AMNIOCENTESIS DYSMELIA IN RATS

Br. J. exp. Path. (1972) 53, 435

AMNIOCENTESIS DYSMELIA IN RATS

A. M. LOVE AND T. H. VICKERS

From the Medical School, University of Queensland, Herston, Queensland, Australia 4006

Received for publication April 25, 1972

Summary.-Amniocentesis performed on Day 15j of gestation caused dysmeliain 85 of 143 offspring of Sprague-Dawley and Wistar strain rats. Almost alldefects were of reduction type, only 3 hind limb postaxial supernumerary digitsbeing seen. Defect intensity graded from longitudinal splitting of the phalanges,through phalangeal suppression, to very severe growth retardation of thelong limb bones and the limb girdle. The lesions in the 2 strains were indis-tinguishable from each other. Subcutaneous haemorrhages were frequentlypresent and could take the form of blebs. Hind limbs were more frequentlyand, on average, more severely affected than fore limbs. Left-sided defectswere more severe than right-sided ones. Sections of tibiae from defects ofintermediate severity showed impaired endosteal, periosteal and endochondralosteogenesis.

The total limb defects may be regarded as a combination of (a) retardedgrowth in long bones, (b) arrested or aberrant differentiation of the bones inthe digital rays and (c) destruction of digital structures by haemorrhage andpossibly localized ischaemic tissue necrosis. Among the associated defectswere cleft palate, anomalies of the abdominal wall, genitourinary tract defectsand short umbilical cord. Some aspects of the possible part played by thechorion and amnion in limb defect production have been reviewed and thetentative conclusion reached that the foetal membranes do not significantlycompress the foetus.

DYSMELIA is one of the more prominent features of the experimental amnio-centesis syndrome in rats. It was first noted by Poswillo and Roy (1965) andPoswillo (1966) following amniotic sac puncture (Trasler, Walker and Fraser,1956) on Day 151 of gestation. They ascribed the anomalies to the pressureexerted on the foetus by the amniotic membrane after the fluid had escaped.Kendrick and Field (1967) confirmed the observation and showed that the natureand incidence of the lesions were not significantly altered by adrenalectomy sothat they could not be regarded as a corticosteroid effect, the result of a maternalstress reaction. DeMyer and Baird (1969) re-examined the problem and con-sidered that intrauterine immobility consequent on oligohydramnios was thebasis for the anomalies. Most recently Poswillo and Sopher (1971) havesuggested that hypervolaemia or hypoxia, by leading to foetal hypertension,play some part in defect induction.

In the absence of more detail about the form of these defects, it seems to uslittle better than speculation to discuss their mechanism. The present reportaims at providing more of this background information.

MATERIALS AND METHODS

Sprague-Dawley and Wistar rats drawn from closed colonies maintained by randombreeding were used. The animals were mated overnight and those showing spermatozoa in


vaginal smears the following morning were considered to have been fertilized at 06.00 hour.Food and water were provided ad libitum.

Amniocentesis under direct vision using a 0 7 mm diameter needle was performed at14.00 hour (368 hour gestation) under ether anaesthesia. Two techniques were employed.The uterus was either pierced right through, producing entry and exit holes in the amnion(Technique A), or it was punctured once right through and the membranes were abraded fromthe exit to the entry hole as the needle point was withdrawn (Technique B). In the firstmethod gentle pressure on the uterus for 5 seconds facilitated escape of the amniotic fluid;no pressure was applied following membrane abrasion. The foetuses in only one horn weretreated, the opposite side acting as control. The side to be treated was determined beforeoperation using a table of random numbers. The young were taken at 21 days gestation,the examination including methylene blue staining of the cartilaginous models of the bones,with alizarin preparations and paraffin sections in selected animals.

RESULTS

Amniocentesis (Technique A) was employed on 19 Sprague-Dawley and 19Wistar mothers and all but 3 had viable young in the treated horn at full term.Tables I and II summarize the general findings. The procedure caused death in

TABLE 1.-Effect of Amniocentesi8 on Foetal SurvivalViable foetuses Viable foetusesat laparotomy at term (%)

Rat Pregnant A rA

strain does Untreated Treated Untreated TreatedSprague-Dawley 18* 92 92 90 (98) 62t (67)

Wistar 19 116 124 116 (100) 81t (65)Total 37 208 213 206 (99) 143 (67)

* 19 mothers used but limb examination of one litter unsatisfactory.t From 16 mothers as 2 mothers had no viable foetuses in treated horn.t From 18 mothers as one mother had no viable foetuses in treated horn.

