Early luteal phase administration of mifepristone inhibits preimplantation embryo development and viability in the rhesus monkey

It is generally believed that progesterone is essential for inducing the changes in oviduct and uterus necessary for embryo viability and implantation in a number of mammalian species. The aim of this study was, in the rhesus monkey, to examine in conception cycles with and without early luteal phase antiprogestin (mifepristone; RU 486) treatment: (i) the growth status of preimplantation embryos and (ii) the implantation ability of the preimplantation embryo after transfer to a synchronous-cycle surrogate recipient. A total of 43 proven fertile rhesus monkeys were randomly placed in the control (group 1, n = 18) and mifepristone (group 2, n = 25) groups. All monkeys cohabited with proven fertile male monkeys on cycle days 8-16 and were injected with vehicle alone [benzyl benzoate:olive oil, 1:4 (v/v), s.c.] for group 1 and with mifepristone (2 mg/kg body weight s.c.) for group 2, on day 2 after the presumed day of ovulation. A total of 12 preimplantation embryos [premorula (n = 1), morula (n = 2), zona- encased (n = 7) and zona-free (n = 1) blastocysts and degenerate embryos (n = 1)] were recovered from 17 ovulatory, mated cycles in group 1 on day 6 after ovulation. In group 2, of the 23 ovulated cycles, 12 preimplantation embryos [premorula (n = 2), morula (n = 7), zona-encased blastocyst (n = 1), and degenerate embryos (n = 2)] were retrieved. Despite no significant difference in the recovery rate between the two groups, early luteal phase RU 486 exposure induced delay (P < 0.01) in preimplantation embryo growth, primarily at the morula-blastocyst transition stage. Nine of the embryos from group 1 and seven of the embryos from group 2 recovered on day 6 were transferred to naturally synchronized, non-mated and untreated surrogate recipients. In group 1, five embryos implanted (55%) and, of these, three (60%) gave rise to live infants through natural delivery; implantation was assessed from extension of the cycle (i.e. no menstrual bleeding) and rise in concentrations of oestradiol and progesterone from day 10 of conception; rectal palpation was performed on cycle day 50 to confirm clinical pregnancy. In group 2, however, there was not a single case of establishment of pregnancy following transfer of embryos retrieved from mifepristone-exposed monkeys. Thus, preimplantation embryos recovered from RU 486-exposed monkeys failed to establish evolutive implantation and pregnancy, while significant (P < 0.02) success was observed in transfers of embryos from the control group. We postulate that progesterone-mediated actions are involved in mediating the growth and viability of preimplantation-stage embryos in the rhesus monkey.      

Anti-nidatory effect of a single, early post-ovulatory administration of mifepristone (RU 486) in the rhesus monkey

The hypothesis that post-coital administration of mifepristone(RU 486) as a single dose in the early luteal phase can be aneffective anti-nidatory strategy was tested using the rhesusmonkey as the experimental model. Incidence of pregnancy, vaginalbleeding patterns, profiles of menstrual cyclicity and of serumlevels of progesterone and oestrogen were examined followingadministration of RU 486 as a single dose of 10 mg/kg and 2mg/kg body weight on the second day after ovulation. In controlmonkeys (group 1; n = 5) receiving the vehicle alone (benzylbenzoate: olive oil, 1: 4, v/v) there was a 60% pregnancy rate.Following s.c. administration of RU 486 at both doses, no pregnancywas recorded in a total of 33 treatment cycles in 12 monkeys.Five monkeys received RU 486 at 10 mg/kg s.c. (group 2) in threeconsecutive cycles. All animals had complete inhibition of implantation;in addition, the treatment cycle length was prolonged (P <0.001) due to an extension of the luteal phase. The subsequentfollicular phase was unaffected. Mild, premature vaginal bleedingduring the luteal phase was recorded in five treatment cycles,3–5 days after drug application. Though the serum profilesof progesterone and oestrogen in these monkeys showed markedindividual variations, there was a characteristic progesteronerebound about 18–20 days after drug administration. Monkeysin group 3 were given RU 486 at 2 mg/kg, s.c. either for threeconsecutive cycles (group 3a; n = 4) or for two consecutivecycles (group 3b; n = 3). Premature luteal phase vaginal bleedingoccurred only in four treatment cycles, within 2–6 dayspost-treatment. An increase in both the duration (P < 0.001)and degree (P < 0.001) of menstrual flow as compared withthe pre-treatment cycles was recorded in six treatment cyclesof three monkeys in group 3. These animals did not have prematureluteal phase vaginal bleeding. Collectively, 100% protectionagainst pregnancy with no change in the cycle length was obtainedin all seven monkeys in 18 treatment cycles. Analysis of pooleddata revealed that the subsequent follicular phase, as wellas the ovarian steroid hormone profiles of treatment cycleswere unaffected. Thus, a single application of RU 486 in theearly secretory phase appears to be a potential anti-implantationstrategy for intercepting pregnancy in the primate.     

