / / / I 3.000 12.00 21.00 30.00 39.00 48.00 Exhibit 43. Protein score _X) versus lAie'iqhtecL nutrTrion score, (j?) DEP IND CONST COEFF FRATIO FPROB STD ERR STD ERR STO ERR RSQ VAR VAR A 8 (B) (B) {A) (B) ( Y) N-IDX N-PRQ 2.733 0.2876D-01 27.54 0.0000 0.1649 0.5480D-02 0.4941 0.3296 THE AND ARE USED TO PLOT THE REGRESSION LINE; THE IS USED WHEN A PLOT POINT COVERS DATA POINTS 4 .800 / L / / / / t f 4.200 / / / 1 1 / 1 1 1 1 1 3.600 / / T f f i l l / / i i / / / / 3.000 - . 1 1 1 1 1 1 / / / / / / / / 2.400 / / / / / / 1 1. 800 / / I / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / i 7 / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / i / / / / / / / / / / / / / / / / / / / j 3.000 12.00 21.00 30.00 39.00 48.00 jZxhibir*/?. Protein score, (x) versus nutrition index. ^V} 

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