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Immunotherapy and fertility control by immunization against gonadotrophin-releasing hormone
Immunotherapy
and fertility control by immunization
against gonadotrophin-releasing
hormone
G.P. Talwar and S. Sad National
Institute
Current
of immunology,
Opinion
in Immunology
Introduction Gonadotrophin-releasing hormone (GnRH), also known as luteInking hormone-releasing hormone @I-WI-I), is a decapeptide released from the hypothalamus. It regulates the secretion of the gonadotrophins LH and folliclestimulating hormone (FSH) from the pituitary; these gonadotrophins in turn act on the gonads to generate the gametes and sex steroid hormones. GnRH is thus a ‘master’ molecule, regulating a cascade of events alkcting reproduction and steroid hormone production. Immunization against GnRH blocks these processes and, because GnRH is made by both males and females, immunization can be used to inlluence either male or female fertiky. Furthermore, as the molecule is essentially conserved through evolution, immunization against the decapeptide is an effective way of controlling fertility In a variety of mammalian species. This property has been useful in studies dete rmining the efficacy and side effects of vaccines designed to produce antibodies against GnRH. Here, we will review some recent advances In the development of anti-GnRH vaccines, and consider possible *erapeutic applications.
The basis of anti-GnRH vaccines Because GnRH is a short peptide and a ‘self molecule, it must be linked to an Immunogenic carrier proteIn to Induce an Immune response. In early studies (Arimura et al, Endocrindo@ 1973, 93:1092-1103; Fraser et al, Nature 1973, 244:160-161), GnRH was tagged to baine serum albumin. To obtain antibodies reactive with GnRH using these conjugates, Freund’s complete adjuvant (FCA) had to be used. These studies demonstrated the elkacy of immunization for blocking the fertility of both male and female animals. FCA is not suitable for use in humans, however, for whom alternative immunization strategies had to be evolved in which an adequate immune response could be obtained using adjuvants appropriate for humans. The observation that a conjugate
New Delhi, India 1990, 2:733-735
of GnRH with tetanus toxoid as the carrier could Induce anti-GnRH antibodies in mice even when the conjugate was administered with alum, an adjuvant approved for human vaccines, laid the basis for a potential vaccine (Shastri et al, Am J Reprod Immunoll981,1:262-265).
Sites for carrier conjugation and their implications In the past, carbodiimide [l-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride] was used to link GnRH to the carrier protein. As native GnRH does not have free carboxyl or amino groups, linkage is probably effected through the hydroxyl group of serine or tyrosine, or through a carboxylated derivative of histidine. The conjugate made by this method is poorly delined and gives inconsistent results. More successfkl conjugation was obtained by diazorization of the molecule, and coupling through hi&line and tyrosine residues (Pique et al, Immunocbem~ 1978, 15:55-&l; Koch et al, Bidwm B@l?ys Res Commun 1973, 55~616-622). The site at which the carrier is attached has a bearing on the reactivity pattern of the antibodies induced. Nett et reported al (I Clin Erzdxrinol Metub 1973,36:880-885) the Induction of an antibody directed to the amino-terminal end of GnRH when the carboxy-terminal end of GnRH was conjugated to bovine serum albumin. ArImura et al. (AC&ZEnabcrind 1975,78:222-231) confirmed this result and also showed that conjugation of GnRH with the carrier at the amino-terminal amino acid results iri antisera with reactivity against the carboxyterminal end. The Importance of an amide group at the carboxy terminal of the GnRH molecule in the generation of a’ conformation recognized by polyvalent antibodies is highlighted by studies based on two antisera derIvd_ from primates (Singh et al, J SteroidBiochem 1985,23:801402). However, from the reactivity pattern of a bioeifective monoclonal antibody to G&I-I, we (Talwar et al, Proc Nat1 Acud Sci US4 1985, 82:122%1231) concluded that the antigenicity of native GnRH depends on the entire
Abbreviations FCA-Freund’s
complete adjuvant; GnRtbgonadotrophin-releasing hormone; FSl+--follicle-stimulating lHRH--luteinizing hormone-releasing hormone; lT--tetanus toxoid.
