RELAXIN AS AN AETIOLOGICAL FACTOR IN DIABETIC EMBRYOPATHY

1428

clear: mixed lymphocyte culture was negative, there was no infection, no blood products from the donor had been transfused, and cytotoxic antibodies were absent. One possible reason for the graft rejection is that the donor was the father. In the other cases, in which a compatible sibling was used as donor, engraftment was rapid and sustained. The number of children in this series is small and the follow-up is too short for definite conclusions to be drawn. We believe, however, that BMT offers a chance of cure for young patients with severe SCA who must live in countries where excellent conventional care is not available.

TABLE III-SHORT-TERM FOLLOW-UP I

I

I

*After second BMT.

We thank Dr M. De Bruyere and Dr D. Latinne for immunohaematological studies; Dr F. Richard for irradiation; and Prof J. Sonnet for electrophoretic studies of haemoglobin.

TABLE IV-HAEMATOLOGICAL FOLLOW-UP I

I

I

I

Correspondence should be addressed to G. C., Department of Paediatrics, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 1(?--B-1200 Brussels, Belgium. REFERENCES

R, Holbrook T. Sickle cell disease. Ways to reduce morbidity and mortality. Postgrad Med 1987; 81: 265-80. 2. Lucarelli G, Galimberti M, Polchi P, et al. Marrow transplantation in patients with advanced thalassemia. N Engl J Med 1987; 316: 1050-55. 3. Piomelli S, Lemer N, Cohen A, et al. Bone marrow transplantation of thalassemia. N Engl J Med 1987; 317: 964. 4. Johnson FL, Look AT, Gockerman J, et al. Bone marrow transplantation in a patient with sickle cell anemia. N Engl J Med 1984; 311: 780-83. 5. Gini EK, Sonnet J. Use of piracetam improves sickle cell deformability in vitro and in vivo. J Clin Pathol 1987; 40: 99-102. 6. Storb R, Weiden PL, Sullivan KM, et al. Second marrow transplants in patients with aplastic anemia rejecting the first graft: use of a conditioning regimen including cyclophosphamide and antithymocyte globulin. Blood 1987; 70: 116-21. 7. Nzilambi N, De Cock KM, Forthal DN, et al. The prevalence of infection with human immunodeficiency virus over a 10-year period in rural Zaire. N Engl J Med 1988, 318: 276-79. 1. Thomas

engraftment second BMT

confirmed, rejected her graft; a performed on day 62, with uneventful

had been was

recovery.

Outpatient Follow-up The follow-up ranges from 3 to 21 months (median 10 months). All five children are in good general condition and none has had a vaso-occlusive episode or needed blood transfusion since BMT. The electrophoretic pattern of the haemoglobin became similar to that of the donor (two AA, three AS) and in three cases (patients 2, 4, and 5) cytogenetic studies confirmed the donor origin of bone marrow cells (table IV). 12 months after BMT, patient 1 became thrombocytopenic and autoimmune thrombocytopenia was diagnosed by bone marrow examination, detection of antiplatelet antibodies, and isotopic studies showing splenic sequestration and a shortened platelet half-life (25 days). This patient was successfully treated with steroids and azathioprine. No other patient had chronic GVHD.

Hypothesis RELAXIN AS AN AETIOLOGICAL FACTOR IN DIABETIC EMBRYOPATHY

J. R. G. EDWARDS

D. R. NEWALL

Cleft Palate Research Unit, University of Newcastle upon Tyne

DISCUSSION

For children with sickle cell anaemia who live in developed countries, the quality of life is good with preventive therapy (piracetam, folic acid, and vaccination against Streptococcus pneumoniae). In developing countries, however, the morbidity and mortality are much higher Vaso-occlusive episodes, infections, splenic autoinfarction, and chronic organ damage are common.’ The mortality after BMT is still as high as 25%;2so, is such treatment justifiable in SCA? Severity of SCA in a particular child is very difficult to assess. In those with SCA and a thalassaemic trait, the concentration of fetal haemoglobin is sufficient to make vaso-occlusive episodes a rare event; but in those with pure SCA we believe that the incidence of vaso-occlusive episodes is a good indicator of severity. The children we treated by BMT did not yet have chronic organ damage, but we could expect them to do so with four vaso-occlusive episodes a year, even with decent medical care in Zaire. Avoidance of blood transfusions is another important point in a country with a high incidence of human immunodeficiency virus infections.7 In our small series, one patient rejected the first graft but was successfully regrafted from the same donor. The reason for the rejection is not

Relaxin, an insulin homologue, has effects collagen resembling those of certain teratogenic agents. It is suggested that diabetic

Summary

on

embryopathy could be due to disturbances secretion during fetal organogenesis.

of relaxin

INTRODUCTION

IN diabetic pregnancy the fetus is

at excess

risk of

congenital malformations, stillbirths, and neonatal deaths. 1-5 Although the incidence of stillbirth and perinatal death has decreased as control of diabetes in pregnancy has improved, the incidence of malformations has not decreased6 and may have increased.7 Much experimental work has been undertaken to elucidate the cause in mammals. Reviewing it, Mills8 concluded that hyperglycaemia may be important, but that neither insulin nor hypoglycaemia is teratogenic in man. In chick and duck embryos, however, insulin administered into the yolk sac produces a constant and repeatable absence of tail vertebrae (rumplessness) and abnormalities of the facial skeleton, attributed by Landauer9 to direct impingement of insulin upon cells of the primordia of specific parts.

