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Background: Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5-6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment., Methods: This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females)., Results: Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights., Conclusions: The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.

Background: The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates., Methods: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis., Results: At 24 h, 72 of 121 (60%) control oocytes progressed to GVBD compared with 9/34 (27%, P < 0.01), 4/36 (11.1%, P < 0.01) and 0/28 (0%, P < 0.01) oocytes incubated with ORG 9935 at 0.1, 0.5 and 1.0 micromol/l respectively. Similar results were achieved at 24 h with 1.0 micromol/l Cilostamide (2/24 oocytes, 8%, P < 0.01) and 100 micromol/l Milrinone (2/32, 6%, P < 0.01). In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 micromol/l Rolipram for 24 h (43/58, 74%) or 48 h (44/58, 76%)., Conclusions: These experiments establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Thus, PDE3 inhibitors have potential use as contraceptives in primates.

Continual administration of low doses of the antiprogestin ZK 137 316 was previously reported to permit ovarian/menstrual cyclicity, but disrupt endometrial growth in macaques. The contraceptive efficacy of this regimen was tested in female rhesus monkeys (10 per group) treated daily with vehicle (controls), 0.01 or 0.03 mg ZK 137 316 per kg body weight for 30 days before and during continual co-habitation with males of proven fertility. Treatment continued until confirmation of pregnancy or for 5 months after pair-housing with males. Mating and vaginal sperm were evident in all females. A cumulative pregnancy rate of 90% (9/10) was observed in the controls. Of the 10 animals receiving 0.01 mg/kg, four conceived during the first 2 months of pairing (P = 0.06) with no further conceptions. No pregnancies were observed in the 0.03 mg/kg group (P < 0.01). Timely, overt menses occurred at a higher frequency in the 0.01 mg/kg group than the 0.03 mg/kg group. However, corpora lutea were present in ovaries from both groups during the last treatment cycle, indicating that ovarian cycles occurred. Thus, chronic administration of low-dose ZK 137 316 that permits continued ovarian cyclicity and a high incidence of timely menses, prevents pregnancy in non-human primates. This regimen may provide a novel method of contraception for women.

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/ group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality.

Large doses of antiprogestin typically disrupt menstrual cyclicity. A chronic low-dose regimen of the potent new antiprogestin ZK 137 316, which permits continued menstrual cyclicity but alters gonadal-reproductive tract activity, was established. Rhesus monkeys received vehicle (n = 6) or 0.01 (n = 8), 0.03 (n = 8) or 0.1 (n = 5) mg ZK 137 316/kg body weight daily for five menstrual cycles (C-1 to C-5). Oestradiol, progesterone and gonadotrophin profiles were normal during cycles involving vehicle and 0.01 and 0.03 mg ZK 137 316/kg body weight. In the 0.1 mg/kg group, mid-cycle oestradiol and gonadotrophin surges, and subsequent progesterone production, were absent in C-3 and C-5. Ovarian cyclicity was accompanied by timely menstruation in the vehicle and 0.01 mg/kg groups. By C-3, half the animals in the 0.03 mg/kg group and all animals in the 0.1 mg/kg group were amenorrhoeic. A corpus luteum was noted during the mid-luteal phase of C-5 in the vehicle, 0.01 mg/kg and 0.03 mg/kg groups. Large antral and cystic follicles were evident in the 0.1 mg/kg group. Thus, a daily treatment with 0.01 mg/kg ZK 136317 permitted normal menstrual cyclicity in macaques. While the daily administration of 0.03 mg/kg ZK 136 317 allowed ovarian cyclicity, menstruation was disrupted in some animals. Increasing the dose to 0.1 mg/kg antagonized pituitary function and resulted in anovulation and amenorrhoea. A chronic low-dose regimen of the antiprogestin ZK 137 316, which permits normal ovarian/menstrual cyclicity, has potential as a contraceptive in women.

