1169
reductions in plasma cholesterol. processes, induced by infections,
always adjust similarly However, pathological
to
precancerous changes,
compromised immunity,
or
can
jeopardise
the efficiency of the defence of some cells. In such settings, a reduction of plasma low-density lipoprotein (LDL) could, theoretically, impair membrane cholesterol homoeostasis and alter normal bilayer functions such as receptor activity, enzyme responses, and permeability. This view would help to explain the fact that an adverse response to cholesterol reduction is not the general rule. The increase in mortality from violence and other non-cardiac causes in clinical trials is smalP and may be sporadic, and one reason might be that it is only a minority who are susceptible. The matter of increased deaths during cholesterol reduction first arose during and after the WHO clofibrate trial,4 although there was no excess of violent deaths in that large study. The findings in the last reports are, in summary: Ratio of standardised rate, clofibrate/control ,
Cause of death
In trial (5
yr)
After trial (8 yr)
A remarkable and still unexplained finding is the return of mortality ratios to unity or near unity after the end of the trial, in which follow-up was 99-8% complete on the intention-to-treat basis. The adverse effect disappeared once clofibrate was withdrawn--or once reduction of high serum cholesterol was discontinued. Since a marginal increase in non-cardiac deaths also took place in clinical trials with cholestyramine6 and gemfibrozil/ this adverse effect is not drug specific. In my opinion, we should keep an open mind about the safety of lowering high cholesterol and monitor trial experiences carefully. Resolution of the difficulty will probably have to wait until 1995 or 1996 when the first HMG CoA-reductase inhibitor primary prevention trials, with reduction of LDL cholesterol by 30% or more, will be reported. Meanwhile, these legitimate doubts should not be allowed to impede treatment of hypercholesterolaerrida to reduce the increased risk of CHD in such people. Wynn Institute for Metabolic Research, London NW8 9SQ, UK
M. F. OLIVER
Mariuck SB, Shively C. The effects of fat and cholesterol on social behaviour m monkeys. Psychosom Med 1991; 53: 634-42. 2. Oliver MF. Might treatment of hypercholesterolaemia increase non-cardiac mortality. Lancet 1991; 337: 1529-31. 3. Oliver M. Reducing cholesterol does not reduce mortality. J Am Coll Cardiol 1988; 12: 814-17. 4. Report from the Committee of Principal Investigators. A cooperative trial in the primary prevention of ischaemic heart disease using clofibrate. Br Heart J 1978; 40: 1069. 5. Report of the Committee of Principal Investigators. WHO cooperative trial on primary prevention of ischaemic heart disease with clofibrate to lower serum cholesterol: final mortality follow-up. Lancet 1984; ii: 600-04. 6. Lipid Research Clinics Coronary Prevention Trial. I Reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 1984; 251: 351-74. 7. Frick MH, Elo O, Haaps K, et al. Helsinki Heart Study: primary prevention trial with gemfibrozil in middle-aged men with dyslipidemia. N Engl J Med 1987; 317: 1237-45. 1.