TABLE II.-Effect of Amniocentesis on Limb Defect Production inFull Term Treated Foetuses

Rat Number of Dysmelic Foetuses Dysmelicstrain litters litters examined foetuses (%)

Sprague.Dawley 16 14 53 33 (64)

Wistar 18 16 77 52 (68)Total 34 30 130 85 (66)

about one-third of the treated foetuses but there was no significant effect on theyoung in the untreated horn; both strains reacted similarly. Dysmelia waspresent in 66% of the viable treated young distributed among 30 of 34 litters.

TABLE III.-Frequency of Fore Limb Dysmelia and Hind Limb DysmeliaTotal Foetuses with Foetuses with

dysmelic fore limb hind limbRat strain foetuses defects defects

Sprague-Dawley 33 20 32

Wistar 52 25 50Total 85 45 82

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Hind limbs were more frequently involved than fore limbs (Table III). Ofthe 85 dysmelic animals from both strains, at least one hind limb was almostalways abnormal whereas only a half of them had fore limb anomalies. Further-more, in foetuses with both fore and hind limbs affected, hind limb lesions weregenerally more severe than those in the fore limbs (Table IV). It should benoted that the severity rating was subjective and was a composite of the numberof digits involved and the lesion intensity.

TABLE IV.-Defect Severity of Fore Limbs Compared with Hind Limbswhen Both InvolvedHind limbs Hind limbs Hind limbs

more equal to lessRat strain severe fore limbs severe

Sprague-Dawley 8 3 0

Wistar 20 2 0

On comparing lesion frequencies, right and left sides revealed no significantdifference in the Sprague-Dawley animals but a highly significant difference inthe Wistar line (Table V). On the other hand, the severity of the lesions wasgreater on the left side in both strains (Table VI).

TABLE V.-Frequency of Right and Left Sided DysmeliaFore limbs Hind limbs

Foetuses Foetuses Foetuses Foetuses Foetuses Foetuseswith with with with

Rat with right sided left sided with right sided left sidedstrains dysmelia dysmelia dysmelia dysmelia dysmelia dysmelia

Sprague-Dawley 20 16 16 32 26 28

Wistar 25 12 23 50 39 48

TABLE VI.-Comparative Severity of Right and Left Sided DysmeliaFore limbs Hind limbs

A A

Left Left Left Left Left LeftRat > = < > = <

strains right right right right right rightSprague-Dawley 8 0 0 8 3 2

Wistar 6 1 1 23 4 2

The dysmelic pattern was, in general terms, similar in the 4 extremities andskeletal defects could exist even when the paws were externally normal. In suchcases the middle phalanges were longitudinally split, i.e. there was phalangealdichotomy. Dichotomy could occur alone, when 2 discrete bars of cartilagereplaced the phalanx (Fig. 2, digit 4), or it could be combined with synarthrosisof the proximal interphalangeal joint, when the single structure had a bifidextremity (Fig. 2, digits 2 and 3). Deficient chondrification was sometimespresent, especially in the split middle phalanx, even to the point that one cartilagebar might be missing. Pallor of distal and less frequently of other phalanges couldoccur but usually these were normal.

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EXPLANATION OF PLATESGross specimens (viewed from plantar surface unless otherwise stated) and methylene blue

cartilage preparations (viewed from dorsal aspect) are of left hind limbs. Gross photographs andskeletal illustrations each at same magnification with skeletal at slightly higher power.

FIG. 1.-Normal hind paw. (a) Note the length of the digits, the well defined interdigital cleftsand the size and form of the nail areas. Hallux (= digit 1) arrowed. (b) The cartilagemodels of the bones are black, and the ossified areas in the metatarsals are grey and in the longbone shafts are unstained. Compact cartilage blocks comprise the phalanges; the metatarsalheads are rounded. Hallux arrowed.

Fice. 2. Minimal skeletal defect from a grossly normal paw. Middle phalanges of rays 2, 3and 4 are longitudinally split (dichotomy). In digit 4 the 2 halves are discrete structures(long arrow), in digits 2 and 3 they are united basally with their proximal phalanx. Thepreaxial bar in digit 2 is incompletely chondrified (arrow head) as are the phalanges of thehallux and the distal phalanx of ray 5. Small arrow = hallux.

