AU2894797A - Improvement of implantation rates after in vitro fertilization - Google Patents

Description

IMPROVEMENT OF IMPLANTATION RATES AFTER IN VITRO FERTILIZATION

Background of the Invention

This invention relates to a method for the improvement of implantation rates after in vitro fertilization (IVF), for the treatment of infertility and for the treatment and prevention of early pregnancy loss in women with a nitric oxide synthase substrate (L-arginine), a nitric oxide donor or both, alone or in combination with progesterone and/or estrogen. This invention also relates to the novel methods of fertility control based on inhibition of endometrial receptivity, prevention of implantation, and menstrual induction with NOS inhibitors, preferentially iNOS inhibitors, in combination with progesterone antagonists, and/or progesterone synthase inhibitors.

Human in vitro fertilization is surprisingly unsuccessful. The overall birth rate per IVF treatment cycle is approximately 14% in USA (Medical Research Intemationai Society for Assisted Reproductive Technology [SARTJ, The American Fertility Society [1992]. Fertil Steril 5:15), and 12.5% in UK (The Human Fertilization and Embryology Authority. Annual Report, London 1992).

Success is greater when more than one embryo is transferred simultaneously. However, simultaneous transfer of multiple embryos increases the incidence of multiple pregnancy and the possibility of miscarriage and prematurity. The reasons for the low pregnancy rates after IVF are still not completely understood. The quality of both the embryo and the uterine environment affects success. Generally, there is a high rate of spontaneous early abortion in fertile cycles in women. After natural conception, possibly as many as 50-60% of very early pregnancies are lost (Winston ML, Handyside AH [1993], New challenges in human in vitro fertilization. Science 260:932-935). This may be due to both conceptus abnormalities and dysynchrony between embryo and endometrium at the time of embryo transfer.

Most losses may be due to abnormalities of the conceptus or the still inappropriate culture conditions, since the success of embryo transfer after IVF decreases as the time after insemination increases (Winston ML, Handyside AH [1993], New challenges in human in vitro fertilization. Science 260:932-935).

To overcome possible deficiencies in culture media, transfer of oocytes (gamete iπtrafallopian transfer- GIFT) or zygotes directly to the fallopian tube (zygote itrafallopian transfer- ZIFT) has been performed in women with intact oviducts. However, these attempts only slightly increased the fertility and birth rates after IVF (Edwards RG [1995] Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3: 60-67).

The effect of uterine environment on fertility rates after IVF may be equally important. It has been well established that the successful establishment of pregnancy after embryo transfer requires both a healthy blastocyct and a receptive uterus. Embryo transferred to an inadequately primed uterus are unlikely to implant. In all mammals, the endometrium. is receptive for implantation only during the specific period of time after ovulation. This phase of the luteal phase is called "implantation window". In women, the successful implantation may only take place between days 15-20 of a histologically defined 28-day cycle, i.e. during the period of highest progesterone levels (Navot D, Scott RT, Droesch KD, Veeck LL, Hung- Ching Liu, Rosenvaks Z [1991], The window of embryo transfer and efficiency of human conception in vitro. Fertil Steril 55:114-118). The optimum condition for implantation was estimated on days 20-22 of the normal cycle, i.e. 7 days after the LH surge (Bergh PA, Navot D [1992], The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 58: 537-542).

Adequate progesterone priming of the endometrium endometrium is essential for a successful implantation, and treatment with an antiprogestin during the luteal phase will completely prevent implantation {Chwalisz K, Stόckemann K, Fuhrmann U, Fritzemeier KH, Einspanier A, Garfield RE [1995] Mechanism of action of antiprogestins in the pregnant uterus. In Henderson D, Philibert D, Roy AK, Teutsch G (eds) Steroid Receptors and Antihormones. Ann N. Y. Acad Sci 761:202-224). In the fertile cycle, progesterone regulates the transport of the fertilized egg through the oviduct and induces secretory changes required for implantation in the endometrium. Implantation is a precisely timed event in mammals. The secretory endometrial proteins (Beier HM, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992] Dissociation of corpus luteum, endometrium and blastocyst in human implantation research. J Reprod Fert 92:511-523), and probably other intracellular and cell surface proteins, such as integrins, cytokines and growth factors produced by endometrial epithelial cells as a result of progesterone stimulation, are necessary for implantation to take place (Edwards RG [1995] Physiological and molecular aspects of human implantation. Hum Reprod 10, Suppl 2: 1-14).

The asynchrony between embryo and endometrial development has been previously recognized as one of possible causes of implantation failures after IVF (Beier HM, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992] Dissociation of coφus luteum, endometrium and blastocyst in human implantation research. J Reprod Fert 92:511-523). However, no effective methods to increase the implantation rates are available to date. The most advanced stages of human implantation are chracterized by the invasion of trophoblastic cells into the decidua and angiogenesis (Loke YW, King A [1995] Human Implantation. Cell biology and immunology. Cambridge University Press). This stages are also dependent on progesterone, since progesterone antagonists also disrupt early pregnancy (Chwalisz K, Stόckemann K, Fuhrmann U, Fritzemeier KH, Einspanier A, Garfield RE [1995] Mechanism of action of antiprogestins in the pregnant uterus. In Henderson D, Philibert D, Roy AK, Teutsch G (eds) Steroid Receptors and Antihormones. Ann N. Y. Acad Sci 761:202-224). During early pregnancy, an adequate blood flow to the uterus is essential for embryo development. An impaired blood flow to the uterus can jeopardise the establishment of pregnancy (Edwards RG (1995) Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3: 60-67). Patients with an impeded blood flow have been given aspirin to improve their blood flow. Low dose aspirin is thought to increase the prostacyclin to thromboxane A2 ratio and thereby to increase placental perfusion. However, the aspirin effect on uterine blood flow were only marginal (Goswamy RK, Williams G, Steptoe PC [1988], Decreased uterine perfusion - a cause of infertility. Hum. Reprod 3955-959; Wada I, Hsu CC; Williams G, Macnamee MC, Brinsden PR [1994], The benefits of low-dose-aspirin therapy in women with impaired uterine perfusion during assisted conception. Hum Reprod 9:1954-1957).

