evaluation10

Ralph Gräsbeck, together with Olga Imerslund famous finder of hereditary cubilin failure with cobalamin deficiency, here describes the state of the art of infertility and deficiency of micronutrients. He suggests that every case of infertility in primary health care should initially be managed by test treatment with micronutrients, before reproduction specialists are consulted (cf Case Reports No 4,6).

Testis biopsy from a patient with aspermia caused by B12 deficiency due to infestation with fish tapeworm (diphyllobotrium latum)		Testis biopsy from the same patient demonstrating recovery of sperm production after treatment

A review. Deficiency of both vitamin B12 and folate may cause infertility. The vitamins are needed for the replication of DNA and failure to replicate causes maturation arrest. Spermiogenesis and cellulopoiesis in the endometrium suffer in ways analogous to haematopoiesis. Poor diet, malabsorption, fish tapeworm and antagonistic drugs may be responsible. A recently discovered frequent cause of infertility is a mutation in the gene encoding methylenetetrahydrofolate reductase. This is accompanied by increased plasma concentrations of homocysteine. Folate therapy may be efficient. Infertility is probably also caused by analogous mutations in other genes encoding enzymes and transport factors connected with the antimegaloblastic anaemia vitamins. Attention is called to the recently discovered multiligand membrane-associated factors cubilin and megalin which influence not only the membrane transport of vitamin B12 but also other fertility-related factors.

In deficiency of either folate or vitamin B12 (cobalamin) the formation of new cells is inhibited, and signs first appear in tissues with rapid cellulopoiesis. The cause is that the replication of DNA is inhibited whereas the syntesis of RNA and protein is not at all or only slightly affected. The condition is called maturation arrest and it is characterised by large cells that do not divide. Thymin (or more correctly thymidine or thymidylate) fails to receive its methyl group. The methyl donor is a form of folate, and therefore, folate deficiency causes disturbed synthesis of DNA. Lack of cobalamin gives the same disturbance, but indirectly via folate, which is trapped in an inactive form. Lack of cobalamin also causes neurological damage, the cause of which is insufficiently known. Folate treatment in vitamin B12 deficiency increases the neurological damage. Homocysteine accumulates in blood and urine in both cobalamin and folate deficiency. In cobalamin deficiency the concentration of methylmalonate increases, too, also in cerebrospinal fluid. The metabolic processes were recently described in this journal (1).

Folate occurs in many forms. In food there are non-reduced (oxidised) and glutamate-conjugated forms. In human metabolism they are converted to the reduced, central and easily utilisable form tetrahydrofolate (THFA). The role of folate in metabolism is to transfer one-carbon units to a number of acceptor molecules. In cobalamin deficiency the methylated and metabolically inactive form N5-methyl-THFA cannot be converted to THFA and accumulates. This is because an enzyme containing the coenzyme methylcobalamin removes the methyl group from N5-methyl-THFA and adds it to homocysteine which becomes methionine. Homocysteine therefore accumulates when folate or cobalamin is lacking. As mentioned, in cobalamin deficiency methylmalonate accumulates, too. The explanation is that there is another cobalamin-dependent enzyme which catalyses the degradation of methylmalonate to succinate. This reaction participates in energy metabolism, in the catabolism of some amino acids and fatty acids with odd numbers of carbon atoms (2) and has little to do with DNA.

New cells are rapidly formed in the bone marrow and therefore, the disease is usually haematological. One may note that the synthesis of haemoglobin is affected late, whereas red cell formation is hit early with a resulting increase in the indices MCH and MCV. The practise of measuring only haemoglobin, common in some countries in the diagnosis and treatment of megaloblastic anaemia, therefore cannot be recommended. Rapid cell formation also takes place in epithelial tissues. In gastrointestinal mucosa there are changes analogous to those in the bone marrow (sore tongue is a classical sign of pernicious anaemia). Changes in the intestine cause secondary general malabsorption; therefore tests of general absorption should be performed when the deficiency state has been abolished (3).