@ Current
Biology
Ltd MN
0952-7915
hormone;
733
734
Reproduction
molecule and that the N-terminal and C-terminal regions are physically close. Recently, Silversides et al. [ 11, using analogues of GnRH substituted with cysteine at the carboxy and amino terminals to achieve conjugation to the carrier, keyhole limpet haemocyanin, have shown that the specificity of antibodies Induced by GnRH/carrler conjugates is a function both of the orientation of antigen presentation and of the responding animals immune regulatory mechanisms. These conclusions are in agreement with the multideterminan t regulatory model of antigenicity for protein antigens (Benjamin et al, Annu Rev Immunol 1984, 2:67-101). In another study [2], we have linked diphtheria toxoid as carder to GnRH through a functional group created by replacing glycine at residue six by o-lysine, which also increases the biological half-life of GnRH and confers conformational consttaints. The Eamino group of lysine is linked to a spacer, amino caproic acid, which is then conjugated to diphtheria toxoid. This conjugate gives a consistent immunogenic response in rodents and monkeys, probably because the linkage is both flexible and long enough to hold the peptide away from the surface of the carrier.
Control
of male fertility
Immunization of male rodents, rabbits and monkeys against GnRH leads to a block of spematogenesis. It is also accompanied by a drastic reduction in testosterone levels, but the extratesticular requirements for androgens can be met by injection of a slow-release androgen. Iadd et al [3,4] reported that spermatogenesis in male rats and rabbits was suppressed upon immunization with GnRH conjugated to tetanus toxoid (‘IT) and that administration of testosterone-17-trans-4-n-butyl cyclohexane carboxylate as a supplemental androgen restored libido and mating behaviour but did not restore spermatogenesis. All of the GnRH-‘IT immunized animals were infertile. Administration of the testosterone ester at the time of primary imrnumzation and then at lo-week intervals did not restore spermatogenesis, even though the serum testosterone levels in Infertile, GnRI-TT immunized, androgen-treated rats were similar to those in nonimmunized, androgen-treated fertile rats. Ladd et al [3,4] also worked out the dosages of androgen (10 mg/ml> needed to restore libido while keeping the animals infertile.
Control
of fertility
in animals
The ability of the anti-GnRH antibodies to suppress oestrous In female dogs has been demonstrated by the administration of a monoclonal antibody to GnRH in small amounts to female dogs at the iirst signs of heat (Tal-
war et al, 1985). A suppression of the progression of oestrous was indicated by behaviour changes, vaginal cytology and sex hormone profiles. Similar effects can be achieved by active immunization of dogs (Talwar et aA In Hormone Receptors in Growth and Reproduction edited by Saxena BB et al Raven Press, 1984, pp 351-359). A possible application of an anti-GnRH vaccine in animals bred for meat stems from the fact that androgens and their metabolites lower the quality of meat. In the past, diethylstilbesterol was added to animal feed to counteract androgens. However, it accumulates in the fatty tissues, is passed on to the consumer, and has been banned in the USA and other countries. Anti-GnRH immunization offers an alternative solution because it can markedly diminish androgen production in animals bred for meat, with no apparent side effects.
Control
of female fertility
Immunization of baboons and monkeys (Talwar et al, 1984) and marmosets (Hodges and Heam, Nature 1977, 265:746-747) against GnRH leads to an impairment of cyclic&y and to an ovulation block. A similar short-term effect can be obtained by administering monoclonal antibodies. Passive immunization stops ovulation in rats (Talwar et d, 1985) and can terminate pregnancy in mice (Gupta et al, Am J Reprod Immunol Microbial 1985, 7:104-108). The disadvantage of this method is the concomitant decline of the sex steroid hormones. Because the anovulatory cycles during lactational amenorrhoea are ascribable to the suppression of GnRH, another possible use of a GnRH vaccine could be in prolonging the gap between successive pregnancies in postpartum women. This was suggested by the results of Fraser et al [5], who showed that daily lntranasal administration of a GnRH agonist, Buserelin, to women prevents ovulation throughout the period of lactation. A decline in II-I and oestrone levels was noticed with &uppression of ovulation on chronic treatment, although in the iirst month there was an increase in LH and oestrone levels, with light bleeding. No changes were observed in nursing habits. These results illustrate the use of GnRH agonists as a potential method of contraception during the post-partum period. GNRH agonists administered in large amounts and repeated doses down-grade the response of the target cells to the hormone; the vaccine a&eves the same end result by causing inactivation of the hormone with antibodies directed against it. Similar effects have, in fact, been obtained by immunization against GnRH. The advantages of immunization ‘over agonist administration are that the costs are much lower and that daily treatment is not required as immunization is only needed periodically. Neonatal teratology studies in monkeys are underway in our laboratory to determine the safety of post-partum immunization of primates with the anti-GnRH vaccine.