Single monthly administration of the anti-progestagen Org 31710 in users of the 75 microg desogestrel progestagen-only pill: effects on pituitary-ovarian activity

Endocrine and ultrasound effects were studied of an intermittent (every 28 days) oral administration of 150 mg of the anti-progestagen Org 31710 during the continued daily use of 75 microg desogestrel (DSG) for progestagen-only contraception. A randomized, double-blind, placebo-controlled two-centre study was conducted in 50 healthy volunteers. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and progesterone concentrations, and follicle number and size were studied, as well as endometrial thickness, which was assessed by transvaginal sonography at least twice weekly during a single medication cycle (cycle 3-5). Forty-eight women were evaluated (Org 31710, n = 25; placebo, n = 23). Seven ovulations were observed in the treated group versus none in the placebo group. LH concentrations were higher on days 9 and 11 and oestradiol concentrations lower on day 3 in the treated group, irrespective of whether ovulation occurred. No parameter could predict ovulation. Endometrial thickness was greater on cycle days 7-13 and 19 in the treated group. However, within the Org 31710 group, no significant differences were found in volunteers who did or did not ovulate. Observed differences may be attributed to a competitive effect of Org 31710 with progestagen-induced suppression of the pituitary-ovarian axis, altered oestradiol feedback mechanisms, and/or altered receptor availability.     

Human endometrial maturation is markedly improved after luteal supplementation of gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin stimulated cycles

In human cycles stimulated for ovulation with gonadotrophin–releasinghormone (GnRH) agonists and human menopausal gonadotrophin (HMG),a luteal phase defect has been described. To evaluate the influenceon the endometrium, endometrial development in GnRH agonist/HMGstimulated cycles was assessed in cycles with and without lutealphase supplementation. Endometrial histological maturation,ultrastructure and oestrogen receptor (ER) and progesteronereceptor (PR) status were analysed in the mid-luteal phase.Serum concentrations of oestradiol and progesterone were measureddaily from days 1–5 of the luteal phase. Supplementationof the luteal phase was achieved with either human chorionicgonadotrophin or natural progesterone, administered intramuscularlyor intravaginally. In non-supplemented cycles all endometrialfeatures were consistent with an impaired progesterone bioavailability.After supplementation of the luteal phase, fewer signs of lutealphase deficiency were visible, especially with the intravaginalroute of progesterone administration. We concluded that theendometrial parameters confirm the need for luteal support inGnRH agonist/HMG stimulated cycles.     

