Effect of 1-34 human parathyroid hormone upon first trimester placental human chorionic gonadotrophin secretion in vitro: potentiation by epidermal growth factor

Human chorionic gonadotrophin (HCG) secretion by the early placentais under multifactorial control. Epidermal growth factor (EGF)has been reported to be involved in regulating the formationand secretion of HCG by first trimester placental explants inculture. The effect of the amino-terminal fragment of parathyroidhormone (1–34 PTH), a calciotrophic factor upon HCG secretion,and its possible interaction with EGF were examined in thisstudy, both in static cultures and in superfusion, where ithas previously been demonstrated that HCG secretion is spontaneouslypulsatile. Gestational age-dependent effects of 1–34 PTHwere noted in both models. In static cultures, 1–34 PTHstimulated HCG secretion in 7–9 week placenta, in a biphasicfashion, the maximal effect being noted at 10–25 ng/mlconcentrations (250–270%), while at 1 and 100 ng/ml, theeffect was mild. In superfusion, the effect of 1–34 PTHadded overnight was also stimulatory, as shown by the significantlyincreased pulse amplitude and area under the curve. Effectsof 1–34 PTH at 11–14 weeks were inhibitory. In staticcultures at 7–9 weeks, the stimulatory effect of 25 ng1–34 PTH was increased by 70% when EGF (100 ng/ml) wasadded. However at 11–14 weeks, this combined effect wasinhibitory. We conclude that 1–34 PTH has an endocrineeffect on secretion of HCG by the first trimester placentaltissue, and this effect is potentiated by the addition of EGF.     

Pregnancy: Gestational-age-dependent effects of retinoids on HCG secretion by placental explants

Vitamin A (VITA) is considered to be an essential nutrient inboth pregnant and non-pregnant states. It has been suggestedthat VITA, among others, is involved in the process of morphogenesis.In contrast, synthetic derivatives of VITA, specifically Tigasone(etretinate, TIG) and Roaccutane (isotretinoin, ROA), are regardedas major teratogens. Therefore, in the present study we haveexamined the effect of VITA and other retinoids on human chorionicgonado-trophin (HCG) secretion by placental explants in thefirst trimester. Results show that, at 7–9 gestationalweeks, all three compounds had a significant inhibitory effecton HCG secretion. In the case of VITA, this inhibition was time-dependent.A biphasic maximal inhibition was present at 1 µM concentrationswhen the retinoids VITA, TIG and ROA were added for 16 h (52,58 and 57%, respectively; P < 0.01 by one-way analysis ofvariance). In contrast, the addition of the three retinoidsat 1 µM concentrations for 16 h had no significant effecton HCG secretion by placental explants of 11–13 weeksgestational age. In conclusion, both natural and synthetic retinoidsdemonstrate a significant inhibitory effect on HCG secretionby the early placenta (pre-HCG peak). VITA may be involved incausing a plateau and the later decline in HCG secretion. Inhibitionof HCG secretion by retinoids may contribute either directlyor indirectly to their teratogenicity.     

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.     

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.     

Endocrinology: Somatostatin action on rat ovarian steroidogenesis

To date, very few studies on the effect of somatostatin on femalereproductive function have been reported. In our study, we examinedthe effects of somatostatin on (i) androgen biosynthesis usingwhole ovarian dispersates, and (ii) aromatase activity and progesteroneproduction using granulosa cells. Whole ovarian dispersatesobtained from immature rats were cultured for 96 h in serum-freemedium with human chorionic gonadotrophin (HCG; 25 ng/ml) andinsulin (10 µg/ml) in the presence or absence of an increasingconcentration of somatostatin (0.03–3.00 ng/ml). HCG-and insulin-stimulated accumulation of androsterone by thesecells was inhibited significantly by somatostatin. Granulosacells from diethyl-stilbestrol-treated rats were cultured for48 h in serum-free medium with follicle-stimulating hormone(FSH; 20 ng/ml) and FSH plus insulin (1 µg/ml) with orwithout somatostatin (0.03–3.00 ng/ml). Both aromataseactivity and progesterone production stimulated by FSH and FSHplus insulin were significantly inhibited by somatostatin. Somatostatinby itself (1 ng/ml) did not have an effect on any of the evaluatedparameters. The action of somatostatin could be immunoneutralizedand did not influence the plated viable cell mass. These findingsindicate that somatostatin can regulate ovarian steroidogenesisby mediating gonadotrophin and growth factor action on differentovarian cell types.     

