In-vitro development of in-vivo produced rhesus monkey morulae and blastocysts to hatched, attached, and post-attached blastocyst stages: morphology and early secretion of chorionic gonadotrophin

The earliest time of secretion of chorionic gonadotrophin (CG)by primate embryos and its role during preimplantation developmentand implantation are not clearly determined. We cultured in-vivofertilized/developed zona-intact, morphologically normal morulae(n = 11) and early blastocysts (n = 11), freshly recovered (bynon-surgical uterine flushing) on days 5 and 6 of pregnancy,respectively (day 0 = the day following LH surge), from non-superovulatednaturally bred rhesus monkeys (Macaca mulatta). Embryos werecultured for a minimum of 24 days in dishes containing 1 mlof CMRL-1066 supplemented with 20% bovine fetal serum in a humidifiedatmosphere of 5% CO₂ in air at 37°C. The culture mediumwas changed every 48 h. The percentage of hatched blastocysts,developed from morulae and early blastocysts, was 90.9; elapsedtimes (mean ± SEM) were 67.8 ± 4.4 h (morula)and 37.8 ± 3.6 h (blastocyst). The minimum number ofHoechst-stained cells/hatched blastocyst was 531. The mean diameter(± SEM) of cultured embryos increased from 180 µmat the beginning of culture to 374 ± 28 and 450 ±19 µm at the fully expanded and hatched blastocyst stages,respectively. Hatched blastocysts continued to expand (maximumdiameter: 1125 ± 25 µm); after an additional 94–96h they attached firmly to the serum-coated dishes and producedhighly proliferating multinucleate trophectodermal cells, extendingto a maximum diameter of 2–6 mm by 11–21 days ofculture. Biologically active CG in embryo-grown, serial spentmedia samples was measured in a mouse Leydig cell bioassay.The embryonic secretion of CG (ng/ml, mean ± SEM) commencedjust prior to hatching ( 0.014 ± 0.0), increased to 1.7± 0.5 after hatching but prior to attaching, and to 122.7± 45.5 by 5–11, 5108.7 ± 1706.0 by 10–17days, and decreased to 317.0 ± 201.4 by 16–40 daysin culture. These results show firstly that in-vivo producedrhesus monkey morulae and early blastocysts develop in vitroto hatched and attached blastocyst stages, exhibiting extensivetrophectodermal outgrowths. Secondly, the secretion of bioactiveCG commences from low levels during the pre-attaching blastocyststage, and increases exponentially after the attachment andtrophectodermal outgrowth of cultured embryos.     

An aggressive philosophy in controlled ovarian stimulation cycles increases pregnancy rates

To assess the effect of timing of human chorionic gonadotrophin(HCG) administration in ovarian stimulation cycles, the serumoestradiol concentration and follicle profile were comparedwith the clinical pregnancy rate in 582 ovarian stimulation— intra-uterine insemination (OS—IUI) cycles and3917 in-vitro fertilization—embryo transfer (IVF—ET)cycles. The pregnancy rates increased exponentially with increasingoestradiol in both OS—IUI and IVF—ET cycles (R²= 0.720, P < 0.001) but then decreased in OS-IUI cycles whenthe oestradiol concentration exceeded 5000 pmol/l (R² = 0.936,P < 0.004) at HCG administration. In OS—IUI cyclesthe percentage of cycles with three or more mature follicles( 18 mm diameter) increased up to an oestradiol concentrationof 5000 pmol/l then declined, mirroring the pregnancy rate (R²= 0.900, P = 0.01). The exponential increase in pregnancy ratewith increasing oestradiol concentration in IVF—ET cyclessuggests that high oestradiol concentration does not have adeleterious effect on endometrial receptivity. The decreasein pregnancy rate in OS-IUI cycles when oestradiol concentrationexceeded 5000 pmol/l reflected fewer mature follicles, resultingfrom premature administration of HCG to avoid severe ovarianhyperstimulation syndrome (OHSS). We recommend that HCG administrationbe delayed until multiple follicles have reached maturity, andreducing the risk of severe OHSS by converting high risk OS—IUIcycles to IVF—ET, or if funds or facilities are unavailable,transvaginally draining all but four or five mature follicles.     