FIG. 3. Minor paw defect. (a) All digits distoproximally shortened, especially marginal pair.Nail areas retracted and interdigital clefts less obvious. Hallux arrowed. (b) All phalangesabnormal. Note especially dichotomy (rays 3 and 4), absent distal phalanx (ray 3), poorstaining of most other phalanges. Observe synarthrosis of preaxial bar of middle phalanxwith proximal phalanx (long arrow). Short arrow = hallux.

FIG. 4. Moderate paw defect. (a) Digits virtually absent but paw pads still recognizable.Note the ill-defined subcutaneous haemorrhage (arrowed). (b) Only proximal phalanges ofdigits 1-4 remain, digit 2 being dichotomous, digits 2 and 4 synarthrosed to their metatarsus.Metatarsal heads of rays 2, 3 and 4 forked. Distal end of metatarsals 1 and 5 tapered.

FIG. 5.-Severe limb defect. (a) Limb a small elevation of tissue without identifiable features(viewed from dorsal aspect). (b) Miniaturization of limb skeleton. Greatly reducedpelvic girdle (short arrow), femur (long arrow) and tibiofibula (small arrow head). Suppres-sion of tarsal bones (large arrow head). Basal parts of metatarsals 1-4 remain and 3 and 4show distal forking. Metatarsal 1 = fleched arrow.

FIG. 6. Polydactyly. The postaxial accessory digit is represented by a single phalanx(small arrow) at level of fifth metatarsophalangeal joint. It was visible in the gross specimen.Point fusion (long arrow) of rays 1 and 2. Terminal phalanx of hallux is united with thedistal end of phalangeal remnants of digit 2. Dichotomy and synarthrosis of rays 3 and 4.

FIG. 7.- Point fusion. Distal end of ray 1 is fused with head of metatarsal 2 (short arrow).Middle phalanges of rays 4 and 5 approximate distally and share a common distal phalanx(long arrow). Proximal phalanx of digit 5 dichotomous. Phalangeal reduction of all rays.

FIG. 8. Digital ray reduction. Distal extremities of reduced rays 1-4 lie along a straightline and the phalangeal remnant of digit 3 (arrowed) is angled (see text). Phalangealdichotomy ray 5.

FIG. 9.-Digit reduction. The distal contour of the paw is rounded on its postaxial side (seetext). Note subcutaneous haemorrhage on digit 4.

FIG. 10.-Full term foetus in membranes. Membranes closely applied to foetus which showsgeneralized flexion. Note enlarged puncture hole in sac overlying left loin through whichskin bulges (arrowed).

FIG. 11. Necrosis of reduced left hindpaw. The distal, opaque, pale slough (arrowed) isdelineated by a hyperaemic margin. Lanceolate deficiency in skin and musculature ofanterior abdominal wall.

FIc. 12.-Haemorrhagic bleb. A haemorrhage in the dorsal tissues of digits 1 and 2 balloonsthe skin. Soft tissue syndactyly (arrowed) between toes 2 and 3.

FIG. 13.-Membrane retraction with foetal herniation. Foetus lay free in uterine cavity;amnion and chorion retracted and form a wrinkled mass on foetal surface of placenta aroundumbilical cord insertion. Note short umbilical cord. Observe also (a) severe hind limbsuppression, (b) hypoplasia of abdominal wall musculature, (c) small malformed genitaltubercle (arrowed) and (d) kinky tail.

FIG. 14.-Incomplete foetal escape from membranes. Foetal membranes still cover the head.Note dysmelia in fore and hind limbs.

FIG. 15. Tibia from control animal. Note length and breadth of tibial shaft and number oftrabeculae in it.

FIG. 16.-Tibia from moderately reduced limb (same magnification as Fig. 15). Bone shorterand narrower than control due principally to the diaphyseal reduction. Shaft trabeculaefewer and irregular.

FIG. 17.-Proximal end of control tibia (higher power view of Fig. 15). Note depth of hyper-trophic cartilage and number and form of bony trabeculae. Osteoblasts are numerous.

FIG. 18. Proximal end of moderately reduced tibia (higher power view of Fig. 16). Comparedepth of hypertrophic cartilage with Fig. 17 taken at same magnification. Shaft trabeculaeare irregular and contain much cartilage. Osteoblasts are few.

FIG. 19.-Phalangeal dichotomy. The middle phalanges of rays 2, 3 and 4 are split; due to theplantar flexion of digits they have been cut transversely. Dichotomous phalanx of digit 2arrowed.