Kaplan JR,
SIR,-Dr Engelberg’s hypothesis generated much interest, not least in the media. However, the mechanism he suggests-namely, that decreased serum cholesterol leads to decreased exposure of serotonin (5-HT) receptors, which in turn causes a reduced uptake of serotonin from the blood and less serotonin in the brain cells-is unlikely. Brain serotonin is synthesised from tryptophan in the brain and is not sequestered from the blood.1 Furthermore, serotonin receptors are heterogeneous, having many different functions; some, such as the autoreceptors and reuptake receptors, reduce rather than enhance serotoninergic function. A reduced exposure of autoreceptor and reuptake receptors as a consequence of a lower cholesterol would enhance rather than diminish serotoninergic function and would be more likely to oppose aggressive or suicidal behaviour. The outcome of a general increase or decrease in the exposure of 5-HT receptors would thus be difficult to predict. I would like to put forward an alternative hypothesis on the relation between cholesterol and brain serotonin. A reduction in
dietary cholesterol intake is likely to go hand in hand with a reduction in overall dietary fat intake, resulting in a decrease in serum fatty acids. Fatty acids and tryptophan compete for a binding site on serum albumin (tryptophan is normally largely bound to serum albumin).1 Increasing the concentration of serum fatty acids displaces bound tryptophan, which in turn increases brain tryptophan (unbound tryptophan is readily transported into the brain whereas bound tryptophan is not).1 The brain synthesis of 5-HT is unsaturated with tryptophan, and increases or decreases in brain tryptophan lead to respective changes in 5-HT synthesis. A change from a high to a low cholesterol diet may thus reduce the concentration of competing fatty acids, which will in turn lead to greater binding of tryptophan, lowering the availability of free tryptophan for 5-HT synthesis. It would not appear that there is a simple way of reversing, through changes in the diet, this cholesterol-associated deficit in brain 5-HT (given the present ban on tryptophan) without increasing cholesterol intake. However, dietary carbohydrate intake has been shown to increase brain tryptophan in the rat as a result of an insulin-stimulated decrease in plasma aminoacids that compete for transport into the brain with tryptophan.2 Whether increases in carbohydrate intake in man would be sufficient to reverse the low-cholesterol-diet-induced decrease in brain 5-HT function is at present unknown. Biochemical Sciences, Wellcome Research Laboratories, Beckenham BR3 3BS, UK 1.
MARK SALTER
Pogson CI, Knowles RG, Salter M. The control of aromatic aminoacid catabolism and its relationship to neurotransmitter amine synthesis. Crit Rev Neurobiol 1989; 5: 29-64.
2. Fernstrom JD, Wurtman RS. Brain serotonin content: increase
following ingestion of
carbohydrate diet. Science 1971; 174: 1023-30.
Prescribing benzodiazepines to drug misusers SIR,-Recent figures from the Scottish Drugs Misuse Database have revealed benzodiazepines to be the single most misused category of drug in Scotland. This difficulty is striking in Lothian region: during six months from October, 1990, 244 new patients who reported taking benzodiazepines were seen by care agencies in Lothian. The equivalent figures for Greater Glasgow and Forth Valley were 206 and 153, respectively. The modal age group of new patients seen in Lothian was 20-24 years. Intravenous injection of drugs continues and, although risk-taking injecting behaviour has declined noticeably in some cohorts since 1986the database reveals that 7% of new patients continue to inject drugs. In north-west Edinburgh, the predominant benzodiazepine drugs of misuse were diazepam and temazepam. Benzodiazepine drugs effectively treat anxiety when given as short-term treatment. Even when patients take benzodiazepines at normal doses for a long time, there do not seem to be any permanent negative consequences.3 However, we find that drug users in north-west Edinburgh take doses of benzodiazepines well outside the normal range. Oral and intravenous doses of 100-150 mg of temazepam and diazepam are common. Side-effects for those taking high-doses of benzodiazepines include severe behavioural disorders, psychiatric disturbance, and convulsions on withdrawa1.4.sThe long-term effects of excessive benzodiazepine intake remain uncertain. Many benzodiazepines taken by drug users are obtained on prescription. A harm-reduction approach, as adopted in Edinburgh, must include support from non-prescribing agencies.6 When this support is not forthcoming, general practitioners and others fmd themselves with few options other than to maintain drug users on prescription. HIV seroprevalence among drug misusers in some areas of Edinburgh is 33-3% so many of those taking benzodiazepines are HIV infected. In addition to pressures exerted by drug users (irrespective of HIV status) on doctors to prescribe drugs, an added difficulty is that in Edinburgh a relatively small number of HIV-infected drug users (IVDUs) are cared for by an increasingly large network of doctors. Thus, drug users can often obtain increased supplies of drugs for personal use or to be sold for