Physiology: Effects of intermittent antiprogestin RU486 combined with cyclic medroxyprogesterone acetate on folliculogenesis and ovulation

The results of several studies have suggested an inhibitoryeffect of the antiprogestin RU486 on late stages of folliculogenesisand ovulation. To assess the feasibility of using this propertyto inhibit ovulation without losing cycle control, an intermittentadministration of RU486 alternated with medroxyprogesteroneacetate (MPA) was tested in a phase I study. RU486 at a doseof 50 mg/day was given on menstrual cycle days 9–11 and27–29, and 10 mg/day of MPA was given on cycle days 17–26for three consecutive cycles to six Finnish and five Chileanwomen. Blood samples were collected two to three times a weekfor serum progesterone and oestradiol assays in three treatmentcycles. One control cycle and one post-treatment recovery cyclewere also monitored by serum samplings. Ultrasonography wascarried out to measure follicular diameters in the treatmentcycles. In 29 of 32 cycles, bleeding commenced within 3 daysafter the last MPA pill intake. Out of 32 treatment cycles,20 were without luteal activity (serum progesterone <9 nmol/l).Although 12 treatment cycles showed luteal activity (serum progesterone9 nmol/l), a clear rupture of a pre-ovulatory follicle >15mm, verified by ultrasonography, was seen in only one treatmentcycle. During the treatment cycles with luteal activity (serumprogesterone levels 9 nmol/1), serum oestradiol concentrationswere significantly higher on cycle days 9–18 and significantlylower at the end of the cycle compared with the cycles withoutluteal activity. The regimen used in this study disturbed folliculogenesisand ovulation (apparently), and was able to provide good cyclecontrol in the majority of the cycles.     

Reversible suppression of menstruation with progesterone antagonists in rhesus macaques

BACKGROUND: A reliable means of menstrual suppression would greatly improve the quality of life for women. Information is lacking on the direct endometrial effects and appropriate dosages of new antiprogestins that may be useful for this purpose. METHODS: The current work evaluated three different systems in macaque monkeys. First, the range of doses of two relatively new antiprogestins, ZK 137 316 and ZK 230 211, that would block progesterone action directly on the endometrium in artificially cycled, spayed rhesus macaques; second, the direct endometrial effects of ZK 230 211, a type III antiprogestin; and third, investigation of whether endometrial-suppressive doses administered chronically to intact, cycling monkeys could be used for reversible, menstrual suppression. RESULTS: The results in naturally cycling animals showed that ZK 137 316 blocked menstruation in all animals, but doses of 0.05 mg/kg blocked ovulation in 55.5% of animals and doses of 0.1 mg/kg blocked ovulation in 66.6% of the animals. However, all doses of ZK 230 211 that blocked menstruation also blocked ovulation. All progesterone antagonist (PA)-treated animals, regardless of dose, maintained normal follicular phase concentrations of oestradiol and returned to normal menstrual cyclicity within 15--41 days post-treatment.Therefore ZK 137 316, depending on dose, can allow ovulation but block menstruation, while ZK 230 211, a much more potent PA, blocks both ovulation and menstruation at all effective doses. Both PAs block unopposed oestrogenic action on the endometrium through their antiproliferative effects. CONCLUSIONS: Reversible amenorrhoea can be achieved with these two PAs, and they can protect the endometrium from the effects of unopposed oestrogen whether or not ovulation is blocked. Chronic, low dose PA treatment may provide a new option for women who wish to suppress their menstrual periods.     

Controlled preparation of the endometrium with exogenous oestradiol and progesterone: a novel regimen not using a gonadotrophin-releasing hormone agonist.

In women having inactive ovaries, controlled preparation of the endometrium has been achieved with exogenous oestradiol and progesterone. We report on the feasibility and practicality of using a similar regimen for timing transfers of cryopreserved embryos in women whose ovaries have not been suppressed. A total of 91 women having cryopreserved embryos from previous in-vitro fertilization (IVF) attempts received 4 mg/day of oestradiol valerate, starting on cycle day 1 of spontaneous (n = 85) or induced (n = 6) menstruation. A single blood sample was obtained on cycle day 14 for the measurement of plasma progesterone, oestradiol and luteinizing hormone (LH). Vaginal administration of micronized progesterone (300 mg/day) was started on day 15. Cryopreserved embryos were transferred on day 17 or 18 provided that day 14 plasma progesterone remained < or = 0.5 ng/ml, thereby confirming the absence of spontaneous ovulation prior to the administration of exogenous progesterone. Out of 91 cycles studied, plasma progesterone was found to be elevated (> 1 ng/ml) in only three (3.2%). Of the 88 scheduled transfers, 31 did not take place because no embryo survived thawing. In the remaining 57 cycles, 116 embryos were transferred resulting in 10 pregnancies, giving pregnancy and embryo implantation rates of 17.5 and 8.6% respectively. When a positive beta human chorionic gonadotrophin (HCG) titre was obtained, supplementation with oral oestradiol and vaginal progesterone was continued until placental autonomy was achieved. Of the 10 pregnancies, five (50%) were lost during the first trimester (biochemical, n = 1; miscarriage, n = 3; ectopic, n = 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oestradiol plus progesterone treatment increases serum leptin concentrations in normal women