Endocrinology: Daily measurements and in-vitro effects of human chorionic gonadotrophin in the early luteal phase

The purpose of the present study was to analyse daily measurementsof human chorionic gonadotrophin (HCG) in in-vitro fertilization(IVF) cycles and to reproduce the effects of HCG in vitro usinghuman granulosa—luteinized cells from the same patients.The study population consisted of nine women undergoing IVFbecause of tubal infertility in whom blood was drawn every 24h from the day of the ovulatory dose of HCG (10 000 IU) until6 days after ovum pick-up. Granulosa—luteal cells fromthe follicular aspirates were collected and cultured in vitroup to 6 days in the presence of increasing concentrations (0,0.01, 0.1, 1.0 and 100.0 IU/ml) of HCG. Serum progesterone andHCG in vivo as well as progesterone accumulation in vitro ondays 2, 4 and 6, were the main outcome measures. Maximum HCGconcentrations (0.25 IU/ml) were reached the day before ovumpick-up, and continuously decreased until day 6 after ovum retrieval.HCG did not stimulate progesterone production in vitro at anydose tested until day 6 after ovum pick-up. Then, 0.01 IU/mlresulted significantly (P < 0.05) stimulatory compared tocontrols, while 1.0 IU/ml was inhibitory (P < 0.05). It isconcluded that HCG supplementation in an IVF cycle is unnecessaryuntil day 6 after ovum pick-up. On day 6, progesterone productionis stimulated with very low concentrations of HCG.     

Endocrinology: Progesterone alone versus progesterone combined with HCG as luteal support in GnRHa/HMG induced IVF cycles: a randomized clinical trial

Two different regimens of luteal support in gonadotrophin hormone-releasinghormone (GnRH) analoguefhuman menopausal gonadotrophin (GnRHa/HMG)-inducedin-vitro fertilization cycles (IVF) were compared in a randomizedclinical trial. After embryo transfer, either vaginal progesteronealone was administered (n=89, P group), or a combination ofvaginal progesterone and human chorionic gonadotrophin (n=87,P/HCG group). The primary aim of this study was to assess theeffect of the different regimens of luteal support on the pregnancyrate. The secondary aim was to compare oestradiol and progesteroneconcentrations in the luteal phase between the two groups, andassess their effect on the pregnancy rate. A clinical pregnancyrate of 15% was found in the P/HCG group in comparison with26% in the P group (odds ratio 0.49; 99% confidence interval:0.18–1.3). The luteal serum oestradiol and progesteronevalues in the P/HCG group were significantly higher when comparedwith the P group on the 6th, 9th and 12th day after oocyte retrieval(Wilcoxon P<0.001). In accordance with the high oestradiolconcentrations, more cases of ovarian hyperstimulation syndrome(OHSS) were found in the P/HCG group. Oestradiol values on the9th day after oocyte retrieval, presumably the day of implantation,appeared to be higher in women who did not become clinicallypregnant. We conclude that vaginal progesterone alone providessufficient luteal support in GnRHa/HMG induced IVF cycles. Thecombination of vaginal progesterone and HCG as luteal supportleads to significant high luteal oestradiol and progesteroneconcentrations. But a high concentration of oestradiol seemsto have a deleterious effect on the implantation process, resultingin a low pregnancy rate.     

Endocrinology: Luteal function following ovulation induction in polycystic ovary syndrome patients using exogenous gonadotrophins in combination with a gonadotrophin-releasing hormone agonist