1170
profit. In Edinburgh, dementia has been diagnosed among several young IVDUs without HIV, which has been attributed to large-scale benzodiazepine misuse. However, when these effects are identified among HIV-infected IVDUs, health professionals may assume that they result from HIV, without considering any connection with drug misuse. HIV should not overshadow the difficulties of drug misuse. During the next few years, the consequences of extreme forms of benzodiazepine misuse, presently emerging in Scotland, may become all too obvious in the rest of the UK. Edinburgh Drug Addiction 1 Muirhouse Avenue, Edinburgh EH4 4PL, UK
Study,
J. R. ROBERTSON P. J. M. RONALD
1. Information and statistics division, Scottish health service. Scottish drug misuse database statistics: October, 1990-March, 1991. Edinburgh: ISD Publications, 1992. 2. Ronald PJM, Robertson JR, Roberts JJK. Risk taking behaviour on the decline in intravenous drug users. Br J Addict 1992; 87: 115-16. 3. Tyrer PJ. Benzodiazepines on trial. Br Med J 1984; 288: 1101-02. 4. Woods JH, Katz JL, Winger G. Use and abuse of benzodiazepines. JAMA 1988; 260: 3476-79. 5. Robertson JR, Skidmore CA, Roberts JJK. Benzodiazepines and convulsions. Lancet
1988; ii: 504-05. 6. Department of Health, Scottish Home and Health Department & Welsh Office. Drug misuse and dependence: guidelines on clinical management. London: HM Stationery Office, 1991.
Successful in-vitro fertilisation and embryo transfer after treatment with recombinant human FSH SIR,-Follicle-stimulating hormone (FSH) plays an important part in the treatment of infertility. The first pregnancy following the use of extracted human pituitary gonadotropin preparation, containing FSH and luteinising hormone (LH), was reported in 1960.1 Soon afterwards, gonadotropins extracted from postmenopausal urine also proved to be effective2 and became the standard gonadotropin preparation for three decades. In 1985, a preparation of urinary FSH practically devoid of LH activity but still of low purity (specific activity of about 150 IU FSH/mg protein) was successfully used to stimulate follicular development that resulted in pregnancy. We report here a pregnancy obtained by treatment with recombinant human FSH (rhFSH). Because FSH is a complex heterodimeric protein that needs to be glycosylated for biological activity, rhFSH was produced by genetically engineered mammalian cells (Chinese hamster ovary) in which the genes encoding for the hFSH subunits had been inserted .4 This rhFSH has a very high specific activity (at least 10 000 IU/mg protein) and can be self-administered subcutaneously. It has no LH activity and has strong physical, immunological, biological, and pharmacological similarity with natural FSH.4 A 32-year-old woman with primary tubal infertility (bilateral tubal occlusion) for 9 years was enrolled by M. G. in a multicentre clinical trial to assess the efficacy and the safety of rhFSH (GONAL-F; Serono) given subcutaneously to stimulate multiple follicular development in women undergoing in-vitro fertilisation and embryo transfer (IVF-ET). The treatment was started in the midluteal phase by daily injections of 200 ug buserelin. On day 2 of the subsequent menstruation, daily treatment with 225 IU rhFSH was started and follicular development was monitored by assaying serum oestradiol and ultrasound scanning of the ovaries: Serum oestradiol
Day
On
day 7,
(pmo///)
the
daily dose of rhFSH
Follicle size
was
(mm) (R/L)
increased to 300 1U. The
injections were well tolerated. On day 14,24 h after the last injection, 10 000 IU hCG was given, and 37 h later four oocytes were recovered and fertilised in vitro.S Two grade I, two-cell embryos and two grade II, three-cell
replaced in the uterus 2 days later. An ultrasound after days embryo transfer revealed an intrauterine twin pregnancy (two amniotic sacs, both containing fetal heart activity). This experience shows that rhFSH, an FSH preparation totally devoid of LH, can stimulate follicular development. embryos scan
were
28
Fertility Unit, Department of Obstetrics and Gynecology, CHU Vaudois, Lausanne; Medical Department, Ares Serono, CH-1202 Geneva, Switzerland
MARC GERMOND SALVATORE DESSOLE ALFRED SENN ERNEST LOUMAYE COLIN HOWLES VANYA BELTRAMI
1. Gemzell CA, Diczalusy E, Tillinger K-G. Human pituitary follicle-stimulating hormone I: clinical effect of a partly purified preparation. Ciba Found Colloq
Endocrinol 1960, 13: 191. B, Sulimovici S, Rabau E, Eshkol A, L’induction de l’ovulation dans les amenorrhées hypophysaires par un traitment combiné de gonadotrophines urinaires ménopausiques et de gonadotrophines chorioniques. C R Société Fr Gynécol 1962; 5: 30. Shaw RW, Ndukwe G, Imoedemhe DAG, Bernard AG, Burford G. Twin pregnancy after pituitary desensitization with LHRH agonist and pure FSH. Lancet 1985; ii:
2. Lunenfeld
3.