There is a need for further improvement of contraceptive methods. Special attention should be given to further reduction of the total monthly dose and intake frequency of a contraceptive drug. During the last decade several possibilities for using antiprogestins in fertility regulation have been suggested, based either on the inhibition of ovulation or the prevention of implantation after post-coital or early luteal phase treatment (Spitz IM, Bardin CV (1993) Clinical pharmacology of RU 486- an antiprogestin and antiglucocorticoid. Contraception 48: 403-444). However, only post-coital treatment with mifepristone (GlasierA, Thong KJ, Dewar M, Mackie M, Baird DT (1992) Mifepristone (RU 486) compared with high- dose estrogen and progestestogen for emergency postcoital contraception. N Engl J Med :327:1041-4) and its early luteal phase administration on LH+2 (Gemzell-Danielsson K, Westlund P, Johannisson E, Swahn ML, Bygdeman M, SeppQIS (1996) Effect of low weekly doses of mifepristone on ovarian function and endometrial development. Hum Reprod 11: 256-264) have as yet been proven to be effective in women. The contraceptive effects of mifepristone in these approaches are most likely due to its effects on the endometrium. The post-coital treatment with mifepristone can be used only occasionally, otherwise it will interfere with ovulation and induce amenorrhea. The use of mifepristone on LH+2 is rather inconvenient, since the LH surge has to be identified in every cycle. The available data on antiprogestins suggest that there is also a possibility to develop a once-a-month pill for menstrual induction and contraception. Such a method could be used in case of emergency, but also on a regular basis. The antiprogestin RU 486 has been given immediately post- coitum and found to be highly effective in the inhibition of implantation (GlasierA, Thong KJ, Dewar M, Mackie M, Baird DT (1992) Mifepristone (RU 486) compared with high-dose estrogen and progestogen for emergency postcoital contraception. N Engl J Med :327:1041- 4). However, RU 486 does not effectively terminate pregnancy when given alone (i.e without a prostaglandin) during the late luteal phase as a once-a-month contraceptive (Couzinet B, le Strat N, Silvestre L, Schaison G. (1990) Late luteal administration of the antiprogesterone RU 486 in normal women: Effects on the menstrual cycle events and fertility control in a long-term study. Fertil Steril 54:1039-1044). The efficacy of RU 486 alone in terminating pregnancy, irrespective of the dose, is about 80-85% within 10 days after missed menses, about 65% within 56 days of amenorrhea and less than 40% in later stages of pregnancy (Van Look PFA, Bygdeman M (1989) Antiprogestational steroids: a new dimension in human fertility regulation. ln:Oxford Reviews of Reproductive Biology 11:1-60). The administration of RU 486 to pregnant women with amenorrhea of 7 weeks or less induced complete abortion in 65- 85% of the subjects. However, the addition of a prostaglandin increases the efficacy to 95% or more (Van Look PFA, Bygdeman M (1989) Antiprogestational steroids: a new dimension in human fertility regulation. ln:Oxford Reviews of Reproductive Biology 11:1-60, Aubeny E, Baulieu EE (1991) Contragestive activity of RU 486 and oral active prostaglandin combinantion Comptes Rendus de L'Academie des Science 312:539-45). These studies indicate that an additional treatment with a prostaglandin improves the efficacy of antiprogestins to terminate an advanced pregnancy. However, it remains unclear whether the same synergistic effect can be achieved during very early pregnancy, i.e. within the first week of amenorrhea. In addition, prostaglandins are known to induce gastro-intestinal side-effects which may be unacceptable during a regular use for contraception. One of the most exciting recent advances in biology and medicine is the discovery that nitric oxide is produced by endothelial cells and that its is involved in the regulation of vascular tone, platelet aggregation, neurotransmission and immune activation. Nitric oxide is an important mediator of relaxation of the muscular smooth muscle and was formerly known as EDRF (endothelin-derived relaxing factor) (Furchgott RF and Zawadzki JV [1980], The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376; Moncada S, Palmer RMG and Higgs EA [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). Nitric oxide is synthesized by the oxidative deamination of a guanidino nitrogen of L-arginine by at least three different isoforms of a flavin-containing enzyme, nitric oxide synthase (Moncada S, Palmer RMG and Higgs EA [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). Synthesis of nitric oxide has been shown to be competitively inhibited by analogues of L-arginine; NG-nitro-L-arginine methyl ester (L- NAME), NG-monoethyl-L-arginine (LMMA), N-iminoethyl-L-amithine (L-NIO), L-monomethyl- L-arginine (L-NNMA) and L-NG-methylarginine (LNMA) and Nw-nitro-L-arginine (L-NA).

Nitric oxide elevates levels of cGMP (1,4,5-cyclic guanosine monophosphate) within the vascular smooth muscle to produce relaxation and to reduce blood vessels tone (Moncada S, Palmer RMG and Higgs EA [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). Nitric oxide of these compounds been realized. Nitrovasodilators are now classified as nitric oxide donors because they are metabolized or spontaneously release nitric oxide (Moncada S, Palmer RMG and Higgs EA. [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). The long-used nitrovasodilators may be regarded as substitution therapy for a failing physiological mechanism. Nitric oxide is also produced by macrophages and other immune cells.

Three highly related NOS enzymes have been isolated and identified. These include endothelial NOS (e-NOS, type III), neuronal NOS (n-NOS, type II) and inducible NOS (i- NOS, type I) (Knowies RG and Moncada S [1994], Nitric oxide synthases in mammals. Biochem J 298:249-258; Sessa WC. [1994],The Nitric Oxide Synthase Family of Proteins. J Vase Res 1994; 31:131-143; Nathan C [1992], Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 301-3064). The constitutive isoforms e-NOS and b-NOS were originally identified in endothelial and neuronal tissues, respectively, and they rapidly and transiently produce small amounts of NO under basal conditions. The i-NOS isoform is inducible by cytokines or endotoxin (LPS) and it produces large quantities of NO for hours or days in a Ca²⁺-ιndependent manner Cells expressing iNOS do not generate NO under basal conditions The e-NOS form of the enzyme is expressed in endothelial cells, in cardiac myocytes, platelets and some neurones The e-NOS-denved NO is the most important vasodilator It is released in low levels to maintain a constant vasorelaxation and maintain normal blood pressure The n-NOS isoform is thought to act as a neurotransmitter It is thought to be important in mediating such functions as gastrointestinal motility and penile erection

There is a substantial body of evidence from animal expenments that a deficiency in nitπc oxide contributes to the pathogenesis of a number of diseases, including hypertension, atherosclerosis and diabetes (Moncada S, Palmer RMG and Higgs EA [1991], Nitπc oxide; physiology, pathophysiology and pharmacology Pharmacol Rev 43 109-142) There are many recent studies showing that the inhibition of nitnc oxide synthase dramatically increases blood pressure Treatment of pregnant rats and guinea pigs with nitπc oxide synthase inhibitors produce symptoms identical to preeclampsia (Chwalisz K and Garfield

RE [1994], Role of progesterone dunng pregnancy Models of partuπtion and preeclampsia