1429 or near term the mammalian placenta is impermeable insulin. In the rat it is also impermeable during the period of organogenesis, 10 and in man both at termil and at sixteen weeks.12 In the fetal rat organogenesis takes place between days eight and sixteen13-before the appearance of fetal insulin.14 In man Mills and co-workers 15 deduced that diabetic malformations must arise before week seven post-conception. Pancreatic B cells are not present before the tenth week14,16 and extractable insulin until twelve weeks.17 Thus insulin, whether maternal or administered or produced by the fetus, cannot be the cause. Let us therefore consider an alternative explantion, based on the notion that insulin-like substances are present in the feto-placental unit at an appropriate stage in embryogenesis. In biological terms a "like substance" is usually one that has similar

At

to

physiological, pharmacological, or pathological connotations (ie, an analogue). What we have in mind, however, is an insulin homologue-namely, relaxin. PROPERTIES OF RELAXIN

Relaxin

(RLX) is related to insulin through a common ancestral polypeptide resembling pro insulin.18 It has a molecular weight of about 6000, and consists of two non-identical chains of aminoacids, joined by two disulphide bridges, which correspond exactly to those of insulin; the conformation is also similar. Two complementary mutations are necessary to accommodate the sequences in the same type of folding arrangement.’9 Since RLX is a hormone of pregnancy its chemistry and its influence on the physiology of the female organs of reproduction have attracted much attention. 19-22 The usual source is the corpus luteum of pregnancy but it is also found in the decidua and term placenta. RLX also enhances secretion of collagenase and plasminogen from dispersed amnion and chorion cell cultures in vitro. This property may lead to collagen degradation in fetal membrane and eventual rupture at

parturition.23 In the pubic ligament of pregnant guineapigs it causes relaxation with breakdown of fibrous elements.24 There is decomposition of collagen25 causing an abrupt transformation of ligamentous connective tissue from colloid-rich, water-poor, to colloid-poor, water-rich .26-2’ RLX influences the distensibility of the nonpregnant uterus in ovariectomised animals maintained on oestrogen but has little effect on its own; for maximum effect and greatest increase in the collagen framework, it must be combined with oestrogen and progesterone in accurate dosage.28-31 Since collagen is not distensible under normal physiological conditions the increase of the framework is possibly accompanied by an increase in elastin, reorientation of fibre bundles, or modification in the cross-linkage of collagen fibres. As yet, there are no published reports on the effects of RLX on the conceptus. CONTROL OF RELAXIN PRODUCTION

In pregnant women one stimulus to RLX production is chorionic gonadotropin (hCG).32,33 The serum profiles of RLX and hCG are similar;34 moreover, in non-pregnant women injection of hCG during the late follicular phase of the menstrual cycle causes RLX secretion.35 Progesterone must also be considered. In women having caesarean section a linear relation was found between progesterone and RLX concentrations in the serum of blood from the ovarian vein on the side of the corpus luteum.36 There is considerable species difference in serum RLX levels in pregnancy; in the pig a 70-fold increase occurs in the last 24 hours but in women it reaches its peak during the first trimester.’7 This difference raises the possibility that in animals RLX is mainly a hormone of pregnancy whereas in man its effects are related more to fetal

development. RELAXIN A

an

TERATOGEN?

Let us consider the known functions of RLX in relation to extensively studied aspect of development-that of the

embryonic palate. During development the palatal shelves undergo reorientation from their initial vertical position lateral to the tongue to a horizontal alignment cephalad to it and, coming into contact with each and (where relevant) with the nasal septum, fuse to form the secondary palate.38. 41 Swelling of the shelves is generally attributed to production of sulphated proteoglycans in the matrix, and shelf remoulding and direction of movement is physically controlled by concomitantly developed collagen, arranged in a specific way. Experimentally, cleft palate may be produced by drugs that inhibit proteoglycan synthesis, such as cortisone, chlorcyclizine, diazonorleucine, vitamin A, and 5-fluoro-2-desoxyuridine and also by those that cause of fetal abnormalities collagen, of which 0 has been the most extensively (BAPN) aminoproprionitrile studied. BAPN exerts its effect by preventing cross linkage of collagen and elastin by blocking the lysine-to-allysine conversion.’2 This effect is not limited to the palate but involves the whole embryo. Contractile elements, actin and myosin, are synthesised in the palate at the time of shelf movement and it has been suggested that these might be a factor in shelf movement. RLX suppresses the biological parameters of uterine contraction in vitro and inhibits the contractile response of isolated uterine muscle to