The luteal phase was studied in 12 polycystic ovary syndrome(PCOS) patients following ovulation induction using exogenousgonadotrophins combined with a gonadotrophin-releasing hormoneagonist (GnRH-a). Human menopausal gonadotrophin (HMG) was precededby 3 weeks of treatment with GnRH-a (buserelin; 1200 µg/dayintra-nasally) and administered in a step-down dose regimenstarting with 225 IU/day i.m. GnRH-a was withheld the day beforeadministration of human chorionic gonadotrophin (HCG; 10 000IU i.m.). Blood sampling and ultrasound monitoring was performedevery 2–3 days until menses. The luteal phase was significantlyshorter in PCOS patients as compared to eight regularly cyclingcontrols: 8.8 (3.3–11.4) days [median(range)] versus 12.8(8.9–15.9) days (P = 0.01). Median peak values for progesteronedid not show significant differences comparing both groups:52.3 (17.1–510.3) nmol/l versus 43.0 (31.2–71.1)nmol/l, respectively (P = 0.8). The interval between the dayof the progesterone peak and return to baseline was significantlyshorter in the PCOS patients than in controls: 2.5 (0.3–4.9)days versus 4.2 (3.9–10.5) days (P < 0.005). Luteinizinghormone (LH) concentrations during the luteal phase as reflectedby area under the curve were significantly lower in PCOS ascompared to controls: 4.4 (1.6–21.0) IU/l x days and 49.0(27.8–79.6) IU/l x days, respectively (P < 0.001).In conclusion, patients with PCOS may suffer from insufficientluteal phases after ovulation induction using HMG/HCG in combinationwith a GnRH-a. The corpus luteum apparently lacks the supportof endogenous LH and may be stimulated only by the pre-ovulatoryinjection of HCG. Potential involvement of adjuvant GnRH-a medicationor HCG itself in luteal suppression of endogenous gonadotrophinsecretion, and the importance of luteal function for pregnancyrates following treatment, warrant further studies.     

Placental and ovarian hormones in anembryonic pregnancy

The circulating levels of human chorionic gonadotrophin (HCG),pregnancy-associated plasma protein-A (PAPP-A), Schwangerschaftprotein 1 (SP-1), oestradiol and progesterone were measuredin 81 pregnant patients between 4 and 11 weeks gestation, followingin-vitro fertilization and embryo transfer. The patients weredivided as follows: singleton anembryonic pregnancies, n = 22;singleton pregnancies which spontaneously aborted followingthe demonstration of fetal heart activity, n = 7; and normalsingleton pregnancies, n = 52. The levels of all substancesmeasured were significantly reduced in women with anembryoniccompared to those with singleton pregnancies which proceededto term. The serum levels of SP-1, weeks 6–8 (P < 0.01);HCG, weeks 6–8 (P < 0.05); oestradiol, weeks 5–8(P < 0.05) and progesterone, weeks 6–8 (P < 0.05),were lower in anembryonic pregnancies than in those of pregnancieswhich spontaneously aborted. These differences may be a reflectionof the fact that miscarriage, after the demonstration of fetalheart activity, represents fetal demise at a later stage inpregnancy. In anembryonic pregnancies, significant associationswere found between HCG and both oestradiol and progesteronelevels from weeks 6 and 8, suggesting that in the absence ofan embryo, HCG is the prime determinant of steroid synthesisby the corpus luteum.     

Relationship of initial chorionic sac diameter to abortion and abortus karyotype based on new growth curves for the 16th to 49th post-ovulation day

In order to determine whether initial chorionic sac diameteris related to subsequent abortion, abortus karyotype, or birthweight and length, chorionic sac diameter was prospectivelymeasured by transvaginal ultrasound in 700 singleton pregnanciesbefore post-ovulation day 31, the latest day cardiac activitybecomes detectable in normal pregnancy. Results were comparedto values for the 10th to the 90th centiles, determined from227 measurements of in-vitro fertilization and gamete intra-Fallopiantransfer pregnancies. The abortion rate was 23.9% [95% confidenceinterval (CI) 19.2%, 28.6%] when initial chorionic sac diameterwas below the 50th centile, compared to 6.9% (95% CI 4.9%, 9.4%)when equal to or above the 50th centile. Chorionic sac diameterwas below the 50th centile in all anembryonic abortions andin 62% of embryonic abortions. Triploidy, trisomy 47 + 16, ortrisomy 16 and the presence of satellite bodies on chromosome22 were the only abortus karyotypes significantly associatedwith small chorionic sac diameter. Initial chorionic sac diameterwas not associated with birth weight or length. We concludethat chorionic sac diameter is decreased in anembryonic andembryonic abortion and that normal pregnancy outcome may beexpected in 90–95% of pregnancies in which initial chorionicsac diameter is equal to or above average     