BACKGROUND: Previous studies have alluded to a role for both oestradiol and progesterone in the secretion of leptin from fat cells in the human, although direct evidence has yet to be obtained. The study aim was to assess serum leptin concentrations in normally cycling women receiving exogenous oestradiol and progesterone. METHODS: Normally cycling women were investigated in an untreated spontaneous cycle (control, n = 10), a cycle treated with oestradiol (oestradiol cycle, n = 10) and a cycle treated with oestradiol plus progesterone (oestradiol+progesterone cycle, n = 6). Oestradiol was given to the women through skin patches on cycle days 2, 3 and 4, and progesterone intravaginally on cycle days 3, 4 and 5. Serum concentrations of leptin, oestradiol, progesterone, FSH and LH were measured in daily blood samples. RESULTS: During the treatment, serum oestradiol and progesterone concentrations increased significantly. In the oestradiol cycles, leptin concentrations were not affected by treatment and did not differ from those in controls. In the oestradiol+progesterone cycles, leptin concentrations (mean +/- SEM) increased in all women from cycle day 3 (8.6 +/- 1.1 ng/ml) to days 5 (12.2 +/- 1.8 ng/ml, P < 0.01) and 6 (11.9 +/- 2.0, P < 0.05), and were at these points significantly higher than in the control cycles (P < 0.05). The mean percentage increase from day 3 to the peak concentration on days 5 or 6 was 62.6 +/- 6.8%. Leptin concentrations returned to the pretreatment value on day 7, together with the concentrations of oestradiol and progesterone. In the oestradiol+progesterone cycles, leptin concentrations correlated significantly with oestradiol and progesterone concentrations, but not with FSH and LH concentrations. CONCLUSIONS: These results show, for the first time, that leptin secretion can be stimulated in women by the administration of oestradiol plus progesterone. This may explain the increased concentrations of leptin during the luteal phase of the normal menstrual cycle.     

Implantation: Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients

This study was undertaken to investigate an empirical observationthat ‘high responder patients have poorer in-vitro fertilization(IVF) outcome than normal responder patients’. The aimof our study was to analyse the effect of high serum oestradioland progesterone concentrations at the day of human chorionicgonadotrophin (HCG) administration on endometrial receptivityand oocyte—embryo quality in high and normal responderpatients. The IVF patients were divided into two groups: 59high responder patients who voluntarily donated some of theiroocytes, and a control group consisting of 105 normal responderpatients. Both groups were compared in terms of the number andquality of oocytes retrieved, embryos transferred, fertilization,implantation and gestation rates, serum oestradiol and progesteroneconcentrations and the oestradiol: progesterone ratio on theday of HCG injection. To ascertain oocyte—embryo quality,a second control group of 96 women undergoing oocyte donation(receiving oocytes from high responder patients) was considered.To assess the impact of steroid concentrations on endometrialreceptivity, high responder patients were divided into two subgroupsaccording to oestradiol concentration, above or below the minimaloestradiol and progesterone concentrations (mean – SD)in this group. The normal responder patients were divided intotwo subgroups according to oestradiol concentration, above orbelow the maximal oestradiol and progesterone concentrations(mean + SD) in this group. To assess further the relevance ofoestradiol concentration on endometrial receptivity, patientswere divided into different subgroups according to increasingoestradiol concentration, regardless of whether they were highor normal responders. High responder patients had significantlydecreased implantation and pregnancy rates per cycle comparedwith normal responder patients (33.3 versus 16.3 and 11.1 versus5.4% respectively; P < 0.05). The results of 108 embryo transfersin 91 recipients who received oocytes from the high respondergroup showed normal embryo quality. Implantation rates and pregnanciesper cycle were significantly lower in high responder patientswith serum oestradiol concentrations > 1700 pg/ml comparedwith those having oestradiol concentrations 1700 pg/ml, as wellas in normal responder patients with serum oestradiol concentrations2200 pg/ml compared with those having oestradiol concentrations<2200 pg/ml. Considering all the patients together, significantdecreases in pregnancy and implantation rates were observedwhen oestradiol concentrations were >2500 pg/ml comparedwith patients having lower oestradiol concentrations. Our clinicalresults demonstrate that high serum oestradiol concentrationson the day of HCG injection in high and normal responder patients,regardless of the number of oocytes retrieved and the serumprogesterone concentration, are detrimental to uterine receptivitywithout affecting embryo quality.     