prostaglandin F2cl’ When acting with specific

quanta of oestrogen and RLX has actions on collagen that resemble progesterone, those of substances producing cleft palate in laboratory animals. If in normal circumstances a high RLX secretion rate is avoided during the stages of organogenesis, one can imagine that disharmony between the three hormones could give rise to numerous malformations other than cleft palate. Azetidine-2 carboxylic acid, a proline analogue and collagen inhibitor in the chick embryo, decreases epithelial budding in the mouse embryonic lung in vitro;44 and, in pregnant rats on a copper deficient diet, orally administered D-

penicillamine (like BAPN, a lathyrogen) can produce pulmonary abnormalities, cleft palate, and vertebral defects.45 The changes in collagen and the consequent skeletal malformations caused by BAPN are drastically reduced if B hydroxyethylrutosides, believed to protect the activity of lysyl-oxidase, are given immediately afterwards.’6 RLX may have a comparable lathyrogenic effect in man and be a factor in the aetiology of the pulmonary morbidity and ’ mortality in children born to diabetic mothers and account for the severe pulmonary abnormalities often present in spontaneously aborted fetuses that have cleft lip and palate.4’ It may also be the mode of gene expression in cystic fibrosis, and contribute to neural tube defects. Collagen is necessary for the growth of non-tumorigenic cells and for their migration4’ and is important in cell growth.49 Thus it is possible in diabetic embryopathy that the influence of RLX on collagen formation may be the initial pathological process and that involvement of the matrix is a secondary phenomenon. What evidence is there of hormonal disturbances in diabetic pregnancy that might give rise to embryopathy in the way we suggest? Among diabetic women studied before the tenth week of pregnancy Jovanovic et apo found that those in whom diabetes was not well controlled had oestradiol, prolactin, and hCG levels below the range associated with normal pregnancy and that these rose to normal after ultra-careful control of the diabetes with insulin. It is possible therefore that administered insulin, while correcting maternal plasma glucose may, by raising circulating hCG and oestrogen concentrations and thus

1430

RLX, trigger a teratogenic pathway in certain predisposed

embryos. Thus minor changes in fetal endocrine status could be the "disastrous malformation factor" of the Copenhagen school.’ Future research into the biological nature of many structural congenital malformations could, with advantage, be directed towards investigation of the endocrine status of the fetoplacental unit and the influence upon this of the mother’s endocrine status and of the drugs she receives. Correspondence should be addressed to J. R. G. E., Cleft Palate Research Unit, University of Newcastle upon Tyne, 1-4 Claremont Terrace, Newcastle upon Tyne NE1 7RU.

Reviews of Books Preventing Famine: Policies and Prospects for Africa D. Curtis, M. Hubbard, and A. Shepherd. With contributions from E. Clay, H. and C. Goyder, B. Harriss, R. Morgan, and C. Touhnin. London and New York: Routledge. 1988. Pp 250. 30.00. ISBN 0-415007119.

RECURRENT famines are the darker side of the image "out of Africa" over the past decades. The future looks much the same. This very useful book sets out to question why it should be so, at a time when globally the world undoubtedly has the communication and transport resources, as well as the food reserves, to make such disasters preventable. But it is not only a book of analysis. Those faced with the realities of looking for solutions in the field will find a large number of ideas discussed which are relevant for governments, aid agencies, and local groups. Case studies of famines in the Sudan, Ethiopia (1984-85), and Botswana are contrasted with responses to famine in the different situations of India and Bangladesh. The analyses of the Sudan and Ethiopian experiences are the best summaries of events I have seen, and I would single out the handling of the resettlement and villagisation movements in Ethiopia as a balanced account of difficult issues. The authors conclude that, paradoxically, the result of Ethiopian rural policies has been only to increase dependence on