Pregnancy: Reduced circulating placental protein concentrations during the first trimester are associated with preterm labour and low birth weight

Serum concentrations of human chorionic gonadotrophin (HCG),Schwangerschaftsprotein 1 (SP-1), pregnancy-associated plasmaprotein A (PAPP-A), progesterone and oestradiol were measuredat weekly intervals between the fifth (embryo transfer plus3 weeks) and 13th week of gestation during the first trimesterof pregnancies achieved following in-vitro fertilization (IVF)and embryo transfer in a group of women who delivered before(n = 8) or at term (n = 52). Those women who had a preterm deliveryhad significantly lower concentrations of PAPP-A (weeks 7–13;P = 0.0001–0.028) and SP-1 (weeks 6–8 and 10–12;P = 0.004–0.04). After correction of birth weight forsex and gestational age at delivery, preterm delivery was foundnot to be associated with growth retardation. However, comparisonof the circulating concentrations of the substances analysedin mothers who delivered babies of < 85% of the 50th centileof the normal range of birth weight for a given gestationalage and sex, with those who delivered babies of >85% revealedthat the concentrations of HCG (P = 0.012–0.04 on weeks6–9) and SP-1 (P = 0.003–0.03 on weeks 7, 9–13)were significantly lower in the former group. Weak, inconsistentassociations were found between the circulating concentrationsof HCG, SP-1 and PAPP-A and both corrected birth weight andgestational age at delivery. Thus, both the gestational ageat delivery and low birth weight may be related to impairedplacental development/function during the first trimester.     

Effect of insulin on human chorionic gonadotrophin secretion by placental explants

The effect of physiological concentrations of insulin (5–50µU/ml) was tested on human chorionic gonadotrophin (HCG)secretion by first trimester (7–9 weeks) and term placentalexplants using both static and dynamic culture models. In staticcultures, insulin exerted a significant biphasic inhibitoryeffect (80% at 5 µU/ml and 40% at 50 µU/ml) on HCGsecretion by placental explants. At approximate fasting plasmalevels, 25 µU/ml insulin added to superfused explantsfor 8 min also had a rapid inhibitory effect. A delayed inhibitoryeffect on HCG pulsatility was also observed using 25 µU/mlinsulin, with a 2-fold decrease in HCG pulse amplitude and a4-fold decrease in the area under the curve following overnightpre-incubation (P < 0.01). Insulin had no effect in staticcultures at term. The effect of insulin-like growth factor (IGF-I)and fibroblast growth factor (bFGF) on HCG secretion in staticcultures was not statistically significant. In conclusion, physiologicalconcentrations of insulin inhibit HCG secretion in first trimesterplacenta in vitro. This effect is gestational age dependentand specific since it is not mimicked by IGF-I or bFGF. Thus,insulin may be an important modulator of trophoblastic HCG secretionduring early pregnancy.     

Endocrinology: Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys

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) withand without recombinant LH (1: 1; n = 6) following 90 days ofgonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatmentin macaques was evaluated. Human chorionic gonadotrophin (HCG)was administered when six follicles >4 mm were observed.Oocytes were aspirated 27 h later and inseminated in vitro.Chronic Antide reduced serum oestradiol and bioactive LH toconcentrations observed in hypophysectomized rhesus monkeys.Multiple follicular growth required a longer interval followingrecombinant FSH (12 ± 1 days) than recombinant FSH +recombinant LH (9 ± 0.2 days), but the total number offollicles/animal did not differ between groups. The day priorto HCG, oestradiol concentrations were 4-fold less followingrecombinant FSH compared to recombinant FSH + recombinant LH.With recombinant FSH, more oocytes completed meiosis to metaphaseII(51%) and fertilized (89 ± 5%) relative to recombinantFSH + recombinant LH (12 and 52 ± 11% respectively).Follicular growth and maturation in LH-deficient macaques occurredwith FSH alone. Thus, LH is not required for folliculogenesisin primates. Higher fertilization rates following follicularstimulation with FSH alone suggest that the presence of LH withFSH (1: 1) during the pre-ovulatory interval impairs gametogenicevents in the periovulatory period.     