506 4.
Chappel S, Kelton C, Nugent N. Expression of human gonadotropins by recombinant DNA methods. In Genazzam AR, Petraglia F, eds. Proc 3rd Wld Congr Gynecol
Endocrinol 1992: 179 5. Germond M, Senn A, Bonanomi S, et al. Fecondation in vitro et transfert d’embryons. Schweiz Med Wschr 1990; 120: 260
SIR,-We report a
pregnancy achieved after controlled ovarian
superovulation with highly purified recombinant follicle stimulating hormone (rhFSH). For 25 years ovarian stimulation has been based on the application of crude urinary gonadotropin preparations but the future of gonadotropin therapy is likely to lie with recombinant FSH of high biochemical purity and specific bioactivity. The production of FSH recombinant DNA technology guarantees improved batch-to-batch consistency and freedom from protein contaminants. Preclinical studies show that in-vitro and in-vivo bioactivities of natural FSH and highly purified rhFSH (Org 32489; Organon International) are indistinguishable.1 A 29-year-old patient and her 31-year-old husband have been infertile since 1984. In 1990, a ruptured ectopic pregnancy was observed and a salpingectomy by laparotomy was done elsewhere. During laparoscopic examination in 1991, the remaining fallopian tube was found to be severely damaged, while the following tests were normal: ovarian function, as judged by serial measurements of serum FSH, luteinising hormone (LH), oestradiol and progesterone; hysteroscopy; karyotype analysis of both partners; and semen analysis. Subsequently, the couple gave informed consent to participate in an open phase II study evaluating the efficacy of combined buserelin/rhFSH therapy in women undergoing in-vitro fertilisation and embryo transfer (IVF/ET). This study was approved by the Belgian health authorities and the ethics committee of the Free University Hospital, Brussels. rhFSH (Org 32489) was given intramuscularly at a daily dose of 75 IU (standardised as urinary FSH/hMG preparations with the Steelman and Pohley assay) from menstrual cycle day 3 to 11. Treatment was in association with intranasal buserelin 4 x 150 Ilgjdaily started at menstrual cycle day 1. After 9 days of rhFSH treatment, at cycle day 12, serum oestradiol had risen to 2640 pg/ml and nine preovulatory follicles (diameter 15-23 mm) were observed by means of vaginal ultrasonography. Buserelin/rhFSH was then discontinued. 10 000 IU human chorionic gonadotropin (hCG) was given intramuscularly and 36 h later ultrasonographically guided vaginal pick-up was done under local anaesthesia. Nine mature oocytes (type 1.02) were retrieved. Oocytes were incubated with 3000 spenmtozoa/pl taken from a fraction of 106 morphologically normal sperm with 100% progressive motility obtained after a swim-up procedure. Removal of the cumulus complex after 18 h of incubation revealed four oocytes with two pronuclei. After two days of incubation, three four-cell embryos were transferred into the uterus and one four-cell embryo was cryopreserved. The luteal phase was supplemented with vaginally administered micronised natural progesterone 600 mg daily.3 On days 10, 13, and 18 after embryo replacement, serum hCG values were 53, 301, and 1938 IU/1, respectively. An ultrasound scan revealed a live singleton intrauterine pregnancy on day 35 after embryo transfer. During the