Immunotherapy
Therapeutic
applications
and fertility control by immunization
of anti-GnRH
vaccines GnRH anaioguesthat suppress or antagonize the effects of GnRH have found applications in many clinical conditions, such as sex steroid hormone-dependent cancers, endometriosis, etc. That an anti-GnRH vaccine could be used to achieve similar results is demonstrated by our recent data. Rats immunized with the GnRH/diphtheria toxoid vaccine showed marked atrophy of the prostate [2]. Every animal responded by making anti-GnRH antibodies. There was a concomitant decline in testosterone levels in every animal. The effect of antibodies was reversed by native GnRH, but not by an analogue, indicating that the antibodies bind specifically to the native hormone. Although there was a reduction in the size of the testes and other androgen-sensitive secondary reproductive organs, the size of the prostate was more dramaticaLly reduced. A reduction in prostate size has also been observed (unpublished data) in monkeys after immunization with this vaccine. This particular vaccine has completed acute, subacute and chronic toxicology studies and has been approved for human use. Phase I/ Phase II clinical trials have started with this vaccine in patients who have carcinoma of the prostate in two medical institutes in India.
Conclusions Vaccine formulations have been developed to induce a consistent antibody response against GnRH in experimental animals. Immunization aga&t GnRH can be used to regulate fertility in both males and females. In the case of males, libido can be preserved by administration of androgens without spermatogenesis being restored. AntiGnRH vaccination may also be useful in lengthening the period during which ovulation is suppressed in post-parttun women. Of additional interest are the irnmunotherapeutic applications of immunization against GnRH in prostate hypertrophy and other pathological conditions where G&H agonists and antagonists are observed to be clinically effective.
Annotated
references
against CnRH Talwar and Sad
and recommended
reading l
*a
Of interest Of outstanding interest
SLVERSlDESDW,&IEN~,bfISRA V,Qu.UlERI L, MAF’IETOFTRJ, MURPHYBD: A synthetic luteinising hormone releasing hormone vaccine. Conjugation and speciticity trials in Balb/c mice. J Rgn-od Immund 1988, 13:24+261. Reports a novel method of conjugating GnRH to a carrier molecule with high e&iency using cysteine-substituted analogues of GnRH. The specificity of GnRH antiserawas shown to be dependent on the site of conjugation of the peptide to the carrier molecule. 1. 00
MK, SUVGH 0, AIAM A, TALWAR JAYMLUVKAR R, CHAT GP: Semisynthctic anti-LHRH vaccine causing atrophy of the prostate. Prostufe 19139, 143-H. Reports the structure of an anti-GnRHvaccine that cor&tently induces a bioeffective immune response. Immunization leads to atrophy of the prostate. This paper provides the basis for the therapeutic use of antiGnRH vaccines in hypertrophy of the prostate and in androgen-dependent carcinoma of the prostate.
2. l
o
3. 00
IADD A, PRABHUG, TSONCYY, PROBSTT, CHLMG W, TIL+LI R: Active immunization against gonadotrophin rclcasing hormone combined with androgen supplementation is a
promisingantifertilityvaccine
for males. Am J Reprod Im
mund Microbid 1988, 17~121-127. Reports 100% infertilityin rats upon active immunization against GnRH and describes the use of exogenous androgen supplements to restore libido and normal mating behaviour. I29~ 4 TSONG YY, PRABHU G, THAU R: Elkas of long-term immunization against LHRH and androgen treatment on gonadal function. J Rep& Immunol 19t3!9, 15:85-101. Neither short-term nor long-term treatment with androgen alone i&uenced fertilityin rats rendered infertile by immunization against GnRH. 4. l
FRASER HM, DEWARTPJ, Slwnl SK, CoWEN GM, SmW J, MCNEIUY As: Luteinising hormone releasing hormone agonist for contraception in breast feeding women. J Clin Endocrid Mekzbd 1989,69:996-1002. Clinical studies on nine women given a GnRH agonist every day in the post-partum period showed a block in ovulation without significantside effects or changes in nursing behaviour. The study indicates the poterrial of anti-GnRH treatment as an acceptable method of post-partum contraception.