FIG. 20.-Synarthrosis. The tarsometatarsal joint is partially closed (arrowed).FIG. 21.-Point fusion of phalanges. A bar of cartilage (arrowed) unites 2 rays at phalangeal

level.

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The least perceptible gross defect was retraction of the nail areas with shorten-ing of the digits (Fig. 3a). Occasionally the toes were flexed and the interdigitalclefts were less obvious. The accompanying skeletal patterns were similar tothose already mentioned but with a greater tendency for reduction or absenceof phalanges, especially the distal and also those of the marginal rays of the paws(Fig. 3b).

As the severity of the lesions increased and the distal and then middle phalangeswere lost, dichotomy affected proximal bones. In addition, the proximal phalanxof the preaxial and postaxial rays could be absent, in which event the distal endof the corresponding metatarsal bone was tapered (Fig. 4b). With this degreeof deformity, digits were lacking and the distal paw contour was smoothly rounded(Fig. 4a).

When all phalanges were absent, and sometimes when a residue of the proximalphalanx persisted, the metatarsals often showed forking of their distal extremity,which was interpreted as the equivalent of phalangeal dichotomy (Fig. 4b and5b). Comparison of the fore and hind paw lesions showed that dichotomy in thefore limbs was not seen unless all phalanges were missing in the hind.

Once the metatarsal bones were involved, reduction of the long bones of thelimbs appeared. This led to dwarfing of the limb, which Fig. 5a shows in extremedegree. Although little more than blunt elevations of tissue, these stumpspossessed a recognizable femur, tibiofibula and tarsal and metatarsal bones indiminutive form, all as pure cartilage models (Fig. 6b). Growth retardationaffected the pelvic girdle as well. When the skeletal reduction was of a lessergrade, ossification existed in the long bone shafts (Fig. 16).

Polydactyly was seen in 3 specimens. 2 from Wistar and one from the Sprague-Dawley strains; all were postaxial. In 2 the accessory digit was externallyvisible, had a phalanx and was attached at the metatarsophalangeal joint (Fig. 6).The other was represented externally only as fullness on the postaxial margin ofdigit 5; it had 2 imperfectly chondrified phalanges adjacent to the proximalinterphalangeal joint.

Syndactyly, mentioned by Poswillo (1966) and Kendrick and Feild (1967),was found in only 10 deformed paws. In this series it was restricted to softtissues. There was, however, another skeletal anomaly in which contiguous rayswere united distally at a single point. To this lesion the term " point fusion "

has been given, for it cannot be regarded as bony syndactyly in the usual sense ofthe term; Fig. 6 and 7 are examples.

A count of the deformities showed that no ray or group of rays was preferen-tially involved and that it was commoner for more than one ray to be affected ina paw. For the most part, no recurring patterns of digit involvement could bediscerned in paws with multiple lesions, but in a number of limbs there was asuggestion that the intensity of digit suppression was not randomly determined;Fig. 8 displays one such paw. It can be seen that the phalangeal reduction ofdigits 1, 2, 3 and 4 has brought the tips of the truncated rays in line, as if theirdistalward extension had been impeded by some mechanical barrier. Thisimpression is reinforced by the lateral inclination of the phalangeal remnants ofdigit 3. A similar appearance, but with the more usual curvilinear form, isseen in Fig. 9. In this instance it is the postaxial segment of the paw which isaffected.

The severe compression described and illustrated by Poswillo and Roy (1965)

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was not a feature of the foetuses in this series. At most, and then very occasion-ally, the sac was very closely applied to the animal (Fig. 10).

TABLE VII.-Frequency of Haemorrhages in Dysmelic PawsFoetuses Total Limbs

Rat Dysmelic with dysmelic withstrain foetuses haemorrhages limbs haemorrhages

Sprague-Dawley 33 29 86 48

Wistar 52 16 122 23Total 85 45 208 71

Macroscopic haemorrhages in the paws occurred in about a half of the dysmelicanimals and in about one-third of the malformed limbs (Table VII), i.e. sometimesmore than one paw was affected. With an incidence of 56% they occurred morefrequently in Sprague-Dawley paws than in Wistar (19%), and this differencehas significance at better than the 0.01 level. Hind limbs were more often affected,both sides approximately equally. For the most part the haemorrhages weresubcutaneous (Fig. 4a and 9); they were usually ill defined but were sometimessharply circumscribed. In the Wistar strain special attention was paid to thesite of the haematomata. They were more frequent on the dorsal surface alonethan on the ventral surface alone, but about one-third of the paws showedhaemorrhages on both sides. The tissues over and between skeletal rays wereequally affected. In a few paws, the haemorrhages took the form of blebs(Fig. 12).