Ovary and Ovulation: An aminopeptidase inhibitor, bestatin, enhances gonadotrophin-stimulated ovulation in mice

The role of aminopeptidases in follicular growth and/or ovulationin vivo was examined. We injected an inhibitor of cell-surfaceaminopeptidases, bestatin (4 mg/ml, 100 µl), i.p. fourtimes during 2 days into 20 day old female ICR mice, in whichfollicular growth and ovulation were stimulated by pregnantmare's serum gonadotrophin (PMSG, 5 IU) and human chorionicgonadotrophin (HCG, 5 IU). The number of ovulated oocytes wasestimated by counting the number of oocytes in the oviduct 19h after HCG injection. The mean ± SD number of ovulatedoocytes in bestatln-treated mice was significantly higher thanthat in control mice [47.00 ± 18.13 (n = 26) versus 35.90± 10.14 (n = 28), P < 0.01]. To confirm the directeffect of bestatin on the ovary, bestatin (2 mg/ml, 3 µl)or its stereolsomer (2 mg/ml, 3 µl) with very weak inhibitoryactivity was unilaterally injected into the ovarian bursa 24h before the administration of PMSG. As a control, buffer (3µl) was injected into the contralateral bursa. In someexperiments, bestatin (2 mg/ml, 3 µl) was injected justbefore HCG administration. The administration of bestatin viathe ovarian bursa prior to PMSG administration significantlyincreased the number of ovulated oocytes per oviduct from thetreated compared with the contralateral ovary [23.70 ±9.61 versus 17.10 ± 5.83 (n = 25), P < 0.01], whereasits stereoisomer elicited no significant effects. The administrationof bestatin just before HCG administration also had no effect.These findings indicate that membrane-bound peptidase(s) presenton murine ovarian cells is an important regulating factor(s)of follicular growth and/or ovulation.     

Relationship of biochemical pregnancy to pre-ovulatory endometrial thickness and pattern in patients undergoing ovulation induction

In order to assess the relationship between pre-ovulatory endometrialthickness and pattern and biochemical pregnancy, the pregnancyoutcome was retrospectively analysed in 81 patients undergoingovulation induction evaluated by vaginal ultrasound on the dayof human chorionic gonadotrophin (HCG) administration or luteinizinghormone (LH) surge. Biochemical pregnancies occurred in 7/32(21.9%) pregnancies when endometrial thickness was <9 mm,compared to 0/49 when endometrial thickness was 9 mm on theday of HCG administration or LH surge (P < 0.0025). Clinicalabortions occurred in 5/32 (15.6%) pregnancies when endometrialthickness was 6–8 mm, compared to 6/49 (12.2%) when endometrialthickness was 6–8 mm (NS). Endometrial thickness was relatedto the cycle day of HCG or LH surge (r = 0.37, P < 0.001)but was unrelated to oestradiol level on the day of HCG administrationor LH surge (r = 0.12). Biochemical pregnancies were relatedto endometrial pattern (r = – 0.22, P = 0.02) but wereunrelated to maternal age or previous abortions. Clinical abortionswere related to age (r = 0.26, P = 0.01) and to previous abortion(r = 0.25, P = 0.013) but were unrelated to endometrial pattern.Neither biochemical pregnancy nor clinical abortion was relatedto oestradiol or LH levels on the day of HCG administrationor LH surge. These findings suggest that the majority of biochemicalpregnancies do not result from karyotypically abnormal embryos,as do clinical abortions.     

Exogenous steroids and the control of oestradiol secretion by human granulosa-lutein cells by follicle stimulating hormone and insulin-like growth factor-I