5. we
735
and fertility control by immunization
against gonadotrophin-releasing
hormone
G.P. Talwar and S. Sad National
Institute
Current
of immunology,
Opinion
in Immunology
Introduction Gonadotrophin-releasing hormone (GnRH), also known as luteInking hormone-releasing hormone @I-WI-I), is a decapeptide released from the hypothalamus. It regulates the secretion of the gonadotrophins LH and folliclestimulating hormone (FSH) from the pituitary; these gonadotrophins in turn act on the gonads to generate the gametes and sex steroid hormones. GnRH is thus a ‘master’ molecule, regulating a cascade of events alkcting reproduction and steroid hormone production. Immunization against GnRH blocks these processes and, because GnRH is made by both males and females, immunization can be used to inlluence either male or female fertiky. Furthermore, as the molecule is essentially conserved through evolution, immunization against the decapeptide is an effective way of controlling fertility In a variety of mammalian species. This property has been useful in studies dete rmining the efficacy and side effects of vaccines designed to produce antibodies against GnRH. Here, we will review some recent advances In the development of anti-GnRH vaccines, and consider possible *erapeutic applications.
The basis of anti-GnRH vaccines Because GnRH is a short peptide and a ‘self molecule, it must be linked to an Immunogenic carrier proteIn to Induce an Immune response. In early studies (Arimura et al, Endocrindo@ 1973, 93:1092-1103; Fraser et al, Nature 1973, 244:160-161), GnRH was tagged to baine serum albumin. To obtain antibodies reactive with GnRH using these conjugates, Freund’s complete adjuvant (FCA) had to be used. These studies demonstrated the elkacy of immunization for blocking the fertility of both male and female animals. FCA is not suitable for use in humans, however, for whom alternative immunization strategies had to be evolved in which an adequate immune response could be obtained using adjuvants appropriate for humans. The observation that a conjugate
New Delhi, India 1990, 2:733-735
of GnRH with tetanus toxoid as the carrier could Induce anti-GnRH antibodies in mice even when the conjugate was administered with alum, an adjuvant approved for human vaccines, laid the basis for a potential vaccine (Shastri et al, Am J Reprod Immunoll981,1:262-265).
Sites for carrier conjugation and their implications In the past, carbodiimide [l-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride] was used to link GnRH to the carrier protein. As native GnRH does not have free carboxyl or amino groups, linkage is probably effected through the hydroxyl group of serine or tyrosine, or through a carboxylated derivative of histidine. The conjugate made by this method is poorly delined and gives inconsistent results. More successfkl conjugation was obtained by diazorization of the molecule, and coupling through hi&line and tyrosine residues (Pique et al, Immunocbem~ 1978, 15:55-&l; Koch et al, Bidwm B@l?ys Res Commun 1973, 55~616-622). The site at which the carrier is attached has a bearing on the reactivity pattern of the antibodies induced. Nett et reported al (I Clin Erzdxrinol Metub 1973,36:880-885) the Induction of an antibody directed to the amino-terminal end of GnRH when the carboxy-terminal end of GnRH was conjugated to bovine serum albumin. ArImura et al. (AC&ZEnabcrind 1975,78:222-231) confirmed this result and also showed that conjugation of GnRH with the carrier at the amino-terminal amino acid results iri antisera with reactivity against the carboxyterminal end. The Importance of an amide group at the carboxy terminal of the GnRH molecule in the generation of a’ conformation recognized by polyvalent antibodies is highlighted by studies based on two antisera derIvd_ from primates (Singh et al, J SteroidBiochem 1985,23:801402). However, from the reactivity pattern of a bioeifective monoclonal antibody to G&I-I, we (Talwar et al, Proc Nat1 Acud Sci US4 1985, 82:122%1231) concluded that the antigenicity of native GnRH depends on the entire
Abbreviations FCA-Freund’s
complete adjuvant; GnRtbgonadotrophin-releasing hormone; FSl+--follicle-stimulating lHRH--luteinizing hormone-releasing hormone; lT--tetanus toxoid.