Occasionally, small masses of opaque slough with an underlying stratum ofhyperaemia capped the tip of severely deformed paws (Fig. 11). Another infre-quent lesion was adhesion of hind limbs to the tail or, in one animal, to loops ofbowel extruded through a deficiency in the umbilicus.

TABLE VIII.-Summary of Effects of Amniocentesis with MembraneAbrasion (Technique B on Sprague-Dauwley Rats)Number of Viable litters Treated Survivors Extruded

litters at term young % young14 10 107 44 (41) 7

Of particular interest was foetal escape from its amnion (Table VIII), acomplication almost restricted to foetuses whose amnion had been abraded.The foetus lay free in the uterine cavity, the retracted chorion and amnion beinga wrinkled mass of tissue surrounding the placental end of a much shortenedumbilical cord (Fig. 13). Most of these foetuses had very severe hind limbdefects. In one animal, the process was incomplete and the head was still en-veloped in the membranes (Fig. 14).

Other associated defects.-Included among the other defects were thickeningof the neck tissues, cleft palate, midline lentiform deficiency in the anteriorabdominal wall centred on the umbilical cord insertion, short umbilical cord,diffuse hypoplasia of the abdominal wall musculature so that the viscera werefaintly visible through the skin, defects of the external genitalia, hydroureter andhydronephrosis and various patterns of tail anomaly.

Histology.-In section, tibiae from moderately reduced limbs were shorter

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and thinner than normal. This was coupled with (a) a reduction in the numberand thickness of the shaft trabeculae, (b) a decrease in the number of periostealand endosteal osteoblasts, (c) probably excessive amounts of residual cartilage inthe shaft, (d) slightly exuberant hypertrophic cartilage although resting andproliferating zones appeared normal and (e) depressed vascularization of thehypertrophic cartilage. In sum, the picture was that of impaired endochondral,endosteal and periosteal osteogenesis. These features are shown in Fig. 16 and18, with Fig. 15 and 17 as a control for comparison.

At the microscopic level dichotomous phalanges were paired masses of cartilageseparated by cells of uncertain nature. The flexor tendons were often split alsoover the length of the dichotomy (Fig. 19).

To exclude the possibility that synarthrosis and point fusion of digital rays,as interpreted in cartilage preparations, were artefacts resulting from super-imposition of bones, their existence was confirmed microscopically. Fig. 20 is anexample showing partial union of a metatarsal with its subjacent distal tarsalbone, while Fig. 21 displays focal continuity between the cartilage of adjacentphalanges.

All haematomata showed surrounding granulation tissue formation and aprominent macrophage reaction. The latter was coupled with storage of degradedhaematogenous pigment.

DISCUSSION

Methylene blue staining of skeletal cartilage has revealed graded patterns ofdefects in the post-amniocentesis dysmelic syndrome. The digit lesion is charac-terized by complete or partial splitting of the phalanges, usually the middle andless frequently the proximal; the distal phalanges can be normal or partially ortotally suppressed. When all phalanges of a ray are missing, the metacarpaland metatarsal bones are distoproximally reduced and their distal end may beforked. In the severest defects, this is associated with miniaturization of thelimb long bones and the limb girdle.

Survey of the literature has provided no assistance in explaining how thelesions of amniocentesis in general, and the limb lesions in particular, arise. Inrespect to the pathogenesis of cleft palate, Trasler, Walker and Fraser (1956)considered that loss of amniotic fluid led to constriction of the foetus. Walkerand Fraser (1957) then advanced the idea that the constriction was due to thecontracting uterus pressing on the embryo. Later Walker (1959) suggested thatit might have been the membranes which compressed the foetus at the criticaltime of palate closure. Poswillo and Roy (1965, 1966) extended this concept byattributing to the amnion elastic properties. They suggested that in the normalstate it stretched like an inflated balloon, so that, when punctured, it deflated toembrace the foetus. Foetal movements were thereby restricted, leading in thelimbs to focal necroses which, with subsequent repair, caused intrauterine ampu-tations, ring constrictions, syndactyly and phocomelia. Kendrick and Feild(1967) raised the possibility that the maternal operative trauma caused cortico-steroid release and that the induced cleft palates, and by inference the limb defectswhich they also noticed, were indentical aetiologically with cleft palate whichfollows corticosteroid injections in mice, rabbits and guinea-pigs. They disprovedthe hypothesis. DeMyer and Baird (1969) postulated that all, or most, defectsfollowing amniotic sac puncture result from restricted movement imposed on the