This study first examined the relative activities of 17-hydroxylase,17, 20-lyase and aromatase in human granulosa–lutein cellsby challenging the cells with steroid precursors in the oestradiolbiosynthetic pathway. When cells from four patients were challengedwith precursor steroids on the pathway to oestrogen synthesis(pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesteroneand androstenedione at 5 x 10^–6 M), oestradiol (nmol/l)outputs after 1 day of culture were (median, interquartile range)as follows: 4.1 (2.1– 8.8; pregnenolone), 3.1 (1.7–6.0;progesterone), 12.5 (6.9–18.1; 17hydroxypregnenolone),8.2 (4.1–16.7; 17hydroxyprogesterone) and 251 (140–819;androstenedione). No further increases were seen when the steroidconcentration was increased to 1 x 10^–5 M. Basal oestradiolsecretion was 3.5 (1.6–8.2) nmol/l. We conclude that theconversion of pregnenolone/progesterone to oestradiol by granulosa–luteincells is rate limited by 17-hydroxylase activity but that thesecells are capable of oestradiol secretion (in the nmol/l range)in the absence of androstenedione. In the second part of thisstudy we examined the control of granulosa–lutein oestradiolsecretion by follicle stimulating hormone (FSH) and insulinlikegrowth factor-I (IGF-I) in the presence and absence of exogenousandrostenedione (10^–6 M). Cells were cultured for up to6 days and basal oestradiol (nmol/l) fell dramatically overthis period both in the presence and absence of androstenedione,e.g. from 339 (223–419) (median and interquartile range,cells from five patients cultured in the presence of androstenedione)after 2 days to 14 (7–59) after 6 days. There was no effectof FSH (83/575, 0–160 IU/l) in the absence of androstenedionebut in its presence FSH (96 IU/l) increased oestradiol secretionslightly by 153 ±45 (day 4) and 151 ± 21 (day6; results mean ± SEM, percentage increase over time-matchedcontrols; cells from five patients). In contrast, the effectof IGF-I (30 ng/ml) was to markedly enhance oestradiol secretionto 1099±320% (n = 7) of the control (day 4) in the absenceof exogenous androgen and to 551 ± 184% (n = 5) in itspresence. There was no evidence of any synergistic interactionbetween IGF-I and FSH during the culture period in the absenceof androstenedione. However, there was a synergistic effectfor IGF-I (30 ng/ml)/FSH (96 IU/l) after 6 days in culture inits presence in that oestradiol secretion increased by 1748± 294% (n = 5) compared to 157 ± 21% (FSH) and1211 ± 233% (IGF-I) for the stimulators on their own.However, this effect may be explained, in part, by the increasein cell number provoked by IGF-I over this culture period. Weconclude that (i) under these conditions granulosa cells showedlow 17-hydroxylase activities, (ii) granulosa cells are capableof synthesizing oestradiol in the absence of exogenous androgens,the substrates for the aromatase complex, and (iii) FSH is oflittle importance in stimulating oestradiol secretion from granulosa-luteincells, and the evidence for it positively modulating IGF-I activityis poor.     

Effect of gonadotrophin-releasing hormone and related analogue on human luteal cell function in vitro

The possible direct effect of gonadotrophin-releasing hormone(GnRH) and GnRH agonist (GnRH-A; buserelin) on basal and humanchorionic gonadotrophin (HCG)-stimulated progesterone (P) andcyclic AMP (cAMP) production by cultured human luteal cellswas examined. Luteal cells from the early or mid-luteal phasewere incubated in long-term cultures. They responded to HCGstimulation with a 2- to 3-fold increase in P production anda 2-fold increase in cAMP production. The addition of GnRH (10^–7and 10^–5 M) or GnRH-A (10^–7 and 10^–5 M) tothe medium had no effect on either basal or HCG-stimulated secretion.These results indicate that both GnRH and GnRH-A have no directeffect on human luteal steroidogenesis in vitro.     

Steroid production in cultured thecal cells obtained from human ovarian follicles

During follicular maturation there is a co-ordinated hormonalregulation of the theca and granulosa cells. It is generallybelieved that granulosa cell proliferation and differentiationare promoted mainly by follicle stimulating hormone (FSH) andthat luteinizing hormone (LH) regulates the function of thetheca cells. The aim of the present study was to examine theeffect of LH/human chorionic gonadotrophin (HCG) on steroidproduction in human thecal cells. Isolated follicles (5–20mm) were obtained during the follicular phase of the menstrualcycle in 10 women undergoing gynaecological laparotomy for reasonsunrelated to ovarian pathology. The leading follicle(s) wasexcised and dispersed cells of the theca interna layer wereisolated through combined mechanical and enzymatic techniques.The thecal cells were cultured 4–6 days with and withoutLH/HCG. Medium levels of androstenedione, testosterone and progesteronewere measured by radioimmunoassay. Isolated thecal cells, culturedfor 6 days, showed a high sensitivity to stimulation by LH/HCG.Steroid secretion was highest during days 0–2 and thendeclined gradually. LH/HCG stimulated steroid production ina dosedependant way with the maximal stimulatory effect of LHat a concentration of 1–10 ng/ml, and of HCG at 0.01–0.1IU/ml. The important question, especially in clinical situations,of the optimal level of LH for normal follicular maturation,remains to be answered. The present study is compatible withthe view that thecal cell steroidogenesis in vivo is close tomaximally stimulated by normal basal LH levels.     

