New methods for the regulation of implantation

CONTRACEPTION

NEW METHODS

FOR THE REGULATION

R.J. Aitkenl

OF IMPLANTATION

and M.J.K.

Harper2

1 Human Reproduction Unit World Health Organization 1211 Geneva 27 2

Health

The University of Texas Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78284

ABSTRACT

This paper summarises the efforts of the World Health Organization to identify new methods of fertility control that would act by preventing or disrupting implantation. It reviews three major fields: (A) (H) (C)

The disruption of luteal function by inhibiting the early luteotrophic activity of the blastocyst; The disruption of luteal function by interfering with progesterone receptors in the endometrium; and The disruption of luteal function with prostaglandins, steroids and other compounds acting directly on the corpus luteum.

Accepted

for publication

July

11, 1977

RJA is a short-term consultant and MJKH was formerly Scientist in the World Health Organization's Human Reproduction Unit, Geneva.

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INTRODUCTION The successful initiation of blastocyst attachment is dependent upon the precise synchronization of endometrial, embryonic and ovarian function during the first days of pregnancy. The finely balanced nature of the mechanisms controlling nidation render this stage of development partiAs a consecularly susceptible to contraceptive attack. quence, the World Health Organization has attempted, through a series of Consultations held in Geneva, to identify new Three high approaches to the inhibition of implantation. priority research areas have been selected. (A)

The disruption of early luteotrophic

luteal function by inhibiting activity of the blastocyst;

(B)

The disruption of luteal function by interfering progesterone receptors in the endometrium,

(C)

The disruption of luteal function with prostaglandins, steroids and other compounds acting directly on the corpus luteum.

The purpose of this communication is to review rationale and theoretical background behind each of approaches to fertility control. The

Disruption of Luteal Function by Inhibiting Early Luteotrophic Activity of the Blastocyst

the

with

the these

the

One of the first functions an eutherian conceptus has to perform is to prevent the death of the corpus luteum at cycle or pseudothe end of the oestrous cycle, menstrual pregnancy. In animals possessing a uterine luteolysin such as the guinea pig, hamster, pig, rabbit, rat, or sheep, the action of the blastocyst is essentially antiluteolytic The nature of this antiluteolytic stimulus is (1, 2). unknown, but a pregnancy specific antigen detected in sheep plasma, myometrium, corpus luteum and embryo as early as day 8 of pregnancy has been implicated in this role (3). In women and monkeys, the early conceptus exerts a direct luteotrophic effect on the ovaries, as well as a possible antiluteolytic influence (4). The luteotrophic factor is thought to be chorionic gonadotrophin. In pregnant women, hCG levels start to rise about one day after implantation (5, 6) and quickly stimulate a rise in plasma progesterone levels (7, 8). A similar increase in the plasma concentration of progesterone has been observed during early pregnancy in the rhesus monkey (9), in association with implantation (10) and the anpearance of chorionic gonadoRecent evidence trophin in the peripheral circulation (11).

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has also been obtained in the rabbit (12, 13), rat (14) and mouse (15) to suggest that the foeto-placental units of other mammalian species may also produce gonadotrophic hormones. Any substance produced by the blastocyst which plays a vital part in the processes of implantation and luteal maintenance forms a logical point of attack for a contraceptive agent. By blocking the action of such factors, it should be possible to arrest pregnancy at the attachment stage without affecting the normal course of the menstrual cycle. In order to achieve this objective, immunogens are being developed for the active immunization of women against chorionic gonadotrophin. When either hCG or the B-subunit of this molecule is used as antigen, the resulting antibodies cross-react with LH (16). This is a consequence of the considerable amino acid homology in the a-subunit and NH>terminal portion of the B-subunit of each molecule (17-19). Recent studies have therefore employed natural or synthetic fragments of the hCG-specific COOH-terminal peptide of the B-subunit (16). Peptide fragments have been found which induce the formation of antibodies in baboons that crossreact with hCG, BhCG, baboon CG and the immunizing peptide, but not with human LH (V. Stevens, personal communication: (16)). An alternative approach towards the production of a specific immunogen has been described by Bahl (20). Partial reduction and S-alkylation of the S-subunit was found to induce a conformational change in t!le structure of the molecule leading to a preferential loss of hLH and the retention of hCG activity. The specificity of such preparations for hCG was improved by conjugation with tetanus toxoid. Talwar et al. (21) have also described the immunological properties o? aconjugate prepared from 'processed' f3hCG and tetanus toxoid. This conjugate elicited the formation of anti-hCG and anti-tetanus antibodies when injected into goats, mice, monkeys, rabbits and a human subject. A low degree of cross-reactivity with human LH was observed in certain cases, however, suggesting the need to develop more specific immunogens (20, 21). Another approach to fertility regulation involves the development of antihormones to compete with hCG for receptor sites on the corpus luteum. These compounds might be administered in the event of a missed menses, or on a oncea-cycle basis at the expected time of menstruation, to induce luteal regression. Yang, Samaan and Ward (22, 23) have recently reported the existence of an LH receptor-binding inhibitor (LHRBI) in aqueous extracts of luteinized rat A similar LH/hCG receptor-binding inhibitor has ovaries. recently been identified in extracts of pig corpus luteum (24).