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foetus by the oligohydramnios. For dysmelia specifically, they hypothesizedthat the short umbilical cord held the limbs in close contact with the amnion andcaused retrogression of them by pressure atrophy or by " a more mysteriouschemical inhibitor manufactured by the amnion which under normal circum-stances would act to prevent adhesion of amnion and foetus ". In their latestcontribution Poswillo and Sopher (1971) have proposed that the dysmelic lesionsresult from hypertensive phenomena provoked by either hypoxia or hypervo-laemia, secondary to changes in the intrauterine physical environment.

It is apparent that most authors link loss of amniotic fluid with development ofthe deformities; the divergence of opinion lies in the pathway of defect induction.The amnion chemical inhibitor theory is only speculative and no factual evidencehas been advanced in support of it. We feel, too, that causally linking dysmeliato a short umbilical cord, as DeMyer and Baird have done, is to attribute to 2lesions a sequential relationship for which there is little material support. Bothare undoubtedly due to oligohydramnios, but whereas the cord shortening reflectsthe reduced mobility of the foetus in the terminal stages of gestation, the limblesions show every sign of dating from the period immediately following sacpuncture.

Walker's (1959) investigation requires extended presentation for it is the onlysystematic study designed to elucidate the influence of the membranes on thethe induction of developmental defects. By comparing the effect of (a) conven-tional amniocentesis with that of (b) amniocentesis plus hysterotomy and (c)hysterotomy without amniocentesis, Walker showed that the first 2 procedurescaused cleft palate in mice and the third no palatal deformity. From this and afurther series of similar experiments, he concluded that (i) cleft palate formationis dependent on loss of amniotic fluid, (ii) compression caused by the uterus is notnecessarily involved in the development of cleft palate and (iii) compression of thefoetus may be due to the amnion-yolk sac membrane. Unfortunately a fourthexperiment to test whether membranes were necessary for cleft palate induction,i.e. release of the foetus from both membranes and uterus, did not provide sufficientsurvivors for conclusions to be drawn. It follows that the part played by themembranes in causing developmental defects is not unequivocally established bythis work.

There is still a fifth experiment which may be performed to investigate therelationship of membranes to defect induction, viz releasing the foetus from itsmembranes but not from the uterus. Obviously, amniocentesis is carried out atthe same time. Although it caused a high mortality and comparatively few ofthe survivors escaped from their membranes, all of them showed limb defectswhich were among the most severe in the series.

A further point bearing on the elastic properties of the foetal membranes atthe time of the operation may be mentioned. It can be confirmed by directobservation that the amnion and chorion do not contract significantly when theyare punctured at Day 151 of gestation. After the fluid escapes they lie in flaccidfolds and there is no evidence of foetal compression if hysterotomy precedes thepuncture. It would seem to us, from this and the evidence of the previousparagraph, that for dysmelia at least membrane pressure is not essential for defectinduction and if it plays any part at all in modelling the full term lesion, it islikely to be a minor one.

By contrast, it is very likely that the haemorrhages seen in full term paws do

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contribute significantly to the genesis of the defects. Large as many are at Day21, they would be relatively and absolutely larger at earlier stages of pregnancyfor organizationi would reduce their size. In this regard, it may be noted thathaematomata are already present 16 hours after sac puncture at a time whenphalangeal organogenesis is still in progress (unpublished observation).

The role of the haemorrhages is probably dual, depending on when and howrapidly they develop. Should they appear early, they would interfere withorganogenesis of the phalanges by disrupting the tissues at the apical parts of thedeveloping digits. On the other hand, their appearance subsequent to phalangealdifferentiation could produce physical destruction of already differentiatedtissues. It is evident that both events may overlap and their end result may besimilar. Pesides local disruptive effects, haemorrhages can lead to tissue necrosisby impeding the blood supply of peripheral and distal structures, and we believethat this is a major factor in slough production.