Fertilization and early embryology: Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon

Of 48 spare human pre-embryos achieving the expanded blastocyststage, 22 (45.6%) secreted significant amounts of human chorionicgonadotrophin (HCG) (>5 IU/l/day). Of these, nine remainedintrazonal, seven partially hatched and six fully hatched. Embryonicproduction of HCG in vitro appeared to be time-dependent, startingafter a certain minimum time (160 h post-insemination) and risingexponentially, with maximal HCG production around day 10. Hatchingwas not a prerequisite for HCG secretion, since similar amountswere produced by intrazonal blastocysts. Blastocysts derivedfrom abnormally fertilized oocytes also began secreting HCGexponentially but secretion was delayed and the upper limitof maximum HCG secretion rate was comparatively low. The actualamount of HCG is thought to reflect the number of viable trophectodermcells producing the hormone. HCG doubling times for blastocystsin vitro were rapid when compared to implanting blastocystsof a similar age in vivo, with 19/22 (86.4%) blastocysts havinga doubling time of < 10 h. Provided a pre-embryo can secreteHCG and maintain an adequate doubling time, sufficient HCG shouldbe produced for initial stages of embryonic recognition in vivo.Since intrazonal blastocysts are capable of fulfilling bothof these criteria, the limiting factor in realizing their fullpotential may be escaping from the zona pellucida.     

Endocrinology: A comparison of two starting doses of human menopausal gonadotrophin for follicle stimulation in unselected patients for in-vitro fertilization

Ovarian responses and embryology data were compared in patientsundergoing in-vitro fertilization following follicular stimulationusing long course gonadotrophin-releasing hormone (GnRH) analogue/humanmenopausal gonadotrophin (HMG) in which the initial daily dosewas two (150 IU) or three ampoules (225 IU) maintained for aminimum of 7 days. Group 1 (n = 31; centre A) patients weretreated with a starting dose of two ampoules, while group 2(n = 46; centre A) patients were treated chronologically immediatelybefore group 1 with a starting dose of three ampoules per day.Group 3 (n = 74; centre B) patients were treated with threeampoules per day simultaneously with group 1. There was no differencein the distributions of patient ages or reasons for treatmentbetween the three groups. Group 1 required longer treatmentbefore the plasma oestradiol attained 250 pg/ml than did boththe other groups (group 1, 9.0; group 2, 6.9; group 3, 6.7 days;P < 0.01), and this resulted in a longer follicular phasefor group 1 (mean: 14.5 days compared with 12.7 and 12.8 forgroups 2 and 3 respectively; P < 0.05). The numbers of follicles>16 mm in diameter at human chorionic gonadotrophin (HCG)administration and the numbers of eggs and embryos were allsignificantly lower (P < 0.04) in group 1, and cycle cancellationsdue to insufficient ovarian responses were higher (P < 0.02)in group 1. There was no difference in the numbers of ampoulesused, the oestradiol concentration at HCG, the fertilizationand pregnancy rates or the incidence of hyperstimulation syndromein the three groups. The lower starting dose, therefore, yieldedinferior responses without significant reduction in the HMGrequirement.     