Long oestradiol replacement in an oocyte donation programme

The objective of this study was to optimize, in terms of endometrialreceptivity (embryo implantation), the limits of unopposed administrationof oestrogens beyond 35 days in an in-vitro fertilization (IVF)and ovum donation programme. Oocytes donated by 182 women undergoingIVF were distributed among 186 women treated by ovum donation.Five groups of recipients were established according to theduration of oestradiol valerate administration, in a ‘prolongedfollicular phase’ protocol, before embryo replacement,employing oestradiol valerate at increasing doses up to 6 mg/day.Gonadotrophin-releasing hormone analogues (GnRHa) were simultaneouslyadministered in ovulatory patients. The dosage of oestradiolvalerate was maintained until oocytes were available for inseminationand subsequent transfer. Donors and recipients were equallydistributed among groups in terms of age and cause of infertility.There was no difference among groups in serum oestradiol concentrationthe day in which progesterone was added to obtain a secretorytransformation of the endometrium. An analysis of the ovum donationcycles showed no difference among groups in pregnancy and implantationrates after the replacement of a similar number of embryos.Successful implantation was observed even after 100 days ofunopposed oestradiol valerate administration. Break-throughbleeding increasingly appeared according to the duration ofoestrogen replacement. These clinical observations provide evidencethat the concept of ‘prolonged follicular phase’oestrogen replacement for ovum donation can be maintained, atleast as long as 15 weeks. However, because of the high (>44%)incidence of break-through bleeding after 9 weeks, it is advisableto stop oestrogen treatment at this point. This protocol enormouslyfacilitates the chances of synchronization between donor andrecipient in an anonymous oocyte donation programme.     

Chronic treatment of cycling rhesus monkeys with low doses of the antiprogestin ZK 137 316: morphometric assessment of the uterus and oviduct

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.      

Regulation of corpus luteum function

The effects have been studied of different ovulation inductionregimens [either domiphene citrate or buserelin in combinationwith human menopausal gonadotrophin (HMG)] on the circulatingconcentrations of progesterone, oestradiol, relaxin and humanchorionic gonadotrophin (HCG) during the first trimester ofpregnancy. Ovulation induction with clomiphene resulted in elevatedconcentrations of gonadotrophins in both phases of the cycle,while during ovulation induction with buserelin, gonadotrophinconcentrations were elevated in the follicular phase only. Theconcentrations of all corpus luteum products were greater inclomiphene pregnancies compared with spontaneous pregnancies,but only oestradiol and relaxin concentrations were greaterin clomiphene pregnancies compared with buserelin pregnancies.The concentrations of HCG were significantly reduced in clomiphenepregnancies compared to natural pregnancies. Relaxin concentrationswere significantly higher from 7 weeks gestation in buserelincompared with spontaneous pregnancies, while progesterone, oestradioland HCG concentrations were not consistently different. Consistentassociations were found between relaxin and HCG concentrationsin spontaneous pregnancies and between the concentrations ofrelaxin and both progesterone and oestradiol in pregnanciesachieved after ovulation induction. These data suggest that(i) given the similarity in the circulating concentrations ofHCG, the relatively lower circulating gonadotrophin concentrationsduring the luteal phase of the cycle of conception result inreduced circulating concentrations of oestradiol and relaxin;while in the case of relaxin this effect is partially reversible,there is no evidence that this is so for oestradiol; (ii) synthesisof progesterone in the corpus luteum is less affected by lowerconcentrations of gonadotrophins during the luteal phase; (iii)ovulation induction with clomiphene results in pregnancies withlower concentrations of HCG, suggesting that trophoblast functionmay be impaired; and (iv) corpus luteum function is linked withplacental steroidogenesis.     