reductions in plasma cholesterol. processes, induced by infections,
always adjust similarly However, pathological
to
precancerous changes,
compromised immunity,
or
can
jeopardise
the efficiency of the defence of some cells. In such settings, a reduction of plasma low-density lipoprotein (LDL) could, theoretically, impair membrane cholesterol homoeostasis and alter normal bilayer functions such as receptor activity, enzyme responses, and permeability. This view would help to explain the fact that an adverse response to cholesterol reduction is not the general rule. The increase in mortality from violence and other non-cardiac causes in clinical trials is smalP and may be sporadic, and one reason might be that it is only a minority who are susceptible. The matter of increased deaths during cholesterol reduction first arose during and after the WHO clofibrate trial,4 although there was no excess of violent deaths in that large study. The findings in the last reports are, in summary: Ratio of standardised rate, clofibrate/control ,
Cause of death
In trial (5
yr)
After trial (8 yr)
A remarkable and still unexplained finding is the return of mortality ratios to unity or near unity after the end of the trial, in which follow-up was 99-8% complete on the intention-to-treat basis. The adverse effect disappeared once clofibrate was withdrawn--or once reduction of high serum cholesterol was discontinued. Since a marginal increase in non-cardiac deaths also took place in clinical trials with cholestyramine6 and gemfibrozil/ this adverse effect is not drug specific. In my opinion, we should keep an open mind about the safety of lowering high cholesterol and monitor trial experiences carefully. Resolution of the difficulty will probably have to wait until 1995 or 1996 when the first HMG CoA-reductase inhibitor primary prevention trials, with reduction of LDL cholesterol by 30% or more, will be reported. Meanwhile, these legitimate doubts should not be allowed to impede treatment of hypercholesterolaerrida to reduce the increased risk of CHD in such people. Wynn Institute for Metabolic Research, London NW8 9SQ, UK
M. F. OLIVER
Mariuck SB, Shively C. The effects of fat and cholesterol on social behaviour m monkeys. Psychosom Med 1991; 53: 634-42. 2. Oliver MF. Might treatment of hypercholesterolaemia increase non-cardiac mortality. Lancet 1991; 337: 1529-31. 3. Oliver M. Reducing cholesterol does not reduce mortality. J Am Coll Cardiol 1988; 12: 814-17. 4. Report from the Committee of Principal Investigators. A cooperative trial in the primary prevention of ischaemic heart disease using clofibrate. Br Heart J 1978; 40: 1069. 5. Report of the Committee of Principal Investigators. WHO cooperative trial on primary prevention of ischaemic heart disease with clofibrate to lower serum cholesterol: final mortality follow-up. Lancet 1984; ii: 600-04. 6. Lipid Research Clinics Coronary Prevention Trial. I Reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 1984; 251: 351-74. 7. Frick MH, Elo O, Haaps K, et al. Helsinki Heart Study: primary prevention trial with gemfibrozil in middle-aged men with dyslipidemia. N Engl J Med 1987; 317: 1237-45. 1.