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Enzymatic cleavage of selected carbohydrate residues from hCG has also been used in an attempt to produce a competitor lacking the luteotrophic properties of the parent moiecule (25-28). It has been found that removal of the sugars beyond sialic acid (galactose, N-acetyl glucosamine and mannose) does not affect the receptor-binding activity of hCG, but does destroy its ability to induce cyclic AMP accumulation in porcine granulosa cells (29). The potential of these derivatives as antigonadotrophins is demonstrated by their ability to inhibit hCG-induced progesterone secretion by porcine and monkey granulosa cells in vitro (30, 31). Important questions concerning the E vivohalf-life and immunogenicity of these molecules now await investigation. The Disruption of Luteal Function by Interfering Progesterone Receptors in the Endometrium

with

High affinity receptors for progesterone have been identified in the uterine cytosol of the calf, guinea pig, In a hamster, human, mouse, rabbit, rat and sheep (32-38). majority of species the progesterone receptor exhibits an association constant for progesterone of lo9 - 10" litre/mol. and sedimentation coefficients of 6-7s or 4-5s depending or. the salt concentration used in the density gradient buffer (32, 33). The formation of the steroid/receptor complex is both heat and acid labile (39) and appears to involve the formation of S-H groups, since sulph dry1 blocking agents like paraCu Y + and Zn2+ hydroxymercuribenzoate, (39, 40) inhibit binding activity. The 6-7s receptor of all species is characterized by a highly specific affinity for progesterone (41, 42). Detailed comparisons of the ligand binding specificities of receptor molecules isolated.from guinea-pig, human, rabbit and sheep uteri have revealed clear species differences, The binding characteristics of human, however (42, 43). rabbit and sheep receptors were found to be very similar, a result which should have an important bearing on our choice of a suitable animal model for screening potential antiprogestins. Comparative studies have also been carried out on the ligand specificities of progesterone receptors isolated from different tissues within species. In the guinea pig, for example, progesterone receptors have been identified in the cervix and vagina which closely resemble the uterine binding component (44). Similarly, the myometrial and endometrial progesterone receptors are thought to be very similar in such species as the guinea pig, human and sheep (39, 45, 46).

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The synthesis of the 6-75 receptor is under hormonal control. The uterine concentration of this receptor declines dramatically following ovariectomy, but rapidly increases again in response to subsequent oestrogen treatment (32, 39, 47). In contrast to the stimulative effect of oestrogen, progesterone induces a marked decline in the number of uterine receptor sites (47). Determination of uterine 6-7s receptor concentration during the oestrous cycle of the hamster and guinea pig has revealed characteristic changes which reflect the cyclical fluctuations in ovarian steroid production (36, 48). Hence, receptor concentration is maximal during prooestrus when plasma oestrogen levels are high: and minimal during dioestrus when oestrogen levels are low and progesterone is being secreted by the corpora lutea. Similarly, the concentration of binding sites in human myometrial and endometrial tissue reaches a maximum during the late proliferative phase as a conseauence of the preovulatory rise in ovarian oestrogen secretion (49, 50). Oestrogen administration to postmenopausal women also induces the appearance of a 6-7s progesterone-binding receptor in myometrial cytosol (39). Both RNA and protein synthesis appear to be involved in the oestrogen-induced increase in 6-7s receptor concentration (47, 51). The suppressive effect of progesterone treatment is associated with the disappearance of the 6-7s binding component from the uterine cytosol, and may be the result of an inhibition of uterine RNA synthesis (52) or the destruction of the receptor with proteolytic enzymes. In addition to the 6-7s binding component, there is some evidence for another category of receptor sedimenting at about 4s. In the normal cyclic human uterus, for example, progesterone receptors have been identified in both the endometrium and myometrium with sedimentation coefficients of 3.7-3.8s C-50, 53). The pregnant guinea pig uterus also possesses a 5s progesterone receptor which contrasts with the 7s binding component induced by oestrogen (54). It has been suggested that the oestroqen-induced 6-7s component represents a complex of the true receptor and another oestroqendependent macromolecule (33). The manipulation of receptor function offers great promise for the development of new contraceptive agents. Antiprogestins are currently being sought which posses the ability to compete with endoqenous progesterone for uterine binding sites, but which lack progstational activity. Affinity labelling antiproqestins have been described by Warren (55) which irreversibly bind the receptor molecule by the Such a compound is 16a-bromoformation of covalent bonds. acetoxy-progesterone, an alkylatinq agent which suffers from However, the disadvantage of being a potential carcinogen.