@ Current
Biology
Ltd MN
0952-7915
hormone;
733
734
Reproduction
molecule and that the N-terminal and C-terminal regions are physically close. Recently, Silversides et al. [ 11, using analogues of GnRH substituted with cysteine at the carboxy and amino terminals to achieve conjugation to the carrier, keyhole limpet haemocyanin, have shown that the specificity of antibodies Induced by GnRH/carrler conjugates is a function both of the orientation of antigen presentation and of the responding animals immune regulatory mechanisms. These conclusions are in agreement with the multideterminan t regulatory model of antigenicity for protein antigens (Benjamin et al, Annu Rev Immunol 1984, 2:67-101). In another study [2], we have linked diphtheria toxoid as carder to GnRH through a functional group created by replacing glycine at residue six by o-lysine, which also increases the biological half-life of GnRH and confers conformational consttaints. The Eamino group of lysine is linked to a spacer, amino caproic acid, which is then conjugated to diphtheria toxoid. This conjugate gives a consistent immunogenic response in rodents and monkeys, probably because the linkage is both flexible and long enough to hold the peptide away from the surface of the carrier.
Control
of male fertility
Immunization of male rodents, rabbits and monkeys against GnRH leads to a block of spematogenesis. It is also accompanied by a drastic reduction in testosterone levels, but the extratesticular requirements for androgens can be met by injection of a slow-release androgen. Iadd et al [3,4] reported that spermatogenesis in male rats and rabbits was suppressed upon immunization with GnRH conjugated to tetanus toxoid (‘IT) and that administration of testosterone-17-trans-4-n-butyl cyclohexane carboxylate as a supplemental androgen restored libido and mating behaviour but did not restore spermatogenesis. All of the GnRH-‘IT immunized animals were infertile. Administration of the testosterone ester at the time of primary imrnumzation and then at lo-week intervals did not restore spermatogenesis, even though the serum testosterone levels in Infertile, GnRI-TT immunized, androgen-treated rats were similar to those in nonimmunized, androgen-treated fertile rats. Ladd et al [3,4] also worked out the dosages of androgen (10 mg/ml> needed to restore libido while keeping the animals infertile.
Control
of fertility
in animals
The ability of the anti-GnRH antibodies to suppress oestrous In female dogs has been demonstrated by the administration of a monoclonal antibody to GnRH in small amounts to female dogs at the iirst signs of heat (Tal-
war et al, 1985). A suppression of the progression of oestrous was indicated by behaviour changes, vaginal cytology and sex hormone profiles. Similar effects can be achieved by active immunization of dogs (Talwar et aA In Hormone Receptors in Growth and Reproduction edited by Saxena BB et al Raven Press, 1984, pp 351-359). A possible application of an anti-GnRH vaccine in animals bred for meat stems from the fact that androgens and their metabolites lower the quality of meat. In the past, diethylstilbesterol was added to animal feed to counteract androgens. However, it accumulates in the fatty tissues, is passed on to the consumer, and has been banned in the USA and other countries. Anti-GnRH immunization offers an alternative solution because it can markedly diminish androgen production in animals bred for meat, with no apparent side effects.
Control
of female fertility
Immunization of baboons and monkeys (Talwar et al, 1984) and marmosets (Hodges and Heam, Nature 1977, 265:746-747) against GnRH leads to an impairment of cyclic&y and to an ovulation block. A similar short-term effect can be obtained by administering monoclonal antibodies. Passive immunization stops ovulation in rats (Talwar et d, 1985) and can terminate pregnancy in mice (Gupta et al, Am J Reprod Immunol Microbial 1985, 7:104-108). The disadvantage of this method is the concomitant decline of the sex steroid hormones. Because the anovulatory cycles during lactational amenorrhoea are ascribable to the suppression of GnRH, another possible use of a GnRH vaccine could be in prolonging the gap between successive pregnancies in postpartum women. This was suggested by the results of Fraser et al [5], who showed that daily lntranasal administration of a GnRH agonist, Buserelin, to women prevents ovulation throughout the period of lactation. A decline in II-I and oestrone levels was noticed with &uppression of ovulation on chronic treatment, although in the iirst month there was an increase in LH and oestrone levels, with light bleeding. No changes were observed in nursing habits. These results illustrate the use of GnRH agonists as a potential method of contraception during the post-partum period. GNRH agonists administered in large amounts and repeated doses down-grade the response of the target cells to the hormone; the vaccine a&eves the same end result by causing inactivation of the hormone with antibodies directed against it. Similar effects have, in fact, been obtained by immunization against GnRH. The advantages of immunization ‘over agonist administration are that the costs are much lower and that daily treatment is not required as immunization is only needed periodically. Neonatal teratology studies in monkeys are underway in our laboratory to determine the safety of post-partum immunization of primates with the anti-GnRH vaccine.