Atrial natriuretic peptide, oestradiol and progesterone in women undergoing spontaneous and gonadotrophin-stimulated ovulatory cycles

The objective of this study was to examine the relationshipbetween the concentrations of oestradiol and progesterone onthe one hand and atrial natriuretic peptide (ANP) concentrationson the other, during the follicular and luteal phases of spontaneousand gonadotrophin-stimulated ovulatory menstrual cycles. A totalof 27 ovulatory women undergoing either a spontaneous (n = 9)or a gonadotrophin-stimulated (n = 18) cycle were selected forinclusion in this study. In comparison with spontaneous cycles,gonadotrophin-stimulated cycles had increased peak follicularoestradiol (mean ± SE; 937 ± 150 versus 195 ±18 pg/ml; P < 0.05) and midluteal progesterone (mean ± SE; 44.0 ± 7.4 versus 14.1 ± 2.4 ng/ml; P <0.05) concentrations. There were no differences in the circulatingANP concentrations between the follicular and luteal phasesof the menstrual cycle. Despite the increased oestradiol andprogesterone concentrations following gonadotrophin stimulation,no difference in ANP concentrations was seen between stimulatedand spontaneous cycles. There was no correlation between circulatingconcentrations of oestradiol, progesterone (at physiologicaland supraphysiological concentrations) and ANP throughout themenstrual cycle.     

The luteal phase after in-vitro fertilization and related procedures

To evaluate any beneficial effect of progesterone supplemen–tationduring the luteal phase of GIFT or IVF cycles stimulated byclomiphene citrate and HMG, two random prospective studies wereperformed. In the first study, a group of patients receiveda luteal phase supplement of 50 mg natural progesterone i.m.daily from the day of oocyte retrieval onwards. Initial resultson 168 patients indicated that the pregnancy rate was similarin patients with or without progesterone supplements. No differenceswere found between the two groups in an analysis of pregnantand failed cycles. In a second study two different protocolsof luteal phase sup–plementation after Buserelin–HMGstimulation were com–pared: natural progesterone in combinationwith oestradiol valerate (50 patients) or HCG supplements (41patients). A 32% pregnancy rate per cycle was encountered inboth groups. Endometrial biopsies, taken during the luteal phasefrom patients who did not undergo embryo replacement, revealedretarded endometrial development in most of the biopsies.     

Serum concentrations of oestradiol-17beta, progesterone, relaxin and chorionic gonadotrophin during blastocyst implantation in natural pregnancy cycle and in embryo transfer cycle in the rhesus monkey