coming

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333-72. 25. Chihal HJ, Espey LL Utilization of the relaxed symphysis pubis of guinea pigs for clues to the mechanism of ovulation. Endocrinology 1973; 93: 1441-45. 26. Crelin ES The development of hormonal response of the autotransplanted interpubic joint in mice Anat Rec 1963, 146: 149-57. 27 Steinetz BG, Manning JP, Butler M, Beach V Relationship of growth hormone, steroids and relaxin in the transformation of pubic joint cartilage to ligament in hypophysectomized mice. Endocrinology 1965; 76: 876-82 28. Hisaw FL Jr, Hisaw FL. Effect of relaxin on the uterus of monkeys ( Macaca mulatta) with observations on the cervix and symphysis pubis. Am J Obstet Gynecol 1964; 89: 141 29. Schofield BM. The increased extensibility of pregnant myometrium. J Physiol 1966; 182: 690-94 30 Wiqvist N. Immediate and prolonged effects of relaxin on the spontaneous activity of the mouse and rat uterus Acta Endocrinol Sup 1959; 46: 15-32 (suppl) 31. Cullen BM, Harkness RD Effects of ovariectomy and of hormones on collagenous framework of the uterus Am J Physiol 1964; 206: 621-27 32 Sherwood OD. Regulation of relaxin levels in the peripheral blood during pregnancy and at parturition in pigs, rats and human beings In-vivo studies. In Bigazzi M, Greenwood FC, Gasparri F, eds. Biology of relaxin and its role in the human. Amsterdam Excerpta Medica, 1983. 219-33 33. Goldsmith LT, Weiss G The control of relaxin secretion: In-vitro studies In Bigazzi M, Greenwood FC, Gasparri F, eds. Biology of relaxin and its role in the human Amsterdam Excerpta Medica, 1983 234-43

Quagliarello J, Szlachter N, Steinetz BG, Goldsmith LT, Weiss G. Serial relaxin concentrations in human pregnancy. Am J Obstet Gynecol 1979, 135: 43-44. 35. Thomas K, Loumaye E, Ferin J. Relaxin in non-pregnant women during ovarian stimulation. Gynaecol Obstet Invest 1980; 11: 75-80. 36. Weiss G, O’Byrne EM, Steinetz BG. Relaxin: a product of the human corpus luteum 34.

of pregnancy. Science 1976; 194: 948-49. 37. Eddie LW, Lester A, Bennett G, Bell RJ, Geier M, Johnston PD, Niall HD. Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin Lancet 1986, i. 1344-45. 38 Greene RM, Pratt RM. Developmental aspects of secondary palate formation J Embryol Exp Morphol 1976; 36: 225-45. 39. Fraser FC. Animal models for cranio-facial disorders. In Melnick M, Bixler D, Shields ED, eds Aetiology of cleft lip and cleft palate New York Liss, 1980 1-4. 40. Diewerts WM. The rate of craniofacial growth in palatal shelf elevation In. Pratt RM, Christansen RL, eds. Current research trends in prenatal craniofacial development. Amsterdam: Elsevier, 1980. 165-86. 41. Ferguson MWJ. Developmental mechanisms in normal and abnormal palate formation with particular reference to the aetiology, pathogenesis and prevention of cleft palate Br J Orthodont 1981; 8: 115-37. 42. Pinnel SR, Martin GR. The cross-linking of collagen and elastin Enzymatic conversion of lysine in peptide linkage to allysine by an extract from bone Proc Natl Acad Sci USA 1968; 61: 708-14. 43. Sanborn BM, Nishikon NW, Weisbrodt NW, Sherwood OD. Relaxin affects uterine myosin phosphorylation and related activities. In. Bigazzi M, Greenwood FC, Gasparri F, eds. Biology of relaxin and its role in the human. Amsterdam Excerpta Medica, 1983. 125-27. 44. Alescio T. Effect of a proline analogue, azetidine-2-carboxylic acid, on the morphogenesis in vitro of mouse embryonic lung. J Embryol Exp Morphol 1973, 29: 439-51. 45 Kilburn KH, Hess RA Neonatal deaths and pulmonary dysplasia due to Dpenicillamine in the rat. Teratology 1982; 26: 1-9. 46. Joneja MG, Wiley MJ. Inhibition of &bgr; aminoproprionitrile (BAPN) induced skeletal terarogenesis by the flavanoid B-hydroxyethylrutosides (HR) in hamster foetuses.

Teratology 1982, 25: 59-63. H, Kraus BS. Visceral variations and defects associated with cleft lip and J palate in human foetuses a macroscopic description. Cleft Palate 1964; 1: 99-115 Postlethwaite AE, Seyer JM, Kang AH Chemotactic attraction of human fibroblasts to type I, II and III collagens and collagen-derived peptides Proc Natl Acad So

47. Kitamura 48.

USA 1978; 75: 871-75. 49. Klemman HK, Hewitt AT, Grotendorst GR, et al. Role of matrix components in adhesion and growth of cells In. Pratt RM, Chnstiansen RL, eds Current research trends in prenatal craniofacial development. Amsterdam. Elsevier, 1980 277-95 50. Jovanovic L, Peterson BB, Saxena BB, Dawood YM, Saudek CD. Feasibility of maintaining normal glucose profiles in insulin-dependent pregnant diabetic women. Am J Med 1980; 68: 105-12