Kaplan JR,
SIR,-Dr Engelberg’s hypothesis generated much interest, not least in the media. However, the mechanism he suggests-namely, that decreased serum cholesterol leads to decreased exposure of serotonin (5-HT) receptors, which in turn causes a reduced uptake of serotonin from the blood and less serotonin in the brain cells-is unlikely. Brain serotonin is synthesised from tryptophan in the brain and is not sequestered from the blood.1 Furthermore, serotonin receptors are heterogeneous, having many different functions; some, such as the autoreceptors and reuptake receptors, reduce rather than enhance serotoninergic function. A reduced exposure of autoreceptor and reuptake receptors as a consequence of a lower cholesterol would enhance rather than diminish serotoninergic function and would be more likely to oppose aggressive or suicidal behaviour. The outcome of a general increase or decrease in the exposure of 5-HT receptors would thus be difficult to predict. I would like to put forward an alternative hypothesis on the relation between cholesterol and brain serotonin. A reduction in
dietary cholesterol intake is likely to go hand in hand with a reduction in overall dietary fat intake, resulting in a decrease in serum fatty acids. Fatty acids and tryptophan compete for a binding site on serum albumin (tryptophan is normally largely bound to serum albumin).1 Increasing the concentration of serum fatty acids displaces bound tryptophan, which in turn increases brain tryptophan (unbound tryptophan is readily transported into the brain whereas bound tryptophan is not).1 The brain synthesis of 5-HT is unsaturated with tryptophan, and increases or decreases in brain tryptophan lead to respective changes in 5-HT synthesis. A change from a high to a low cholesterol diet may thus reduce the concentration of competing fatty acids, which will in turn lead to greater binding of tryptophan, lowering the availability of free tryptophan for 5-HT synthesis. It would not appear that there is a simple way of reversing, through changes in the diet, this cholesterol-associated deficit in brain 5-HT (given the present ban on tryptophan) without increasing cholesterol intake. However, dietary carbohydrate intake has been shown to increase brain tryptophan in the rat as a result of an insulin-stimulated decrease in plasma aminoacids that compete for transport into the brain with tryptophan.2 Whether increases in carbohydrate intake in man would be sufficient to reverse the low-cholesterol-diet-induced decrease in brain 5-HT function is at present unknown. Biochemical Sciences, Wellcome Research Laboratories, Beckenham BR3 3BS, UK 1.
MARK SALTER
Pogson CI, Knowles RG, Salter M. The control of aromatic aminoacid catabolism and its relationship to neurotransmitter amine synthesis. Crit Rev Neurobiol 1989; 5: 29-64.
2. Fernstrom JD, Wurtman RS. Brain serotonin content: increase
following ingestion of
carbohydrate diet. Science 1971; 174: 1023-30.
Prescribing benzodiazepines to drug misusers SIR,-Recent figures from the Scottish Drugs Misuse Database have revealed benzodiazepines to be the single most misused category of drug in Scotland. This difficulty is striking in Lothian region: during six months from October, 1990, 244 new patients who reported taking benzodiazepines were seen by care agencies in Lothian. The equivalent figures for Greater Glasgow and Forth Valley were 206 and 153, respectively. The modal age group of new patients seen in Lothian was 20-24 years. Intravenous injection of drugs continues and, although risk-taking injecting behaviour has declined noticeably in some cohorts since 1986the database reveals that 7% of new patients continue to inject drugs. In north-west Edinburgh, the predominant benzodiazepine drugs of misuse were diazepam and temazepam. Benzodiazepine drugs effectively treat anxiety when given as short-term treatment. Even when patients take benzodiazepines at normal doses for a long time, there do not seem to be any permanent negative consequences.3 However, we find that drug users in north-west Edinburgh take doses of benzodiazepines well outside the normal range. Oral and intravenous doses of 100-150 mg of temazepam and diazepam are common. Side-effects for those taking high-doses of benzodiazepines include severe behavioural disorders, psychiatric disturbance, and convulsions on withdrawa1.4.sThe long-term effects of excessive benzodiazepine intake remain uncertain. Many benzodiazepines taken by drug users are obtained on prescription. A harm-reduction approach, as adopted in Edinburgh, must include support from non-prescribing agencies.6 When this support is not forthcoming, general practitioners and others fmd themselves with few options other than to maintain drug users on prescription. HIV seroprevalence among drug misusers in some areas of Edinburgh is 33-3% so many of those taking benzodiazepines are HIV infected. In addition to pressures exerted by drug users (irrespective of HIV status) on doctors to prescribe drugs, an added difficulty is that in Edinburgh a relatively small number of HIV-infected drug users (IVDUs) are cared for by an increasingly large network of doctors. Thus, drug users can often obtain increased supplies of drugs for personal use or to be sold for