Cells are rapidly formed in the gonads, in the endometrium and in the testicles. As early as in 1962 Watson (4) reported in Lancet that vitamin B12 deficiency, documented by low concentrations of cobalamin in serum and seminal fluid, causes male infertility, and subsequent correspondence in the journal supported this contention. In 1976 C.-J. Johansson and the author performed testicular biopsies in cases of vitamin B12 deficiency and observed nearly absent spermiogenesis, which returned after cobalamin treatment (3).* In 1967 Jackson et al. (5) reported that female infertility may be caused by cobalamin deficiency, verified by later reports (6, 7).

Since the discovery of folic acid, this vitamin has been used to cure and prevent megaloblastic anaemia of pregnancy. It has long been known that lack of various vitamins may cause foetal damage, and folate deficiency may produce neural tube defects, spina bifida etc. Low birth weight is clearly connected. In developing countries folate deficiency is common in low socioeconomic groups, and one-third of all women in the world are said to suffer from it (2). To prevent deficiency, folate is therefore given routinely to pregnant women. However, there are few reports of female infertility due to lack of folate (8). Incidentally, early miscarriage is difficult to distinguish from failure to conceive. On the other hand, there are reports on successful folate treatment of male infertility (9).

On the whole, there are few publications on infertility due to lack of the antipernious anaemia vitamins. The explanation is possibly that doctors routinely give the patients a cocktail of vitamins and minerals (at present zinc is popular, 10) without trying to reveal the basic cause, which may be expensive.

Vitamin-poor diet is an obvious cause of vitamin deficiency. An ordinary Scandinavian diet usually contains sufficient quantities of folate and vitamin B12, and generally only vegans get cobalamin deficiency because the vitamin is absent in plants. On the other hand, vegetables are rich in folate. Low folate intake may be due to poor food habits, few ingredients, especially lack of vegetables. Such factors often act among the aged. In the English-speaking countries vegetables are often thoroughly boiled or baked, resulting in extraction and destruction of the vitamins. If a substantial part of the energy intake comes from alcohol, folate deficiency may result; the most impressive megaloblastic anaemia due to folate deficiency seen by the author was is a male who subsisted on vermouth. Ethanol may also be a folate antagonist, as are some drugs. Outright antagonists are methotrexate and trimethoprim, but also antiepileptics may have that effect. Vitamin deficiences of various kinds also occur in connexion with general malabsorption, coeliac disease, sprue and in maldigestion, especially exocrine pancreatic insufficiency. The fish tapeworm is known to inhibit vitamin B12 absorption and cause deficiency, but the parasite is nowadays rare. As mentioned, vitamin deficiency may cause secondary malabsorption, in the author's experience usually weak.

What is mentioned above is common knowledge (2). Vitamin deficiency in a wide sense may be the result of genetical or metabolical errors or weaknesses. Recently attention has been drawn to a folate-related enzyme methylenetetrahydrofolate reductase, MTHFR. It catalyses the conversion of N5,10-methylene-THFA to N5-methyl-THFA (mentioned above), which in turn is converted to THFA. The gene has a variant, the mutant C677T. In homozygotes the enzyme activity is reduced with 70 per cent and in heterozygotes with 35 per cent. Nineteen per cent of infertile males had this gene, twice as often as controls. In homozygotes the homocysteine concentration is increased. Folate therapy lowers the concentration and improves fertility (11). That the homocysteine level of healthy persons drops after folate treatment was shown as early as in 1988 by Brattström et al. (12). However, one should bear in mind that the patient may suffer from cobalamin deficiency and that folate treatment would increase the neurological damage, including impotence.