Z Geburtsh u Pennat 198'170-180) Preeclampsia is characterized by increased blood pressure and peπpheral vascular resistance, fetal growth retardation, proteinuπa and edema In humans, histopathologic and clinical (fetal growth retardation, fetal death) evidence indicate that reduced placental perfusion is the eariiest and most consistent change observed in preeclampsia (Roberts JM and Redman CWG [1993], Pre-eclampsia. more than pregnancy-induced hypertension 341 1447-1451, Fπedman EA [1988],

Preeclampsia a review of the role of prostagiandms Obstet Gynecol 71 122-137)

The L-arginine-NO system is present in the uterus (Garfield RE and Yallampalli C [1993] Control of myometπal contractility and labor In. Basic Mechanisms Controlling Term and Preterm Birth ed K Chwalisz, RE Garfield, Spπnger-Veriag, New York, pp 1-29, Chwalisz K and Garfield RE [1994], Antiprogestins in the Induction of labor. Ann New York Acad Scie 734.387-413; Buhtmschi I, Yallampalli C, Dong Y-L and Garfield RE [1995], Involvement of a nitπc oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility duπng pregnancy Am J Obstet Gynecol 172:1577-1584, Sladek SM, Regenstrin AC, Lykms D et al [1993 ], Nitnc oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy Am J Obstet Gynecol 169-1285-1291) This system plays an important role in control of uteπne contractility, pregnancy maintenance, onset of labor and also fetal perfusion L-arginine and nitπc acid caused a rapid and substantial relaxation of spontaneous activity of the utenne stnps from rats at mid to near term gestation {Buhimschi I, Yallampalli C, Dong Y-L and Garfield RE [1995] Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol 172:1577-1584; Garfield RE and Yallampalli C. [1993] Control of myometrial contractility and labor. In: Basic Mechanisms Controlling Term and Preteπn Birth, ed: K. Chwalisz, RE Garfield, Springer-Veriag, New York, pp. 1-29; Sladek SM, Regenstrin AC, Lykins D. et al. [1993] Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 169:1285-1291; Natuzzi ES, Ursell PC, Harrison M. et al [1993], Nitric oxide synthase activity in the pregnant uterus decreases at parturition. Biochem Biophys Res Commun 194:108-114; Jennings RW, MacGillvray TE and Harrison MR. [1995], Nitric oxide inhibits preterm labor in the rhesus monkey. J Mat Fet Med 2:170-175).

The expression of NOS enzymes in the rat uterus was studied with immunoblotting with monoclonal antibodies. i-NOS and e-NOS were detected in the uterus (myometrium) The uterine i-NOS enzyme decreased in the uterus during labor at term and preterm in animals treated to deliver prematurely. Opposite changes were observed in the cervix (Buhimschi I, AH M, Jain V, Chwalisz K and Garfield RE. [1996], Differential regulation of nitric oxide in the uterus and cervix during pregnancy and labor. Human Reproduction [in press]).

NOS is also present in placental tissues and uterine arteries. The trophoblast invasion of uteroplacental arteries in relation to the NO synthase isoform expression was studied in pregnant guinea pigs by means of immune- and histochemistry as compared to arterial dilatation. A pronounced dilatation of uteroplacental arteries begins at mid-pregnancy and progresses until term (Nanaev A, Chwalisz K, Frank H-G, Kohnen G, Hartung C-H and Kaufmann P. [1995], Physiological dilation of uteroplacental arteries in the guinea pig depends upon nitric oxide synthase activity of extravillous prophoblast. Cell Tissue Res:282: 407-421). This study demonstrates that dilatation of uteroplacental arteries can be seen when invading trophoblast cells coexpressing endothelial (eNOS) and macrophage (iNOS) nitric oxide synthase are found in the vicinity of the vessels, i.e. prior to trophoblast invasion of the arterial walls. Conrad et al.,(1993), localized NOS to the syncythiotrophoblast cell layer in human placenta (Conrad KP, Vill M, Mcguire PG, Da// WG, Davis AK [1993], Expression of nitric oxide synthase by syncythiotrophoblast in human placental villi, FASEB J 7:1269-1276). Morris et al., (1993), demonstrated both calcium-dependent and calcium- independent activity in human placental villi and the basal plate (Morris NH, Sooranna SR, Eaton BM, Steer PJ (1993) NO synthase activity in placental bed and tissues from normotensive pregnant women. Lancet 342:679-680), and Myatt et al (1993), showed that placental villous tree synthesized a calcium-dependent-isoform of the NOS (Myatt L, Brockman DE, Langdon G, Pollock JS [1993], Constitutive calcium-dependent isoform of nitric oxide synthase in the human placenta villous vascular tree. Placenta 14:373-383; Myatt L, Brockman DE, Eis AL, Pollock JS [1993] Immunohistochemical totalization of nitric oxide synthase in the human placenta. Placenta 14: 487-495). In addition, Buttery et al., (1994) showed that endothelial NOS at term was localized in the endothelium of umbilical artery and vein and in the placental syncythiotrophoblast (Buttery LDK, McCarthy A, Springall A et al., [1994], Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at feto-matemal interface. Placenta 15: 257-267). Furthermore, Moorhead et al., (1995) have shown that NADPH diaphorase (non-specific reaction to identify nitric oxide synthase) was in various uterine components during early pregnancy (Moorhead CS, Lawhun M, Nieder GL [1995], Localization of NADPH diaphorase in the mouse uterus during the first half of pregnancy and during an artificially-induced decidual cell reaction. J Histochem Cytochem 43:1053-1060). Finally, Toth et al., (1995) demonstrated that NOS activity was present in the first trimester human placental homogenates (Toth M, Kukor Z, Romero R, Hertelendy F [1995], Nitric oxide synthase in first trimester human placenta: Characterization and subcellular distribution. Hypertens Pregnancy 14/3: 287-300).

These studies suggest that nitric oxide is an important factor regulating placental blood flow and myometrial quiescence during pregnancy. However, there are no studies published to date which demonstrate the detrimental effects of NOS inhibition on implantation or the beneficial effects of nitric oxide donors or substrates on implantation after IVF or in women with early pregnancy loss. In contrast, Haddad et al„ (1995) suggested that the increased nitric oxide production is associated with early embryo loss in mice and that iNOS inhibitors can by used to treat early abortion (Haddad EK, Duclos AJ, Baines MG [1995], Eariy embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med 182:1143-51).

Objects of the Invention

It is an object of the invention to provide a method for the improvement of implantation rates after IVF comprising administering a nitric oxide substrate and/or donor in mammals. It is another object of the invention to provide a method for treatment and prevention of infertility or early pregnancy loss comprising administering a nitric oxide substrate and/or donor in early pregnant mammals.