Nevertheless, we are unable to believe that the haemorrhages could havebeen responsible for the finely sculptured form of phalangeal dichotomy. Theprecisioni with which the phalanges are split, taken in conjunction with theremarkable simyilarity of the lesions in different paws and in different digits of thesame paw, demand as explanation for dichotomy some exquisitely localizeddisturbance of phalangeal differentiation with the middle phalanges the mostsusceptible of the bones. That this susceptibility is real is shown by reference tothe fore and hind limb patterns. Although there is a differential of about 12hours (Milaire, 1965) between fore and hind limb development, with fore limbleading, dichotomy is rarely seen in forepaw distal phalanges even when it ispresent in the more proximal parts of the hindpaw digital rays. By contrast,dichotomy of the middle and proximal phalanges in the forepaw accompaniesthe severer hind limb defects.

The mechanism of phalangeal splitting is uncertain. Tentatively, we suggestthat transient pressure on the growing tips of the digital rays shortly afteramniocentesis possibly lies at its base. The effect may be partly nutritional,resultant on impeded blood supply, and partly mechanical. Mechanical con-straint acting at the free end of the developing digit might also contribute to thevolar inclination which, as has been noted, is frequently present.

Impeded blood supply, but on a wider front, is probably the cause of theoverall dwarfing of the long bones and limb girdles, for there is no evidence thatthey have been disorganized. The process is purely one of growth arrest orretardation. It can be seen in the methylene blue preparations that the cartilagemodels of bones show little advance on the stage of development attained, whenthe ischaemia becomes effective.

There is other evidence that haemorrhages are not wholly responsible forinitiating the defects. Table VII shows that haematomata occurred 3 times asfrequently in Sprague-Dawley young as in Wistar. In spite of this, defectfrequency and severity were comparable in the 2 strains.

Limb malformations with haemorrhages and haemorrhagic blebs follownumerous teratogenic techniques and Petter et al, (1971) have summarized these.Among them is intra-amniotic injection of drugs; for exanmple, Davies and Robson(1970) have reported limb defects after vasopressin, noradrenaline and adrenalineadministered by this route. It is clear that in introducing the agent foetalmembranes must be punctured, with the risk of fluid drainage. If this occurs,

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444 A. M. LOVE AND T. H. VICKERS

amniocentesis may be partly or wholly responsible for any observed anomaly.The possibility of an amniocentesis component has already been acknowledged

in relation to induction of cleft palate by intra-amniotic cortisone. Both Walkerand Fraser (1957), discussing their own experiments, and Kalter and Warkany(1959), reviewing the work of Jost (1956), have commented on it.

Because this element of ambiguity will always exist when a drug is introducedinto the sac mechanically, especially if precautions are not taken to avoid leakage,an indicator of amniotic fluid loss is desirable. In rats, phalangeal dichotomymay provide such an index, if the injection is given around Day 15- and if dysmeliais part of the induced syndrome. It is a very striking lesion and for the reasonsgiven, we believe it is unlikely to be due to the disruptive effect of haemorrhagesper se.

This work was carried out during the tenure of a Medical Postgraduate ResearchScholarship (A.M.L.) granted by the National Health and Medical ResearchCouncil of Australia.

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drenaline on the Mouse Foetus. Br. J. Pharmac., 38, 446.DEMYER, W. & BAIRD, I. (1969) Mortality and Skeletal Malformations from Amnio-

centesis and Oligohydramnios in Rats: Cleft Palate, Club Foot, Microstomia andAdactyly. Teratology, 2, 33.

JOST, A. (1956) The Age Factor in Some Prenatal Endocrine Events. Ciba FoundationColloquia on Aging, 2, 18.

KALTER, H. &; WARKANY, J. (1959) Experimental Production of Congenital Malformationsin Mammals by Metabolic Procedure. Physiol. Rev., 39, 69.

KENDRICK, F. J. & FEILD, L. E. (1967) Congenital Anomalies Induced in Normal andAdrenalectomized Rats by Amniocentesis. Anat. Rec., 159, 353.

MILAIRE, J. (1965) Organogenesis. Ed. R. L. DeHaan & H. Ursprung. New York:Holt, Rinehart and Winston. p. 283.

PETTER, C., BOURBON, J., MALTIER, J.-P. & JOST, A. (1971) Production d'Hemorragiesdes extremites chez le Foetus de Rat soumis 'a une Hypoxie in Utero. C.R. Acad. Sc. (Paris), 272, 2488.

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