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CONTRACEPTION recent studies with other compounds, e.g. R2323 (13-ethyl-17hydroxy-18,19-dinor-17a-4,9,ll-pregnatrien-2O-yn-3-one~ Roussel-Uclaf), indicate that the development of non-toxic antiprogestins should be possible (56). These compounds could be used on a once-a-cycle basis to saturate the large number of free receptor-sites present in the endometrium during the late proliferative phase. Alternatively, antioestrogens could be administered shortly after the termination of menstruation to inhibit the oestrogen-induced synthesis of progesterone receptors. One advantage of this approach is that several ootentiallv useful antioestrogens are currently available, e.g. RU16117 (lla-methoxy-19-nor-17a-1, 3,5(10)-pregnatrien-20-yne-3,17-dial; Roussel-Uclaf) (56). The Disruption of Luteal Function with Prostaglandins, Steroids and Other Compounds acting directly on the Corpus Luteum Prostaglandin

Fza

(PGF2c0

In a number of domestic and laboratory animals, removal of the uterus significantly extends the life span of the corpus luteum (cow, guinea pig, hamster, pig, rabbit, rat, sheep) (1). This consequence of hysterectomy is thought to be due to the removal of a uterine luteolytic factor which is normallv released at the end of the luteal phase and induces luteal regression. There is now a substantial body of evidence indicating that the uterine luteolysin is PGF2a (57). This compound induces luteolysis in cows, guinea pigs, hamsters, mares, monkeys, pigs, rabbits, rats and sheep (1, 57, 58). In contrast, uterine luteolysins do not appear to play a major role in the regulation of human luteal function since neither hysterectomy (59) nor systemically administered PGF2a (60) influence corpus luteum activity in women. Luteolysis has been observed, however, following either the intraovarian injection of this compound (611, or the exposure of luteinized human granulosa cells to PGFza in vitro (62). Since human ovarian tissue is capableofsynthesizing PGF2a (63), it is possible that normal luteal regression is induced by prostaglandins manufactured within the corpus luteum ifself. The mechanism by which PGF2a induces luteal regression has been the subject of much disagreement, possibly as a An influence of PGFzu on result of swecies differences. the vascularization of the corpus luteum has been suggested bv exoeriments on the sheep (64) and rabbit (65). However, when &uce and Hillier (66j administered PGF2a to rabbits, they observed a marked fall in plasma progesterone concentration several hours before any vascular changes could be Evidence has also been obtained in detected in the ovaries. rats to suggest that such vascular changes are secondary to the initiation of luteolysis by PGF2a (67). The fact that

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PGF2a is luteolytic -in vitro suggests a direct biochemical effect on the corpus luteum. In rats and mice, an important step in PG-induced luteolysis appears to be the activation of an enzyme, 20a-hydroxysteroid dehydrogenase (20a OHSDH), which catalyses the conversion of progesterone to a biologically less active derivative, 20a-dihydroprogesterne OHSDH by PGFza is closely (68). The induction of 20a associated with a loss of LH binding sites from the corpus lutea but, once again, these changes occur some time after the initial inhibition of progesterone secretion (69). The mechanism by which PGFza initiates luteolysis is poorly understood. The process can be antagonised in vivo (70) and in vitro (62, 71) by the administration of gEiiadotrophins, EdHenderson and McNatty (72) have suggested that PGF2a directly inhibits the activation of adenyl cyclase by receptor bound LH. Oestradiol-17f? Oestrogenic compounds have been found to induce luteolysis in both laboratory (guinea pig, hamster, rabbit, rat) and domestic (cow, pig, sheep) animals (1, 73). Systemically administered oestrogens are also known to exert a powerful antifertilitv effect in women when iniected post-coitallv, and there is-some evidence to support-a luteblytic mode of action (74-76). Not only does oestrogen administration during the luteal phase bring about a decline in plasma progesterone levels and a shortening of the cycle (74, 77), but also oestrogen crystals implanted within the human corpus luteum induce local luteolysis (78). Although neither Oriol-Bosch and Cortes (73) nor Board et al. (79) could detect any sign of luteolysis following theadiiiinistration of oestradiol benzoate and diethylstilbestrol respectively, the dose and/or potency of the oestrogens used in these experiments was lower than those in which a luteolytic or contraceptive effect was observed. The administration of luteolytic agents for the induction of menstruation is an attractive approach to fertility regulation. There is an urgent need for research on the normal mechanism of luteal regression, particularly in primates, with a view to identifying new approaches for the induction of luteolysis. It is possible that both prostaglandin analogs and oestrogens have some potential in this respect, although the harmful side effects associated with the administration of these compounds represents a major obstacle to their future use.

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