It is a further object of the invention to provide a method for improvement of implantation and treatment and prevention of infertility or early pregnancy loss comprising administering a nitric oxide substrate and/or donor in which progesterone or a progestagenic agent in combination with a nitric oxide substrate and/or donor is used.

It is another object of this invention to provode a method in which a progestational agent and an estrogenic agent are administered in combination with a nitric oxide substrate and/or nitric oxide donor for the for improvement of implantation and treatment and prevention of infertility or early pregnancy loss female mammals.

It is another object of this invention to provide a method for fertility control comprising administering antiprogestin (e.g. mifepristone, ORG 31710, ORG 33 628, J867, CDB 2914, ZK 137316) and/or progesterone synthase inhibitor (e.g. epostane, trilostane) in combination with an nitric oxide synthase inhibitor.

It is another object of this invention to provide a method for fertility control comprising administering an antiprogestin and/or a progesterone synthase inhibitor in combination with an nitric oxide synthase inhibitor for once-a-month contraception.

It is another object of this invention to provide a method for fertility control comprising administering an antiprogestin in combination with an nitric oxide synthase inhibitor for termination of eariy pregnancy.

A further object is the provision of pharmaceutical compositions useful in practicing the methods of this invention. Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art. Summary of the Invention

In a method aspect, this invention relates to a method of improving implantation rates after IVF or treating of early pregnancy loss which comprises administering to an individual manifesting the symptoms thereof one or both of a nitric oxide substrate and a nitric oxide donor, alone or in combination with progesterone alone or in further combination of an estrogen in amounts effective to ameliorate the symptoms thereof, the amount of the nitric oxide synthase substrate and nitric oxide donor or both being effective to increase implantation and birth rates by raising the blood level of circulating L-arginine in a female to whom the composition is administered to at least 10 to 500 μmole above the normally 50-

1000 μmole circulating levels or raise nitric oxide donor levels to about 10 nM to 100 μM

(micromolar), the amount of the progestational agent administered being bioequivalent to

10-300 mg of injected progesterone, and the amount of the estrogen being bioequivalent to approximately 2 mg per day of estradiol (Progynova ^R, Schering).

In a method aspect, this invention relates to a method of fertility control which comprises administering to a female mammal a nitric oxide inhibitor and an antiprogestin, alone or in combination with a progesterone synthase inhibitor. In one embodiement, this fertility control is effected prior to implantation. A nitric oxide inhibitor, preferentially an iNOS inhibitor, can be used in combination with an antiprogestin for the inhibition of implantation after administration during the periovulatory period (LH peak +/- 4 days), as emergency contraception (i.e. within 72 hours of an unprotected intercourse), for the inhibition of endometrial receptivity using continous daily treatment, for the inhibition of endometrial receptivity using once-a-week treatment, for menstrual induction (i.e. within the first week of amenorrhea of the fertile cycle), or regularly for menstrual induction every 28 +/- 3 days.

In a product aspect, this invention relates to a pharmaceutical composition comprising at least one of a nitric oxide synthase substrate (L-arginine) and a nitric oxide donor (e.g. sodium nitroprusside or glyceryl trinitrate), alone or in further combination with one or more of a progesterone/progestin and/or estradiol/estrogen with the amount of the nitric oxide synthase substrate, a nitric oxide donor or both per unit dosage being equivalent to either raise the blood level of circulating L-arginine to least 10 to 500 μM above the normally 50 to 1000 μM circulating or raise nitric oxide donor levels to about 10 nM to 100 μM, the amount of the estrogen being bioequivalent to about 2 mg of estradiol (e.g. Progynova ^R, Schering) with the amount of the progesterone bioequivalent to 5 to 300 mg of injected progesterone. In a product aspect, this invention relates to a pharmaceutical composition comprising at least one of a nitric oxide synthase inhibitor, preferentially an iNOS inhibitor (e.g. L-

NMMA, L-NIO, L-NIL, aminoguanidine, AMT, 4-methyl-2-aminopiridine, 6,4-dimethyl-2- aminopiridine, or 2-aminopiridine) in an effective dose in combination with an antiprogestin alone or/and in further combination with a progesterone synthase inhibitor.

Detailed Disclosure

The methods of this invention improve the implantation and birth rates after IVF and treat early pregnancy loss in pregnant female, who is manifesting the symptoms thereof.

Because the low implantation and birth rates and early pregnancy loss are produced by or aggravated by inadequate uterine blood supply to the conceptus due to insufficient or subnormal nitric oxide synthesis, both nitric oxide synthase substrates, e.g., L-arginine and nitric oxide donors, e.g., sodium nitroprusside, nitroglycerin, glyceryl trinitrate, SIN-1, isosorbid mononitrate isosorbid dinitrate and diethylenetriamine/NO (DETA/NO), are useful for ameliorating the symptoms thereof and, in one respect of this method of this invention, a combination of both are employed.

An additive effect is achieved when a progestagenic and/or and estrogenic agent is administered concurrently with a nitric oxide substrate and/or nitric oxide donor. In the case of a female mammal, a progestagenic agent can be administered concurrently with or in lieu of an estrogen.

Thus, the method aspect of this invention and the pharmaceutical composition aspect of this invention employs either or both of a nitric oxide substrate and a nitric oxide donor and, optionally one or more of an estrogen (e.g., Progynova ^R, Schering) or a progestin (e.g. progesterone or hydroxyprogesterone caproate [Proluton^R Depot), etc.).

In the method aspect of this invention regarding the fertility control, a synergistic or additive effect is achieved when a progesterone receptor antagonist (antiprogestin) and/or a progesterone synthese inhibitor is administered concurrently with a nitric oxide synthase inhibitor, preferentially an iNOS inhibitor. Examples of dosage ranges of typical NO-substrates and NO-donors (per os) are:

total dose:

L-Arginine 500 mg - 10 g p. o.

Sodium nitroprusside 500 - 2000 μg/kg/day

Nitroglycerin 0.5 - 10 mg

Isosorbid mononitrate 10 - 100 mg

Isosorbid dinitrate 10 - 100 mg

Examples of combinations of active agents which can be administered concurrently with a nitric oxide substrate and/or nitric oxide donor are the following estrogens and progestins and typical oral dosage ranges active agents of the estrogen and progestin with the nitric oxide substrate or donor

Estrogens: A daily dose bioequivalent to about 1 to 2 mg per day, e.g., Premarin ^R, Wyeth-Ayerst, 0.625 mg/day, estradiol 25-100 μg/day transdermally or vaginal estradiol gels or creams.