Immunotherapy
Therapeutic
applications
and fertility control by immunization
of anti-GnRH
vaccines GnRH anaioguesthat suppress or antagonize the effects of GnRH have found applications in many clinical conditions, such as sex steroid hormone-dependent cancers, endometriosis, etc. That an anti-GnRH vaccine could be used to achieve similar results is demonstrated by our recent data. Rats immunized with the GnRH/diphtheria toxoid vaccine showed marked atrophy of the prostate [2]. Every animal responded by making anti-GnRH antibodies. There was a concomitant decline in testosterone levels in every animal. The effect of antibodies was reversed by native GnRH, but not by an analogue, indicating that the antibodies bind specifically to the native hormone. Although there was a reduction in the size of the testes and other androgen-sensitive secondary reproductive organs, the size of the prostate was more dramaticaLly reduced. A reduction in prostate size has also been observed (unpublished data) in monkeys after immunization with this vaccine. This particular vaccine has completed acute, subacute and chronic toxicology studies and has been approved for human use. Phase I/ Phase II clinical trials have started with this vaccine in patients who have carcinoma of the prostate in two medical institutes in India.
Conclusions Vaccine formulations have been developed to induce a consistent antibody response against GnRH in experimental animals. Immunization aga&t GnRH can be used to regulate fertility in both males and females. In the case of males, libido can be preserved by administration of androgens without spermatogenesis being restored. AntiGnRH vaccination may also be useful in lengthening the period during which ovulation is suppressed in post-parttun women. Of additional interest are the irnmunotherapeutic applications of immunization against GnRH in prostate hypertrophy and other pathological conditions where G&H agonists and antagonists are observed to be clinically effective.
Annotated
references
against CnRH Talwar and Sad
and recommended
reading l
*a
Of interest Of outstanding interest
SLVERSlDESDW,&IEN~,bfISRA V,Qu.UlERI L, MAF’IETOFTRJ, MURPHYBD: A synthetic luteinising hormone releasing hormone vaccine. Conjugation and speciticity trials in Balb/c mice. J Rgn-od Immund 1988, 13:24+261. Reports a novel method of conjugating GnRH to a carrier molecule with high e&iency using cysteine-substituted analogues of GnRH. The specificity of GnRH antiserawas shown to be dependent on the site of conjugation of the peptide to the carrier molecule. 1. 00
MK, SUVGH 0, AIAM A, TALWAR JAYMLUVKAR R, CHAT GP: Semisynthctic anti-LHRH vaccine causing atrophy of the prostate. Prostufe 19139, 143-H. Reports the structure of an anti-GnRHvaccine that cor&tently induces a bioeffective immune response. Immunization leads to atrophy of the prostate. This paper provides the basis for the therapeutic use of antiGnRH vaccines in hypertrophy of the prostate and in androgen-dependent carcinoma of the prostate.
2. l
o
3. 00
IADD A, PRABHUG, TSONCYY, PROBSTT, CHLMG W, TIL+LI R: Active immunization against gonadotrophin rclcasing hormone combined with androgen supplementation is a
promisingantifertilityvaccine
for males. Am J Reprod Im
mund Microbid 1988, 17~121-127. Reports 100% infertilityin rats upon active immunization against GnRH and describes the use of exogenous androgen supplements to restore libido and normal mating behaviour. I29~ 4 TSONG YY, PRABHU G, THAU R: Elkas of long-term immunization against LHRH and androgen treatment on gonadal function. J Rep& Immunol 19t3!9, 15:85-101. Neither short-term nor long-term treatment with androgen alone i&uenced fertilityin rats rendered infertile by immunization against GnRH. 4. l
FRASER HM, DEWARTPJ, Slwnl SK, CoWEN GM, SmW J, MCNEIUY As: Luteinising hormone releasing hormone agonist for contraception in breast feeding women. J Clin Endocrid Mekzbd 1989,69:996-1002. Clinical studies on nine women given a GnRH agonist every day in the post-partum period showed a block in ovulation without significantside effects or changes in nursing behaviour. The study indicates the poterrial of anti-GnRH treatment as an acceptable method of post-partum contraception.
5. we
735