Rapid diagnosis of early ectopic pregnancy in an emergency gynaecology service--are measurements of progesterone, intact and free {beta} human chorionic gonadotrophin helpful?

The clinical usefulness of measuring serum concentrations ofprogesterone, human chorionic gonadotrophin (HCG) and the free-subunit of HCG in distinguishing between early viable and non-viablepregnancy, before an accurate ultrasound diagnosis is possible,was evaluated in a prospective study of patients presentingto our emergency gynaecology service with a clinical suspicionof ectopic pregnancy. Patients were selected on the basis ofinitial HCG concentrations; samples with HCG 25–10 000IU/I were later analysed for progesterone and free HCG. Of the181 patients studied, 38 (21%) had an ectopic pregnancy, 108(60%) had a spontaneous abortion and 35 (19%) had a viable intra-uterinepregnancy. Concentrations of HCG and free HCG in the group withviable pregnancies were significantly higher than in the groupwith ectopic pregnancy (P < 0.001) and than those destinedto miscarry (P < 0.01). Progesterone concentrations werealso significantly higher in the viable versus the ectopic andthe spontaneous abortion groups (P < 0.001 in each case).Despite these highly significant differences there was a degreeof overlap such that it was impossible to devise a cut-off levelfor any hormone analysed, either singly or in combination, whichwould offer a clinically useful predictor of outcome.     

Endocrinology: Pre-ovulatory progesterone growth rate in patients treated with gonadotrophin-releasing hormone agonist and outcome of in-vitro fertilization

The present study was undertaken to assess whether the increasein serum progesterone concentration following the administrationof human chorionic gonadotrophin (HCG) may have predictive valueon the in-vitro fertilization (IVF) success rate. Progesteroneconcentration on the day of HCG administration and the increasein progesterone concentration on the following day were evaluatedin 140 consecutive patients undergoing IVF with embryo transfer.Stimulation protocol in all study patients entailed intranasaladministration of short-acting gonadotrophin-releasing hormoneagonist (GnRHa) buserelin and human menopausal gonadotrophin.A pregnancy rate of 37.2% was achieved when at least three embryoswere transferred. The only significant difference between conceptionand non-conception cycles was found in serum progesterone concentrationsafter HCG administration (P < 0.01), whereas the mean progesteroneconcentration on the day of HCG did not differ. No differencein other hormonal or cycle parameters was observed. The increasein progesterone concentration was significantly greater in thegroup of patients who achieved pregnancy than in the group whodid not (2.2 ± 0.2 versus 1.6 ± 0.1 ng/ml, respectively;P < 0.01). A critical breakpoint in serum progesterone wasarbitrarily determined at 1 ng/ml. An increase in progesteroneconcentration 1 ng/ml when three or more embryos were transferredwas associated with a positive predictive value for pregnancyof 40.4% (sensitivity of 94.7%), whereas a negative predictivevalue of 86.7% was obtained when this value was <1 ng/ml.These findings indicate that an adequate rise in serum progesteronefollowing HCG administration provides useful information aboutthe possible outcome of the treated cycle.     

Increasing progesterone secretion in human granulosa-luteal cells induced by human follicular fluid