1170
profit. In Edinburgh, dementia has been diagnosed among several young IVDUs without HIV, which has been attributed to large-scale benzodiazepine misuse. However, when these effects are identified among HIV-infected IVDUs, health professionals may assume that they result from HIV, without considering any connection with drug misuse. HIV should not overshadow the difficulties of drug misuse. During the next few years, the consequences of extreme forms of benzodiazepine misuse, presently emerging in Scotland, may become all too obvious in the rest of the UK. Edinburgh Drug Addiction 1 Muirhouse Avenue, Edinburgh EH4 4PL, UK
Study,
J. R. ROBERTSON P. J. M. RONALD
1. Information and statistics division, Scottish health service. Scottish drug misuse database statistics: October, 1990-March, 1991. Edinburgh: ISD Publications, 1992. 2. Ronald PJM, Robertson JR, Roberts JJK. Risk taking behaviour on the decline in intravenous drug users. Br J Addict 1992; 87: 115-16. 3. Tyrer PJ. Benzodiazepines on trial. Br Med J 1984; 288: 1101-02. 4. Woods JH, Katz JL, Winger G. Use and abuse of benzodiazepines. JAMA 1988; 260: 3476-79. 5. Robertson JR, Skidmore CA, Roberts JJK. Benzodiazepines and convulsions. Lancet
1988; ii: 504-05. 6. Department of Health, Scottish Home and Health Department & Welsh Office. Drug misuse and dependence: guidelines on clinical management. London: HM Stationery Office, 1991.
Successful in-vitro fertilisation and embryo transfer after treatment with recombinant human FSH SIR,-Follicle-stimulating hormone (FSH) plays an important part in the treatment of infertility. The first pregnancy following the use of extracted human pituitary gonadotropin preparation, containing FSH and luteinising hormone (LH), was reported in 1960.1 Soon afterwards, gonadotropins extracted from postmenopausal urine also proved to be effective2 and became the standard gonadotropin preparation for three decades. In 1985, a preparation of urinary FSH practically devoid of LH activity but still of low purity (specific activity of about 150 IU FSH/mg protein) was successfully used to stimulate follicular development that resulted in pregnancy. We report here a pregnancy obtained by treatment with recombinant human FSH (rhFSH). Because FSH is a complex heterodimeric protein that needs to be glycosylated for biological activity, rhFSH was produced by genetically engineered mammalian cells (Chinese hamster ovary) in which the genes encoding for the hFSH subunits had been inserted .4 This rhFSH has a very high specific activity (at least 10 000 IU/mg protein) and can be self-administered subcutaneously. It has no LH activity and has strong physical, immunological, biological, and pharmacological similarity with natural FSH.4 A 32-year-old woman with primary tubal infertility (bilateral tubal occlusion) for 9 years was enrolled by M. G. in a multicentre clinical trial to assess the efficacy and the safety of rhFSH (GONAL-F; Serono) given subcutaneously to stimulate multiple follicular development in women undergoing in-vitro fertilisation and embryo transfer (IVF-ET). The treatment was started in the midluteal phase by daily injections of 200 ug buserelin. On day 2 of the subsequent menstruation, daily treatment with 225 IU rhFSH was started and follicular development was monitored by assaying serum oestradiol and ultrasound scanning of the ovaries: Serum oestradiol
Day
On
day 7,
(pmo///)
the
daily dose of rhFSH
Follicle size
was
(mm) (R/L)
increased to 300 1U. The
injections were well tolerated. On day 14,24 h after the last injection, 10 000 IU hCG was given, and 37 h later four oocytes were recovered and fertilised in vitro.S Two grade I, two-cell embryos and two grade II, three-cell
replaced in the uterus 2 days later. An ultrasound after days embryo transfer revealed an intrauterine twin pregnancy (two amniotic sacs, both containing fetal heart activity). This experience shows that rhFSH, an FSH preparation totally devoid of LH, can stimulate follicular development. embryos scan
were
28
Fertility Unit, Department of Obstetrics and Gynecology, CHU Vaudois, Lausanne; Medical Department, Ares Serono, CH-1202 Geneva, Switzerland
MARC GERMOND SALVATORE DESSOLE ALFRED SENN ERNEST LOUMAYE COLIN HOWLES VANYA BELTRAMI
1. Gemzell CA, Diczalusy E, Tillinger K-G. Human pituitary follicle-stimulating hormone I: clinical effect of a partly purified preparation. Ciba Found Colloq
Endocrinol 1960, 13: 191. B, Sulimovici S, Rabau E, Eshkol A, L’induction de l’ovulation dans les amenorrhées hypophysaires par un traitment combiné de gonadotrophines urinaires ménopausiques et de gonadotrophines chorioniques. C R Société Fr Gynécol 1962; 5: 30. Shaw RW, Ndukwe G, Imoedemhe DAG, Bernard AG, Burford G. Twin pregnancy after pituitary desensitization with LHRH agonist and pure FSH. Lancet 1985; ii:
2. Lunenfeld
3.