Investigators of fertility have been interested in the content of folate and cobalamin in seminal fluid. Wallock et al. (13) found an increased proportion of inactive (or rather not readily mobilisable) folates (N5-methyl-THFA, etc.) in smokers. Smoking is considered to reduce the quality of sperm and to decrease the folate stores. Seminal fluid contains a folate-binding protein which may play a role but it has not been much examined. Semen also contains transcobalamin (14, 15). This protein stimulates the uptake of cobalamin in tissues, much like intrinsic factor in the intestine, and is probably needed for the uptake of vitamin B12 by the spermatozooa and their precursors. Congenital lack of it causes megaloblastic anaemia appearing soon after birth. The disease is successfully treated with frequent injections of cobalamin. The author does not know of cases reported from Scandinavia, but before the disease was discovered he predicted its existence (16). In principle, low content of transcobalamin in semen (or its receptor megalin, vide infra) would lead to infertility.

A detour follows, as the topic is of interest in Scandinavia and intriguing findings have recently been made: More than 40 years ago, Olga Imerslund in Norway and the author with collaborators in Finland described the syndrome selective vitamin B12 malabsorption with proteinuria. About 250 cases have been reported in the literature, in Scandinavia most from Finland and Norway, none from Sweden (where at least 2 cases have been diagnosed, however) (17). The interest in the disease is today fairly high as mutations in the gene coding for the multiligand receptor cubilin has been shown to be the cause of the Finnish cases (but not the Norwegian patients) (18). Cubilin and megalin were recently described by a Danish-French team Søren Moestrup, Pierre Verroust and collaborators (19). The two factors participate

in the membrane transport of several different substances. Cubilin corresponds to the ileal receptor for the vitamin B12-intrinsic factor complex, but is also found in other tissues including kidney, where it mediates the reabsorption of albumin from primary urine. Lack of function here explains the proteinuria in selective malabsorption. Megalin and cubilin seem to act in concert and the list of their ligands (substrates) is long: lipoproteins, vitamin D binding protein, transcobalamin etc. The factors were originally discovered in experiments on the yolk sack, thus participating in the development of the embryo, and also play a role in the gonads, e.g. in the seminal vesicles, and vitamin D is a steroid. Disturbances in these membrane transport factors may be suspected of causing infertility and many other pathologic conditions.

Finally, it is worth mentioning failure to synthesise from cobalamin its coenzymes (2). Such congenital defects have been described, but not from Scandinavia. In analogy with the case of the folate enzyme MTHFR, the cobalamin apoenzymes may be assumed to be affected. Though no relation to infertility has yet been reported, one need not have the gifts of a prophet to predict -as exemplified above (16) - that such cases will be discovered.

Many interesting connexions have thus been reported between infertility and lack of or disturbances in the antimegaloblastic anaemia factors and additional ones are to be expected. They are worth noting and represent interesting research topics, relevant also outside fertility medicine. The reader is reminded of the studies indicating that high homocysteine concentrations in blood entail increased risk for cardiovascular disease.

In a routine case of infertility, diving deeply into the basic biochemical mechanism may be rather expensive, however. The author would therefore find it acceptable if doctors as first aid in infertility prescribe a cocktail of vitamins and minerals and abstention from tobacco and alcohol.

The article was ordered as a chapter in an book "Encyclopaedia of Food and Nutrition" and the authors made great efforts to collect data, e.g. on the cobalamin-content of foodstuffs. German corresponding encyclopaedias served as a model. When the manuscript had been submitted, the publishers announced that the book would not be published and suggested publication as an article in a new journal, which was also done. As the book was assumed to be widely distributed, the fertility investigator Dr. Carl-Johan Johansson and myself did not publish the results of the testicular biopsies elsewhere. The pictures in the article are from a case of tapeworm anaemia. In addition, I have diapositive slides of another patient suffering from genuine pernicious anemia. In this patient, the cobalamin injections also restored potency. Altogether I remember 4 similar patients were studied, but my colleague is deceased and publication is no longer possible. The incident with the publishers prompts me to suggest to invited authors to include in their contracts the stipulation that the article ordered must also be published.