Pregnancy: Corpus luteum failure in ectopic pregnancy

The endocrinology of ectopic pregnancy was studied in orderto investigate the origin of the discordance in the circulatingamounts of human chorionic gonadotrophin (HCG) and those ofoestradiol and progesterone. Serial maternal blood samples wereobtained at 4–9 weeks gestation from 93 patients who becamepregnant following in-vitro fertilization and embryo transferincluding 10 ectopic, 21 anembryonic and 62 normal singletonpregnancies. The samples were analysed for HCG, Schwangerschaftprotein-1 (SP-1), pregnancy-associated plasma protein-A (PAPP-A),progesterone and oestradiol. In ectopic pregnancies, concentrationsof all substances analysed were significantly reduced comparedto singleton pregnancies from 5 weeks gestation (P < 0.05–0.001)but they were not significantly different from those of anembryonicpregnancies. In ectopic pregnancies, associations were foundbetween the concentration of both HCG and SP-1 and those ofprogesterone and oestradiol. No associations were found betweenPAPP-A and any other substances analysed. This may be due toinsensitivity of the PAPP-A assay; alternatively PAPP-A concentrationsmay be differentially reduced in ectopic pregnancy. These findingssuggest that progesterone and oestradiol are derived from thecorpus luteum in early ectopic pregnancy but that the corpusluteum fails rapidly and the dominant source of both hormonesbecomes the trophoblast as early as 5 weeks.     

Pregnancy: The expectant management of early pregnancies of uncertain site

The role of expectant management was evaluated in 80 women inwhom clinical examination, including vaginal ultrasound, hadfailed to identify the location of an early pregnancy. In 45cases, spontaneous resolution of the pregnancy products occurred.A normal intra-uterine pregnancy was diagnosed in 12 patients.A total of 23 patients underwent active therapeutic measuresdue to an ectopic pregnancy (n = 16) or a spontaneous abortion(n = 7). The effectiveness of different diagnostic measuresto identify patients suitable for expectant management was analysed.In 33/34 patients (97%) with a relative daily human chorionicgonadotrophin (HCG) change of <–5%, and a serum progesteroneconcentration of <20 nmol/l, spontaneous resolution of thepregnancy products occurred. Among 46 cases, with a relativedaily HCG change of >–5% and/or serum progesterone>20 nmol/l, active therapeutic measures were carried outin 22 cases (48%), a normal intra-uterine pregnancy was diagnosedin 12 cases (26%) and spontaneous resolution of the pregnancyproducts occurred in 12 cases (26%). In conclusion, the combinationof a single progesterone assay and serial HCG determinationsretrospectively identified early pregnancies of uncertain locationin whom expectant management was a safe management option.     

Endocrinology: Insulin-like growth factor-I stimulated growth and progesterone production by granulosa--lutein cells. Lack of interaction with physiological concentrations of luteinizing hormone and follicle stimulating hormone

This study examined the effect of physiological concentrationsof insulin-like growth factor-I (IGF-I), follicle stimulatinghormone (FSH) and luteinizing hormone (LH) alone and in combinationon growth and progesterone production by human granulosa —lutein cells. Granulosa—lutein cells were obtained frompatients (n > 5) undergoing in-vitro fertilization (IVF)or gamete intra-Fallopian transfer (GIFT) treatment. Cells werecultured for 2 and 4 days in the presence of physiological concentrationsof human LH (code 68/40, 5IU/1), FSH (code 83/575, 20IU/1),or IGF-I (30 ng/ml) alone and in combination. Medium was changedevery 2 days. No change in cell number (relative to each patient'sown control) was observed after treatment with FSH or LH aloneor in combination at any time. IGF-I alone produced a 117 ±8% and 176 ± 15% (mean ± SEM, n = 5) increasein cell number after 2 and 4 days respectively. This increasewas unaffected by the addition of LH or FSH at any time. Basalprogesterone secretion was variable (1633, 975–2409 nmol/l,median and interquartile range, day 2) and decreased with timein culture (564, 375–1089 nmol/l, day 4). After 2 daysculture progesterone output increased by 116 ± 5% ofcontrol in response to LH and 153 ± 13% (mean ±SEM, n = 5) of control in response to IGF-I. After 4 days, LHand IGF-I stimulated progesterone levels by 279 ± 52%and 264 ± 37% (mean ± SEM, n = 5) respectively.IGF-I stimulated progesterone output was unaffected by the additionof LH or FSH at any time. FSH alone had no effect on progesteroneoutput and did not enhance the stimulation by LH. We concludefirstly that IGF-I stimulates the growth of granulosa—luteincells but this growth is unaffected by LH or FSH; secondly thatprogesterone secretion is stimulated by LH but that seen withIGF-I is secondary to an increase in cell number; thirdly thatFSH and LH do not synergize with IGF-I with regard to progesteronesecretion, and lastly that FSH does not stimulate progesteronesecretion or growth.     