506 4.
Chappel S, Kelton C, Nugent N. Expression of human gonadotropins by recombinant DNA methods. In Genazzam AR, Petraglia F, eds. Proc 3rd Wld Congr Gynecol
Endocrinol 1992: 179 5. Germond M, Senn A, Bonanomi S, et al. Fecondation in vitro et transfert d’embryons. Schweiz Med Wschr 1990; 120: 260
SIR,-We report a
pregnancy achieved after controlled ovarian
superovulation with highly purified recombinant follicle stimulating hormone (rhFSH). For 25 years ovarian stimulation has been based on the application of crude urinary gonadotropin preparations but the future of gonadotropin therapy is likely to lie with recombinant FSH of high biochemical purity and specific bioactivity. The production of FSH recombinant DNA technology guarantees improved batch-to-batch consistency and freedom from protein contaminants. Preclinical studies show that in-vitro and in-vivo bioactivities of natural FSH and highly purified rhFSH (Org 32489; Organon International) are indistinguishable.1 A 29-year-old patient and her 31-year-old husband have been infertile since 1984. In 1990, a ruptured ectopic pregnancy was observed and a salpingectomy by laparotomy was done elsewhere. During laparoscopic examination in 1991, the remaining fallopian tube was found to be severely damaged, while the following tests were normal: ovarian function, as judged by serial measurements of serum FSH, luteinising hormone (LH), oestradiol and progesterone; hysteroscopy; karyotype analysis of both partners; and semen analysis. Subsequently, the couple gave informed consent to participate in an open phase II study evaluating the efficacy of combined buserelin/rhFSH therapy in women undergoing in-vitro fertilisation and embryo transfer (IVF/ET). This study was approved by the Belgian health authorities and the ethics committee of the Free University Hospital, Brussels. rhFSH (Org 32489) was given intramuscularly at a daily dose of 75 IU (standardised as urinary FSH/hMG preparations with the Steelman and Pohley assay) from menstrual cycle day 3 to 11. Treatment was in association with intranasal buserelin 4 x 150 Ilgjdaily started at menstrual cycle day 1. After 9 days of rhFSH treatment, at cycle day 12, serum oestradiol had risen to 2640 pg/ml and nine preovulatory follicles (diameter 15-23 mm) were observed by means of vaginal ultrasonography. Buserelin/rhFSH was then discontinued. 10 000 IU human chorionic gonadotropin (hCG) was given intramuscularly and 36 h later ultrasonographically guided vaginal pick-up was done under local anaesthesia. Nine mature oocytes (type 1.02) were retrieved. Oocytes were incubated with 3000 spenmtozoa/pl taken from a fraction of 106 morphologically normal sperm with 100% progressive motility obtained after a swim-up procedure. Removal of the cumulus complex after 18 h of incubation revealed four oocytes with two pronuclei. After two days of incubation, three four-cell embryos were transferred into the uterus and one four-cell embryo was cryopreserved. The luteal phase was supplemented with vaginally administered micronised natural progesterone 600 mg daily.3 On days 10, 13, and 18 after embryo replacement, serum hCG values were 53, 301, and 1938 IU/1, respectively. An ultrasound scan revealed a live singleton intrauterine pregnancy on day 35 after embryo transfer. During the