Progestins: A daily dose bioequivalent to 50 - 300 mg of progesterone/day, e.g., hydroxyprogesterone caproate i.m. to provide a weekly dose of thereof of 100 - 1000 mg or tablets or dragees providing an effective oral dose of micronized progesterone, or vaginal gel of progesterone in a daily dose of 2-300 mg/day.

Examples of progestins are listed below

Product Composition Dose

ProlutonR Depot (Schering) Hydroxyprogesterone caproate 250-1000 mg/week i.m. Progesteron-Depot (Jenaphamn) Hydroxyprogesterone caproate 250-1000 mg/week i.m.

Examples of estrogens are listed below:

Product Composition Dose (mg per day)

Climava! (Sandoz) Estradiol valerate 0.5 - 4 mg Progynova (Scheπng) Estradiol valerate 0.5 - 4 mg

Examples of antiprogestins are listed below.

11β-[4-N,N-(dιmethylamιno)phenyl]-17a-hydroxy-17β-(3-hydroxypropyl)-13a-methyl-4,9(10)- gonadιen-3-one,

11 β-(4-acetylphenyl)-17β-hydroxy-17a-(3-hydroxyprop-1 (Z)-enyl)-4,9(10)-estradιen-3-one (EP-A O 190 759), 11β,19-[4-(cyanphenyl)-o-phenylen]-17b-hydroxy-17a-(3-hydroxyprop-1 (Z)-enyl)-4-androsten 3-one (WO-A 93/23020)

11 β, 19-[4-(3-pyπdιnyl)-o-phenylen]-17β-hydroxy-17a-(3-hydroxyprop-1 (Z)-enyl)-4-androsten 3-one (WO-A 93/23020)

Examples of iNOS inhibitors are listed below.

Dose

L-NMMA (NG-monomethyl-L-argtnιne), 1-1000 mg/day

L-NIO (L-N⁵-(1-ιmιnoethyl)ornithιne), 1-1000 mg/day

L-NIL (L-N⁶-(1-ιmιnoethyl)lysιne, 1-1000 mg/day

Aminoguanidine, 1-1000 mg/day

AMT (2-amιno-5,6-dιhydro-6-methyl-4H-1,3-thiazιn) (Abbott), 0 1-100 mg/day

4-methyl-2-amιnopyπdιne, 0.1-100 mg/day

6,4-dιmethyl-2-amιnopyπdιne, 0 1-100 mg/day

2-amιnopyrιdιne, 0 1-100 mg/day

The pharmacologically active agents employed in this invention can be administered in admixture with conventional excipients, i e., pharmaceutically acceptable liquid, semi- liquid or solid organic or inorganic camers suitable, e.g., for parental or enteral application and which do not deleteπously react with the active compound in admixture therewith Suitable pharmaceutically acceptable camers include but are not limited to water, salt solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglyceπdes, pentaerythπtol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.

For parental application, particularly suitable are solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories, transdermal patches, and vaginal gels, creams and foams. Ampoules are convenient unit dosages.

In a preferred aspect, the composition of this invention is adapted for ingestion.

For enteral application, particularly suitable are unit dosage forms, e.g., tablets, dragees or capsules having talc and/or or carbohydrate carrier or binder or the like, the carrier preferably being lactose and/or com starch and/or potato starch; particulate solids, e.g., granules; and liquids and semi-liquids, e.g., syrups and elixirs or the like, wherein a sweetened vehicle is employed. Sustained release compositions can be formulated including those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.

Suitable for oral administration are, inter alia, tablets, dragees, capsules, pills, granules, suspensions and solutions. Each unit dose, e.g., each table spoon of liquid or each tablet, or dragee contains, for example, 5 - 5000 mg of each active agent.

Suitable for transdermal application are inter alia transdermal patches and gels.

Solutions for parenteral administration contain, for example, 0.01 - 1% of each active agent in an aqueous or alcoholic solution.

The nitric oxide substrate and/or donor can be administered as an admixiture with an estrogen and/or progestational agent and any other optional active agent or as a separate unit dosage form, either simultaneously therewith or at different times during the day from each other.

The combination of active agents is preferably administered at least once daily (unless administered in a dosage form which delivers the active agents continuously) and more preferably several times daily, e.g., in 2 to 6 divided doses. The typical dose is about 0.5 to 1000 mg of each active agent, although some less active agents, e.g., L-arginine, require much higher oral dosages, e.g., 500 to 10,000 mg, and others, e.g., sodium nitroprusside, require lower doses, e.g., 500 -2,000 μg/kg/day. Doses for nitroglycerine typically are orally 2.6 mg 2 x daily; sublingually, 0.8 mg 1-4 x daily; and transdermally, 0.2 - 0.5 mg/hr. The preferred administration form is a transdermal application. Since the LD50 dosages of most of these active agents is known in the prior art, a lower dosage regimen can be initiated and the dosage increased until a positive effect is achieved or a higher dosage regimen can initially be employed, e.g., in a crisis situation, and the dosages regulated downward as relief from the symptoms is achieved. Combinations of agents can be employed either continuously or sequentially.

In humans, both L-arginine and progesterone (or bioequivalent of another progestin) should be given in a ratio which produces blood plasma levels of 50 - 5000 μmolar L- arginine, 10 - 100 nmolar estradiol and 100 to 1000 nmolar of progesterone .

The nitric oxide synthase inhibitor, preferentially iNOS inhibitor, can be administered as an admixiture with an antiprogestin and/or progesterone synthase inhibitor (e.g. epostane, trilostane) and any other optional active agent or as a separate unit dosage form, either simultaneously therewith or at different times during the day from each other.

Brief Description of the Drawings

Various other objects, features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:

Figure 1: Figure 1 shows the effects of L-NAME (NOS inhibitor) on implantation in rats.

The animals were treated with 25 and 50 mg/animal/day L-NAME administered via continuous s.c. infusion from day 1 post coitum (p.c.) until day 7 p.c The autopsy was performed on day 12 p.c. Figure 2: Figure 2 shows the effects of L-NAME (NOS inhibitor) on implantation in rats.

The animals were treated with 25 and 50 mg/animal/day L-NAME administered via continuous s.c. infusion on days 5-7 (p.c). The autopsy was performed on either day 9 p.c. or day 12 p.c.

Figure 3: Figure 3: shows the effects of L-NAME (NOS inhibitor; 50 mg/animal/day; continuous s.c. infusion) alone, low-dose onapristone (0.3 mg/animal, s.c. injection in oil) alone and of the combination L-NAME (50 mg/animal/day; continuous s.c. infusion) plus low-dose onapristone (0.3 mg/animal, s.c. injection in oil) on implantation in rats. The animals were treated on days 5-8 p.c, i.e. during early implantation. The autopsy was performed on day 9 p.c. Note a significant (p< 0.05) increase in pathological implantation sites (small and blood-stained implantation sites) and the reduction in size and weight of the implantation sites after treatment with L-NAME plus onapristone.