During in-vitro fertilization (IVF) cycles, a large bolus of human chorionic gonadotrophin (HCG) is used to induce periovulatory events, but the efficacy of lower doses is undefined. Following follicular stimulation in rhesus monkeys, oocyte nuclear maturation, IVF, granulosa cell luteinization and corpus luteum function were compared after injection of 100, 300 or 1000 IU recombinant HCG or 1000 IU urinary HCG. Bioactive HCG rose to peak concentrations within 2 h that were proportional to the dose administered (100 < 300 < 1000 IU, recombinant HCG = urinary HCG). The duration of surge values (>100 ng/ml) was also dose-dependent (0 h, 100 IU; 24 h, 300 IU; >48 h, 1000 IU, recombinant and urinary HCG). While the proportions of oocytes resuming meiosis and undergoing IVF were similar among groups, fewer animals yielded fertilizable oocytes following 100 and 300 IU (five of nine) compared to 1000 IU recombinant and urinary HCG (nine of 10). Peak values of serum progesterone in the luteal phase were similar, but declined 2 days earlier after 100 and 300 IU relative to 1000 IU recombinant and urinary HCG. Thus, 3-10 fold lower doses of HCG elicit low amplitude surges of short duration that induce periovulatory events such as re-initiation of oocyte meiosis and granulosa cell luteinization. However, oocyte fertilization and luteal function may optimally require surges of higher amplitude and longer duration similar to those produced by standard doses of 1000 IU recombinant or urinary HCG.

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.

Both follicle stimulating hormone (FSH) and luteinizing hormone (LH) are proposed requirements for follicular growth and steroidogenesis; however, the role of LH in primate folliculogenesis is unclear. Follicular stimulation by recombinant human FSH (n = 5) with and without recombinant LH (1:1; n = 6) following 90 days of gonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatment in macaques was evaluated. Human chorionic gonadotrophin (HCG) was administered when six follicles > or = 4 mm were observed. Oocytes were aspirated 27 h later and inseminated in vitro. Chronic Antide reduced serum oestradiol and bioactive LH to concentrations observed in hypophysectomized rhesus monkeys. Multiple follicular growth required a longer interval following recombinant FSH (12 +/- 1 days) than recombinant FSH+recombinant LH (9 +/- 0.2 days), but the total number of follicles/animal did not differ between groups. The day prior to HCG, oestradiol concentrations were 4-fold less following recombinant FSH compared to recombinant FSH+recombinant LH. With recombinant FSH, more oocytes completed meiosis to metaphase II (51%) and fertilized (89 +/- 5%) relative to recombinant FSH+recombinant LH (12 and 52 +/- 11% respectively). Follicular growth and maturation in LH-deficient macaques occurred with FSH alone. Thus, LH is not required for folliculogenesis in primates. Higher fertilization rates following follicular stimulation with FSH alone suggest that the presence of LH with FSH (1:1) during the pre-ovulatory interval impairs gametogenic events in the periovulatory period.

The actions of the mid-cycle gonadotrophin surge to convert the pre-ovulatory follicle into the corpus luteum are not well understood in primate species. In experiments titrating the surge duration required in macaque monkeys during artificial in-vitro fertilization (IVF)-related cycles, short (< or = 14 h) LH surges similar to those in rodents and domestic animals failed to initiate peri-ovulatory events. Attenuated (24 h) surges, one-half the duration of spontaneous surges in primates, reinitiated oocyte meiosis and initial luteinization of granulosa cells. However, only surges of > or = 48 h sustained luteal development and function to produce luteal phases of approximately 13 days. Three approaches-immunocytochemistry of steroid receptors, binding of radiolabelled steroid, and reverse transcription-polymerase chain reaction of mRNA-indicate that progesterone receptor (PR), but not oestrogen receptor, is expressed in the macaque corpus luteum. Studies on cells collected before and after the gonadotrophin surge identified a novel action of LH to induce PR expression in luteinizing granulosa cells. Thus, the LH surge requirements vary between non-primate and primate species, as well as between peri-ovulatory events, and the LH surge may promote cellular recognition of paracrine and autocrine factors (e.g. progesterone) that become predominant in the developing corpus luteum.