The effects of the somatostatin analogue octreotide on ovulatory performance in women with polycystic ovaries

The elevated luteinizing hormone (LH) and androgen concentrationscharacteristic of women with polycystic ovaries (PCO) are consideredcrucial factors in their infertility. The somatostatin analogueoctreotide lowers LH and androgen concentrations in women withPCO. The effects of octreotide given concurrently with humanmenopausal gonadotrophin (HMG) were therefore compared withthat of HMG alone in 28 infertile women with PCO resistant toclomiphene. In 56 cycles of combined HMG and octreotide therapythere was more orderly follicular growth compared with the multiplefollicular development observed in 29 cycles in which HMG wasgiven alone (mean number of follicles > 15 mm diameter onthe day of human chorionic gonadotrophin (HCG) administration:2.5 ± 0.2 and 3.6 ± 0.4 respectively; P = 0.026).There was a significantly reduced number of cycles abandoned(>4 follicles > 15 mm diameter on day of HCG) in patientstreated with octreotide + HMG, so that HCG had to be withheldin only 5.4% of cycles compared to 24.1% with HMG alone (P <0.05). The incidence of hyperstimulation was also lower on combinedtreatment. Octreotide therapy resulted in a more ‘appropriate’hormonal milieu at the time of HCG injection, with lower LH,oestradiol, androstenedione and insulin concentrations. Althoughgrowth hormone concentration was similar on both regimens, significantlyhigher insulin growth factor-I concentrations were observedon the day of HCG in women on combined therapy than on HMG alone.     

In-vivo and in-vitro assessment of the influence of ritodrine and oxytocin on the placental secretion of human chorionic gonadotrophin and placental lactogen

The purpose of this study was to evaluate, in vivo and in vitro,the influence of ritodrine and oxytocin on the placental releaseof human chorionic gonadotrophin (HCG) and placental lactogen(HPL). The in-vivo study was performed on maternal sera collectedbefore and 1 h after the onset of either ritodrine treatment(50µg i.v/min; administered to 15 women at risk of prematurelabour) or oxytocin infusion (2 mU i.v./min; administered to21 women for acceleration of slow labour). The in-vitro studywas performed on human term placental explants incubated inthe presence of 4–400 ng ritodrine/ml or 15–1500UiV oxytocin/ml. HCG and HPL were measured by radio-immunoassayon maternal sera and incubation media. Maternal circulatingconcentrations of HCG and HPL remained unaffected after 1 hof ritodrine or oxytocin treatment The in-vitro release of HCGand HPL by placental explants was not modified when ritodrineor oxytocin was added to the incubation media. The lack of influenceof ritodrine and oxytocin on the placental secretion of HCGand HPL suggests that β₂-adrenergic and oxytocin receptorsare not involved in the releasing process.     

Association of early {beta}-human chorionic gonadotrophin values with pregnancy wastage and multiple implantation in a donor oocyte programme