Figure 4: Figure 4: shows the progesterone concentrations in various groups treated with L-NAME (NOS inhibitor; 50 mg/animal/day; continuous s.c. infusion) alone, low-dose onapristone (0.3 mg/animal, s.c. injection in oil) alone and with the combination L-NAME (50 mg/animal/day; continuous s.c. infusion) plus low-dose onapristone (0.3 mg/animal, s.c. injection in oil) as described in Figure 3.

Figure 5: Figure 5 shows the effects of L-NAME (NOS inhibitor; 50 mg/animal/day; continuous s.c. infusion) alone, low-dose onapristone (0.3 mg/animal, s.c. injection in oil) alone and of the combination L-NAME (50 mg/animal/day; continuous s.c. infusion) plus low-dose onapristone (0.3 mg/animal, s.c. injection in oil) on implantation in rats. The animals were treated on days 1-4 p.c, i.e. during the pre-receptive phase. The autopsy was performed on day 9 p.c. Note a significant (p< 0.05) inhibition of endometrial receptivity (no implantation sites in 6/7 animals) and the dramatic reduction in size and weight of the implantation sites (in 1/7 animals) after treatment with L-NAME plus onapristone. Detailed Description of the examples

Experiment 1: Effects of L-NAME on endometrial receptivity and early implantation in rats after treatment on days 1-7 post coitum (p.c.)

In the experiments whose results are shown in Figure 1 , pregnant rats (n= 6/group) were treated with 25 and 50 mg/animal/day L-NAME from day 1 p.c. until day 7 p.c, i.e. during the period of pre-implantation and early implantation (in rats implantation takes place on day 5 p.c. ). The control animals were treated with the vehicle. During autopsy (days 9 [part 1 of the study] and 12 p.c. [part 2 of the study]; n=6/grouρ in each part) the implantation sites were assessed macroscopically, their diameter was measured, and the weights of isolated implantation sites (embryonic and placental tissues) were recorded.

Experiment 2: The effects of L-NAME on early implantation in rats after treatment on days 5-7 p.c.

Figure 2 presents results from a second study in pregnant rats in which L-NAME (25 and 50 mg/animal/day) treatment was performed on days 5-7 p.c, i.e. shortly after nidation had taken place. The autopsy was performed on day 12 p.c. Implantation showing haemorrhagic changes were defined as pathological implantation sites. It can be concluded from these results that the nitric oxide synthase inhibitor L-NAME has a profound effect on implantation. There was a dose-dependent inhibition of the size and weight of the implantation sites. In addition, L-NAME induced pathological changes in the implantation sites (mainly haemorrhages).

Experiment 3: The effects of L-NAME in combination with low-dose antiprogestin (onapristone) on early implantation after treatment on days 5-8 p.c in rats.

The aim of the study was to evaluate whether nitric oxide interacts with progesterone during early implantation in rats. Rats (n=8/group) received the following treatments on days 5-8 p.c: L-NAME (50mg/day; s.c infusion), onapristone (progesterone antagonist) at a sub- abortifacient dose (0.3 mg/day, s.c), L-NAME plus onapristone at equivalent doses and the vehicle (controls). The uterus was removed for morphometric and histological evaluation of implantation sites on day 9 p.c. Results: Both L-NAME and mini-dose onapristone significantly (α=0.05) reduced the diameter and weight of the implantation sites when administered during early pregnancy (Figure 3). The extent of the decidual tissue was reduced after L-NAME and onapristone treatment. However, normal mesometrial and antimesometrial decidualization was maintained. A combination of L-NAME and onapristone dramatically reduced the diameter and weight of implantation sites accompanied by a drastic reduction in the extent of decidualization, the necrosis of decidual cells and embryos. Neither treatment showed any significant effect on the serum progesterone concentrations (Figure 4) which indicates that the effects of L-NAME plus onapristone on implantation were due to a direct effect. Conclusions: NOS inhibition does not alter endometrial receptivity. However, when administered during early pregnancy both NOS inhibition and ultra low-dose onapristone treatment alone attenuated the decidualization process, and retarded the development of implantation sites. A combined treatment led to a synergistic effect and substantially impaired implantation.

Experiment 4: The effects of L-NAME in combination with low-dose antiprogestin (onapristone) on endometrial receptivity after treatment on days 1-4 p.c in rats.

In this study pregnant rats (n=7/group) were treated with L-NAME (50 mg/animal/day, s.c. infusion) with and without low-dose onapristone (0.3 mg/animal/day) during the pre- receptive phase on days 1-4 p.c. The autopsy was performed on day 9 p.c. The results of this study are presented in Figure 5. The control animals (n=7) were treated with the vehicle. In the vehicle control group all animals (7/7) showed normal implantation sites. Treatment with L-NAME alone had little effect on endometrial receptivity for embryo implantation and macroscopically normal implantation sites were found in all animals (7/7) during autopsy. Treatment with onapristone alone led to the reduction in the number of implantation sites, and in the majority of animals (5/7) there were no implantation sites at all. In contrast, treatment with L-NAME plus onapristone completely prevented implantation in 6/7 of animals. In the remaining two animals (2/7), one animal had morhologically normal implantation sites but they were very small in size (Figure 5). The second animal had morphologically abnormal (haemorrhagic), extremely small implantation sites. In can be concluded from this study that a combined treatment of L-NAME plus onapristone prevented implantation (inhibited uterine receptivity to embryo implantation) when administered during the pre-receptive phase. Experiment 5: Spatiotemporal distribution of nitric oxide synthase (NOS) isoforms in the mouse implantation site.

The objective of this study was to determine the presence or absence of the three main NOS isoforms (iNOS, ecNOS and bcNOS) during nidation in mice. Implantation sites from mid-day 4 through mid-day 7 of pregnancy were examined for the presence of NOS isoforms. Mice were anesthetized and implantation sites were removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained using standard immunohistochemical procedures. Polyclonal antibodies (Transduction Laboratories) against iNOS, ecNOS and bcNOS were used. iNOS staining was most apparent in macrophages and monocytes between the inner and outer muscle layers of the myometrium at all time points observed. The decidua also displayed positive staining scattered throughout the tissue. There was an increase in iNOS positive cells towards the implantation sites. ecNOS demonstrated very prominent staining on days 6 and 7 in the primary decidual zone adjacent to the anti-mesometrial pole of the embryo. Staining was also present in myometrial vessels at all time points, as<well as the basal portion of the luminal epithelial cells prior to implantation. Specific staining for bcNOS was located primarily in the outer myometrial muscle layer and within the mesometrium with no major differences in the time frames examined. Negative and positive controls showed the appropriate staining. In conclusion these data indicate that the both iNOS and ecNOS show an increased expression during the peri-implantation period in mice.