The present study was undertaken to assess the temporal association between the profiles of serum concentrations of oestradiol-17beta, progesterone, chorionic gonadotrophin (CG) and relaxin in pregnancies established naturally, and after embryo transfer, as well as in failed pregnancies in rhesus monkeys. In naturally mated cycles (group 1) a conception rate of 75% was obtained. In group 1, the mean day of CG detection in serum was 11.5 +/- 1.9 day post-ovulation, and for relaxin, 9.0 +/- 2.5 day post-ovulation. In group 2, embryo transfer to synchronous, non-mated surrogate recipients was performed; seven embryo transfer cycles yielded three pregnancies which were allowed to continue to term and normal infants were delivered. In embryo transfer cycles the mean day of CG detection was 14.8 +/- 1.8 day post- ovulation, and for relaxin, 11.4 +/- 2.6 day post-ovulation. A delay of about 3 days was observed in the appearance in circulation of CG (P < 0.05) and also of relaxin (P < 0.05) between natural mated and embryo transfer conception cycles. Significant differences (P < 0.05 for progesterone and P < 0.03 for oestradiol) were obtained for the areas under the curves for progesterone and oestradiol between days 12 and 16 in conception cycles compared with failed pregnancies. These data provide the first observation of the normal hormonal signals associated with maternal recognition of transferred embryos during the peri- implantation period, and suggest that the use of such an experimental primate embryo transfer model may help to elucidate components of maternal and embryonic signal-response mechanisms during embryo implantation.      

The effect of RU486 administered during the proliferative and secretory phase of the cycle on the bleeding pattern, hormonal parameters and the endometrium

Seventeen healthy women aged 24–45 years with regularmenstrual periods, proven fertility and not using steroidalcontraceptives or IUD were recruited for the study. The volunteerswere followed during one control, one treatment and one follow-upcycle. Daily morning urine samples were obtained during thecontrol and the treatment cycle. The samples were analysed withregard to pregnanediol glucuronide (P₂-G), oestrone glucuronide(E₁-G), oestradiol (E₂), progesterone (P₄), LH and creatinine.During the entire 3-month study the subjects kept a record ofuterine bleeding and side effects. The subjects received 50mg RU486 daily either on cycle days 7–10 (n = 7) or oncycle days 20–23 (n = 10). An endometrial biopsy was takenon cycle day 10 in the first group and on cycle days 21–28in the second group of patients. Treatment during the proliferativephase caused significant prolongation of the cycle length dueto a delay of the oestrogen and LH surge. However, once theoestrogen concentration started to increase, the remaining partof the cycle was normal. The length of the follow-up cycle wassimilar to that of the control cycle. The morphology of theendometrium did not differ from control samples taken from untreatedwomen at the same time of the cycle. All ovulating women (n= 9) treated in the mid-luteal phase started to bleed on the3rd to 4th day of the treatment. In four of these women thebleeding was scanty and followed by a menstrual-like bleedingat expected time, while in the remaining five volunteers thetreatment bleeding was heavier and not followed by a new bleedinguntil a month later. The duration of the secretory phase was16.5 ± 1.3 days in women with two bleeding episodes and11.8 ± 1.9 days in women with one bleeding episode (P< 0.05). The hormonal parameters were similar in both groupsup to the start of the treatment. In the patients with one bleedingepisode, the treatment was associated with a reduction in progesteroneconcentration, while in the patients with two bleeding episodesthe progesterone concentration remained elevated until the secondbleeding episode. Light microscopic examination of the endometriumrevealed unique changes in the endometrial morphology. The resultsindicate that RU486 acts mainly on the endometrium but a director indirect effect on the corpus luteum cannot be excluded.The age of the corpus luteum may be of importance for its susceptibilityto RU486 treatment.     

Effects of enclomiphene and zuclomiphene on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of small and large ovine luteal cells