An early marker predictive of a viable pregnancy would easethe anxiety associated with positive pregnancy tests after theuse of donor oocytes. We examined the predictive value of anearly serum quantitative human chorionic gonadotrophin (Q-HCG)concentration on pregnancy outcome following oocyte donation.Embryo transfers after oocyte donation resulting in a positiveserum -HCG were examined beginning 9 days after embryo transferfrom those samples assayed in our laboratory (n = 77). Q-HCGconcentrations were measured in our laboratory by an immunoradiometricassay utilizing the first International Reference Preparation.Implantations were defined as the number of gestational sacsvisualized by transvaginal ultrasound 21 days after embryo transfer.Biochemical pregnancies were those with transient elevationsin -HCG concentration but without implantation sites. Spontaneousabortions were characterized by an implantation site with theeventual arrest of development. Ongoing/delivered pregnanciesdeveloped appropriately and proceeded beyond the first trimester.Day 9 Q-HCG concentrations did not differentiate between biochemicalpregnancies/spontaneous abortions and ongoing/delivered pregnancies,although mean ± SD concentrations for biochemical pregnancieswere significantly lower than those for the other groups (P< 0.0001): biochemical pregnancies, n = 18, 5.8 ±8.9 mlU/ml, range 0–35; spontaneous abortions, n = 2,46.0 ± 10.0 mlU/ml, range 39–53; ongoing/deliveredpregnancies, n = 57, 41.5 ± 35.4 mlU/ml, range 0–214.In addition, day 9 Q-HCG concentrations did not differentiatebetween multiple implantations, although the implantation offour sacs had a significantly higher mean Q-HCG concentrationcompared with the implantation of fewer sacs (P > 0.0001):one sac, n = 22, 32.2 ± 21.5 mlU/ml, range 3–78;two sacs, n = 25, 35.8 ± 21.3, range 0–81; threesacs, n = 7, 47.1 ± 37.1 mlU/ml, range 22–126;four sacs, n = 4, 122.3 ± 62.4 mlU/ml, range 76–214.The positive predictive value of a Q-HCG >10 mlU/ml was 0.91(sensitivity 91%, specificity 75%). These initial data suggestthat early day 9 serum Q-HCG determinations do not accuratelyidentify viable pregnancies or multiple implantations. Evenan early negative pregnancy test should be repeated becauseit can be associated with a normal pregnancy.     

Prolactin and gonadotrophin interactions on progesterone formation in cultured human granulosa cells

Human granulosa cells were isolated from preovulatory folliclesduring cycles stimulated with HMG-HCG or clomiphene-HMG-HCGor from unstimulated cycles. The cells were cultwed for 6–8days in medium M199 containing fetal calf serum under 5% CO₂in air. Highly purified human prolactin and human chorionicgonadotrophin were added alone or in combination to the cultures,and the content of steroids in the medium was measured everysecond day, utilizing conventional RIA techniques. In the presenceof HCG the formation of progesterone (P) increased 3–5-foldover the control level with maximal effect after 4 days. Incells derived from clomiphene-HMG-HCG stimulated cycles, prolactinper se did not influence basal P formation but reduced the stimulatoryeffect of HCG. This was only seen in granulosa cells from follicles< 20 mm in diameter. In experiments with Forskolin, an adenylatecyclase activator, P formation was stimulated and the stimulationwas counteracted by the concomitant presence of prolactin, indicatingthat prolactin did not interfere with the LH-HCG receptor. Incells from smaller follicles, or in cells from follicles aspiratedfrom the natural cycle prior to the endogenous LH peak, P formationwas stimulated by HCG but the addition of prolactin did notreduce this stimulatory effect. The results are discussed inrelation to earlier reports on prolactin effects in vitro bothon laboratory animals and human material.     

Endocrinology: Follicle stimulating hormone-granulosa cell axis involvement in the antifolliculotrophic effect of low dose mifepristone (RU486)

This study was designed to assess the involvement of folliclestimulating hormone (FSH)—granulosa and luteinizing hormone(LH)—theca axes in the antifolliculotrophic effect ofmifepristone. Plasma gonadotrophins, including plasma LH bioactivityand pulsatility, oestradiol, testosterone and inhibin concentrations,and follicular growth were monitored in volunteer women treatedwith placebo or mifepristone in two consecutive cycles. Mifepristonewas given either as a single dose of 5 mg (n = 7) when the leadingfollicle had reached a diameter between 12 and 14 mm, or asa multiple dose of 5 mg/day for 3 days, beginning when the leadingfollicle had reached a diameter between 14 and 16 mm (n = 5)or between 6 and 11 mm (n = 5). Following the single dose ofmifepristone, follicular growth and the accompanying increasein plasma oestradiol were arrested at 12 and 36 h respectivelywithout changes in gonadotrophin or testosterone serum concentrations.The 3 day regimen arrested follicular growth and oestradiolrise and decreased plasma inhibin concentrations when follicleswere larger than 12 mm at the onset of treatment. These resultsindicate that the antifolliculotrophic action of mifepristoneis associated with a selective compromise of the FSH—granulosaaxis of dominant follicles that have passed a critical stageof growth.