The results of these studies (both functional and histochemical) indicate that nitric oxide plays a pivotal role during implantation. It seems to be important for placental perfusion during the entire implantation process. Nitric oxide deficiency in the uterus can lead to either resorption of implantation sites or to spontaneous abortions. Therefore, nitric oxide donors or substrates alone or in combination with a steroid hormones (progesterone estradiol) will prove effective for improvement of implantation rates and treatment of infertility and early pregnancy loss. Furthermore, a combination of nitric oxide inhibitors with antiprogestins and/or progesterone synthase inhibitors will be effective in preventing or interrupting pregnancy. Example 1: Improvement of implantation rates after in vitro fertilization with a nitric oxide substrate. To a human female (50 - 90 kg) undergoing IVF, administer L- arginine 0.5 to 20 g of L-arginine per os daily in three divided doses for the first 2-6 weeks of pregnancy or longer.

Example 2: Improvement of implantation rates after in vitro fertilization with a nitric oxide donor. To a human female (50 - 90 kg) undergoing IVF, administer nitroglycerine (5-15 mg/day) transdermally for the first 2-6 weeks of pregnancy or longer.

Example 3: Treatment of infertility with a nitric oxide substrate. To an infertile human female (50 - 90 kg), administer L-arginine 0.5 to 20 g of L-arginine per os daily in three divided doses

Example 4: Treatment of infertility with a nitric oxide donor. To an infertile human female (50 - 90 kg) administer administer nitroglycerine (5-15 mg/day) transdermally.

Example 5: Improvement of implantation rates after in vitro fertilization with a nitric oxide substrate in combination with progesterone. To a human female (50 - 90 kg) undergoing IVF, administer nitroglycerine (5-15 mg/day) transdermally in combination with progesterone (Prolutoπ^R Depot (Schering) 250-1000 mg/week i.m.) for the first 2-6 weeks of pregnancy or longer.

Example 6: Treatment of infertility with a nitric oxide substrate in combination with a nitric oxide substrate. To an infertile human female (50 - 90 kg) administer administer nitroglycerine (5-15 mg/day) transdermally in combination with L- arginine 0.5 to 20 g of L-arginine per os daily in three divided doses.

Example 7: Improvement of implantation rates after in vitro fertilization with a nitric oxide substrate in combination with progesterone and estradiol. To a human female (50 - 90 kg) undergoing IVF, administer nitroglycerine (5-15 mg/day) transdermally in combination with progesterone (ProlutonR Depot (Schering) 250-1000 mg/week i.m.) and estradiol valerate 0.5 - 4 mg/day for the first 2-6 weeks of pregnancy or longer.

Example 8: Postcoital contraception with a nitric oxide synthase inhibitor in combination with an antiprogestin. Administer 0.5-400 mg antiprogestin/day (e.g. mifepristone) in combination with a nitric oxide synthase inhibitor, preferentially iNOS inhibitor, at an effective dose within 72 hours after the intercourse

Example 9: Endometrial contraception with a nitric oxide synthase inhibitor in combination with an antiprogestin using daily administration. Administer an antiprogestin at a daily dose which does not inhibit ovulation (e.g. 0.1 -1 mg/day mifepristone) in combination with a nitric oxide synthase inhibitor, preferentially iNOS inhibitor at an effective dose.

Example 10: Endometrial contraception with a nitric oxide synthase inhibitor in combination with an antiprogestin using once-a-week administration. Administer once-a- week an antiprogestin at a dose which does not inhibit ovulation (e.g. 5-20 mg/week mifepristone) in combination with a nitric oxide synthase inhibitor, preferentially iNOS inhibitor at an effective dose.

Example 11 : Menstrual induction with a nitric oxide synthase inhibitor in combination with an antiprogestin. Administer 0.5-400 mg/day antiprogestin (e.g. mifepristone) in combination with a nitric oxide synthase inhibitor, preferentially iNOS inhibitor, at an effective dose on day 28 +/3 days of the menstrual cycle.

Example 12: Menstrual induction with a nitric oxide synthase inhibitor in combination with a progesterone synthase inhibitor. Administer 1-600 mg/day progesterone synthase inhibitor (e.g. epostane) in combination with an nitric oxide synthase inhibitor, preferentially iNOS inhibitor, at an effective dose on day 28 +/3 days of the menstrual cycle.

Example 13: Menstrual induction with a nitric oxide synthase inhibitor in combination with an antiprogestin and in a further combination with a progesterone synthase inhibitor. Administer 0.5-400 mg antiprogestin/day (e.g. mifepristone) in combination with 1-600 mg progesterone synthase inhibitor (e.g. epostane) an nitric oxide synthase inhibitor, preferentially iNOS inhibitor, at an effective dose on day 28 +/3 days of the menstrual cycle.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims (1)

1. Claims

   A method for improvement of implantation rates in a pregnant female mammal after in vitro fertilization which comprises administering to an afflicted female an amount of nitric oxide synthase substrate, a nitric oxide donor, or both, effective to raise the blood level of circulating L-arginine to at least about 50 - 5000 μmolar above the normally 50 - 1000 μmolar circulating levels and, optionally, also a progestin or, both of an estrogen and a

   10 progestin, in amounts effective to increase the pregnancy rates.

   The method of claim 1, wherein the mammal is a non-pregnant human female suffering from infertility, or a pregnant female suffering from habitual abortions or or a pregnant female exhibiting symptomps of threatening

   L5 abortion.

   3. The method of claim 1 , wherein the nitric oxide substrate is L-arginine.

   4. The method of claim 1, wherein the mammal is a pregnant human female

   20 and a nitric oxide donor is administered hereto.

   5. The method of claim 4, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitrate, SIN-1, isosorbidmonoitrate or isosorbiddinitrate

   25 The method of claim 4, wherein the nitric oxide donor is administered orally.

   7. The method of claim 4, wherein the nitric oxide donor is administered transdermally.

   30 8. The method of claim 1, wherein the mammal is a pregnant human female and the nitric oxide substrate or donor is administered thereto in combination with progesterone.