We examined the effects of enclomiphene and zuclomiphene, aloneand in combination with oestradiol, on basal and gonadotrophin-stimulatedprogesterone secretion by isolated subpopulations of both large(granulosa-lutein) and small (theca-lutein) ovine luteal cells.Isolated large and small luteal cells derived from intact, enucleatedovine corpora lutea were incubated for 48–120 h with orwithout 22R-hydroxycholesterol or pregnenolone (2.5 µM)and a range of enclomiphene, zuclomiphene, and/or oestradiolconcentrations (3–100 µM), both with and withoutovine Iuteinizing hormone (100 ng/ml). Spent media were assayedin duplicate for progesterone content by radioimmunoassay. Enclomiphene,zuclomiphene, and oestradiol exhibited equivalent dose-dependentinhibitory effects on basal and gonadotrophin-stimulated smalland large ovine luteal cell progesterone secretion under allsubstrate conditions. Both cell types became more sensitiveto clomiphene inhibition with increasing time in culture. Incombined treatments, the effects of oestradiol and either enclomipheneor zuclomiphene became additive in longer-term cultures andwere never antagonistic In this model system, (i) clomiphene,like oestradiol, appears to inhibit 3-hydroxysteroid dehy-drogenaseactivity, (ii) both stereoisomers act as oestrogen agonists,(iii) neither demonstrates any anti-oestrogenic properties,and (iv) both large and small luteal cells become more sensitiveto clomiphene inhibition with increasing duration of exposure.     

The role of pre-ovulatory progesterone in the midcycle gonadotrophin surge, ovulation and subsequent luteal phase: studies with RU486 in rhesus monkeys

Conflicting evidence exists on the possible physiological roleof progesterone in the regulation of the midcycle surge of gonadotrophinsduring the normal primate menstrual cycle. We designed the presentstudy based on the availability of a potent antiprogesterone,RU486, that acts by binding to the progesterone receptor withoutinducing progestational activity. Regularly cycling rhesus monkeysreceived daily administration of RU486,10 mg orally (n = 8)or vehicle (n = 5) from the day of the menstrual cycle in whichserum oestradiol was 130 pg/ml or more, and a laparoscopy revealedthe presence of a dominant follicle. While vehicle administrationdid not affect the normal ovulatory pattern nor the hormonalmilieu of the menstrual cycles, RU486 induced marked aberrationsduring the treated cycles. Delay of ovulation with a normalsubsequent luteal phase was observed in three animals. Threeanimals remained anovulatory until the following cycle and twoanimals that ovulated on days 14 and 16 of the treated cycleshad short luteal phases. Analysis of daily FSH, LH, oestradioland progesterone revealed that the administration of RU486 disruptedthe midcycle pattern of gonadotrophins by disrupting them afterthe surge was initiated. Oestradiol surges were not differentfrom controls and in all animals the ascendant levels of progesteronewere interrupted by the administration of the antiprogesterone.This study clearly shows that the pre-ovulatory administrationof RU486, a potent antiprogesterone, alters pre-ovulatory gonadotrophinsecretion, inducing different degrees of menstrual irregularitiessuch as anovulation, delayed ovulations and short luteal phases.Based on these results it is possible to speculate that progesteroneexerts a facilitatory effect (positive feed-back) on the midcyclegonadotrophin peak that induces ovulation in primates.     

A flexible protocol for the induction of recipient endometrial cycles in an oocyte donation programme

Synchronization of the availability of good quality oocytesfrom donors and adequate endometrial maturation of recipientsare very important for the success of an oocyte donation programme.A flexible protocol for the endometrial preparation of recipientsis important in timing embryo transfer between days 17 and 19of the cycle (‘window of receptivity’). The purposeof this study was to evaluate the effect of the length of oestradioladministration to recipients on pregnancy outcome. Oestrogenadministration was 8 mg/day, but its length varied prospectivelyfrom 6 to 27 days, followed by the addition of progesterone(100 mg daily Lm.) for 2–4 days according to the availabilityof good quality oocytes. Pregnancy outcome was evaluated regardlessof age, indication for oocyte donation or number of embryostransferred per patient The pregnancy rate per cycle was comparablewhen oestradiol was administered from 6 to 11 days before progesteroneaddition, while it dropped significantly thereafter. The variationin progesterone administration did not affect pregnancy outcome.These findings provide us with a greater flexibility by allowingus to vary oestradiol administration to recipients from 6 to11 days prior to progesterone, reducing considerably, therefore,the need to cancel embryo transfer because of oocyte unavailability.Thus we can arrange to transfer embryos between days 17 and19 of the recipient's cycle so as to obtain the best possibleclinical outcome.     

Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.