   The method of claim 1, wherein the mammal is a pregnant human female

   35 and the nitric oxide substrate or donor is administered thereto in combination with progesterone and/or estradiol. 10. The method of claim 8, wherein the progestin is progesterone or hydroxyprogesterone caproate

   11. The method of claim 9, wherein the estrogen is estradiol valerate

   12. The method of fertility control with a nitric oxide synthase inhibitor in combination with an antiprogestin

   13. The method of claim 12, wherein the antiprogestin is mifepristone, ORG

   31710, ORG 33 628, J867, CDB 2914, or ZK 137316

   14. The method of claim 12, wherein the treatment is performed postcoitally

   15. A pharmaceutical composition comprising an admixture of (a) a nitric oxide synthesis substrate, (b) a nitric oxide donor or both, and optionally, also at least one of (c) a progestin, (d) and an estrogen in amounts effective to increase the pregnancy rates or ameliorate the symptoms of threatening abortion in a pregnant or infertile female mammal when administered thereto in an amount effective of estrogen equivalent to 1 - 2 mg of estradiol and an amount of the progestin bioequivalent to 50 - 300 mg of injected progesterone and an amount of the nitric oxide synthase substrate, nitric oxide donor or both effective to raise the blood level of circulating L-arginine to at least about 50 - 5000 μmolar above the normally 50 - 1000 μmolar circulating levels or raise the nitric oxide donor levels to about 10nM to 100 μmolar.

   16. The composition according to claim 15, wherein (a) is a nitric oxide synthesis substrate.

   17. The composition according to claim 16, wherein the nitric oxide synthesis substrate (b) is L-arginine.

   18. The composition according to claim 14, wherein (b) is a nitric oxide donor. 19. The composition according to claim 18, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glycerytrinitride, SIN-1, isosorbidmonoitrate or isosorbiddinitrate, etc.

   20. The composition according to claim 15, wherein the progestin (d) is progesterone or hydroxyprogesterone caproate.

   21. The composition according to claim 15, wherein the estrogen (c) is estradiol valerate.

   22. The method of claim 12 wherein the NOS inhibitor is L-NMMA, L-NIO, L-NIL, amionoguanidine, AMT, 4-methyl-aminopiridin, 6,4-dimethyl-2- aminopiridine,or 2-aminopyridine.

   23. The method of claim 12 wherein the pharmaceutical composition comprises and admixture of a) nitric oxide inhibitor, and b) an antiprogestin for fertility control.

   24. The composition according to claim 23 wherein the NOS inhibitor is -NMMA, L-NIO, L-NIL, amionoguanidine, AMT, 4-methyl-aminopiridin, 6,4-dimethyl-2- aminopiridine,or 2-aminopyridine.

   25. A method of improving implantation rates and/or pregnancy rates in a female mammal, comprising administering to a female mammal in whom pregnancy is desired an effective amount of

   a) a nitric oxide synthase substrate, a nitric oxide donor, or both, optionally in combination with b) a progestin, and, c) optionally, in further combination with an estrogen.

   26. A method of claim 25, whereinthe mammal is a non-pregnant human female suffering from infertility, a pregnant female suffering fom habitual abortions, or a pregnant female exhibiting symptoms of impending abortion. 27. A method of claim 25, wherein the nitric oxide synthase substrate is L- arginine.

   28. A method of claim 25, wherein the mammal is a pregnant human female and a nitric oxide donor is administered thereto.

   29. A method of claim 28, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitrate, SIN-1 , isosorbid mononitrate or isosorbid dinitrate.

   30. A method of claim 28, wherein the nitric oxide donor is administered orally.

   31. A method of claim 28, wherein the nitric oxide donor is administered transdermally.

   32. A method of claim 25, wherein the mammal is a pregnant human female and a nitric oxide substrate or donor is administered thereto in combination with a progestin.

   33. A method of claim 25, wherein the mammal is a pregnant human female and a nitric oxide substrate or donor is administered thereto in combination with a progestin and an estrogen.

   34. A method of claim 32, wherein the progestin is progesterone or hydroxy- progesterone caproate.

   35. A method of claim 33, wherein the estrogen is estradiol valerate.

   36. A method of fertility control for a female mammal, comprising administering to a female mammal in whom pregnancy is not desired and a risk for becoming pregnant an effective amount of nitric oxide synthase inhibitor in combination with an antiprogestin.

   37. A method of claim 36, wherein the antiprogestin is mifepristone, ORG 31 10, ORG 33 628, J867, CDB 2914, or ZK 137316. 38. A method of claim 36, wherein the method is performed postcoitally.

   39. A method of claim 25, wherein the mammal is a human female and the amount of the nitric oxide synthase substrate, nitric oxide donor or both administered is effective to raise the blood level of circulating L-arginine in said female to at least about 50 - 5000 μmole circulating levels.

   40. A method of claim 25, wherein the mammal is a human female and the amount of the nitric oxide synthase substrate, nitric oxide donor or both administered is effective to raise the nitric oxide donor level to about 1 - 1000 nmolar.

   41. A method of claim 25, wherein the mammal is a human female and the amount of progestin administered is bioequivalent to 50 - 300 mg of injected progesterone, and the amount of estrogen administered, if any, is bioequivalent to 1 - 2 mg of estradiol.

   42. A pharmaceutical composition comprising an admixture of effective amounts of

   a) a nitric oxide synthesis substrate, a nitric oxide donor or both; and b) a progestin, and optionally, c) an estrogen,

   in amounts effective to increase the pregnancy rates or ameliorate the symptoms of impending abortion in pregnant female mammal or a female mammal with impaired fertility, wherein the amount of nitric oxide synthase substrate, nitric oxide donor or both administered is effective to raise the blood leel of circulating L-arginine in said female to whom the composition is administered to at least about 50 - 5000 μmolar above the normally 50 - 1000 μmole circulating levels and/or is effective to raise the nitric oxide donor level to about 1 - 1000 nmolar; the amount of progestin administered is bioequivalent to 50 - 300 mg of injected progesterone; and the amount of estrogen administered, if any, is bioeqiuvalent to 1 - 2 mg of estradiol.

   43. A composition of claim 42, wherein (a) is a nitric oxide synthase substrate. 44. A composition of claim 43, wherein the nitric oxide synthesis substrate is L- arginine.

   45. A composition of claim 42, wherein (a) is a nitric oxide donor.

   46. A composition of claim 45, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitride, SIN-1 , isosorbid mononitrate or isosorbid dinitrate.

   47. A composition of claim 42, wherein (b) is progesterone or hydroxyprogesterone caproate.

   48. A composition of claim 42, wherein (c) is is estradiol valerate.

   49. A method of claim 25, wherein components (a) and (b) are administered sequentially.

   50. A method of claim 25, wherein components (a) and (b) are administered simultaneously.