Increasing evidence suggests that local ovarian agents playa central role in the regulation of follicular maturation andcorpus luteum formation. In previous studies, we have shownthat porcine follicular fluid induces granulosa cell luteinizationin sow, human and rat. In the present study, the effect wasinvestigated of either human follicular fluid (FF) alone, humanfollicle-stimulating hormone (FSH) alone, or both upon progesteronesecretion of human granulosa-luteal cells. Granulosa-lutealcells were cultured in the presence of either FSH (5, 50 and250 ng/ml), lyophilized FF (50 and 250 µg/ml) or both.Secretion of progesterone increased from a minimum of 2.5-foldto a maximum of 23-fold in the presence of FSH alone and, significantlyless (2-fold) in the presence of FF alone, compared to cellscultured in medium alone. The co-administration of FSH and FFwas significantly more effective than either alone, while additionof both FSH (250 ng/ml) and FF (250 µg/ml) gave maximalprogesterone secretion. In granulosa-luteal cells pre-culturedwith both FSH and FF, subsequent exposure to human chorionicgonadotrophin (HCG) alone increased progesterone secretion 1.6-foldto 11-fold, compared to cells pre-cultured with FSH alone. Theeffect of FF from individual follicles was also studied. FFfrom follicles yielding mature cumulus—oocyte complexeswas 4.2-fold more effective, than FF obtained from folliclesyielding immature cumulus—oocyte complexes in enhancingthe FSH stimulation of progesterone secretion. A pre-cultureof granulosa-luteal cells in FSH plus FF from mature follicleswas 1.8-fold more effective in enhancing HCG-induced progesteronesecretion, compared to FF from immature follicles. This studysuggests that progesterone secretion by granulosa-luteal cellsis regulated by gonadotrophins and other factors accumulatingin FF.     

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.     

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: 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.     

A double-blind cross-over controlled study to evaluate the effect of human biosynthetic growth hormone on ovarian stimulation in previous poor responders to in-vitro fertilization

The effect of exogenous human biosynthetic growth hormone (HGH;12 IU/day; Norditropin, Novo-Nordisk) on the response to ovarianstimulation using a buserelin/human menopausal gonadotrophin(HMG) regimen was assessed in women who had previously showna ‘poor response’ in spite of increasing doses ofHMG. Forty patients were recruited into a prospective double-blindplacebo-controlled study. The serum follicle stimulating hormone(FSH) on day 2–5 of a menstrual cycle (< 10 IU/I) wasused to exclude any peri-menopausal candidates. The urinary24 h GH secretion was normal in all patients. Thirty-three patientscompleted the study with 21 patients having human chorionicgonadotrophin (HCG) in both arms, thus providing a completeset of placebo control data. Of these 21 patients, the administrationof HGH compared to the placebo cycle resulted in increased serumconcentrations of fasting insulin on the 8th (median 3.9 versus5.8 mU/I; P < 0.0005) and13th (median 4.4 versus 5.8 mU/I;P < 0.05) day of HMG in those cycles receiving HGH. After8 days of co-treatment with HGH the number of cohort follicles(14–16.9 mm) was significantly increased, but this changewas not sustained on the day of HCG administration. No statisticaldifference in the serum oestradiol on the 8th day of HMG orday of HCG, length of the follicular phase, total dose of HMGused, or the number of oocytes collected was seen between theplacebo or HGH cycles. This study demonstrates that HGH doesnot improve the ovarian response to ovulation induction in previouspoor responders.     

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on culturedporcine granuiosa cells by employing the aminopeptidase assayusing alanine-p-nitroanilide and histochemical staining usingL-leucyl-β-naphthylamide. Porcine granuiosa cells obtainedfrom follicles 4–5 mm in diameter were cultured for 7days. The aminopeptidase assay showed that the porcine granuiosacell culture had aminopeptidase activity and that this activitywas inhibited in a dose-dependent manner by bestatin which bindsto cell surfaces and inhibits cell surface aminopeptidases.Histochemical staining also indicated that cultured granuiosacells had aminopeptidase activity. Porcine granuiosa cells werecultured in the presence or absence of porcine follicle stimulatinghormone (FSH, 3.125 nmol/1) and/or bestatin (0.4, 4.0 and 40.0µ/ml) for 7 days, and the production of progesterone andoestradiol was measured. In the presence of porcine FSH, theproduction of progesterone and oestradiol by granuiosa cellswas increased significantly by 5- and 2-fold respectively. Theseincreases were enhanced further by bestatin (40.0 (µg/ml).In the absence of porcine FSH, progesterone production was enhancedby bestatin (40.0 µg/ml), whereas no significant effectof bestatin on oestradiol secretion was observed. These findingsindicate that the inhibition of membrane-bound amino-peptidase(s)on the cell surfaces affects the steroidogenesis of granuiosacells, and that these aminopeptidase(s) are important regulatorsof granuiosa cell differentiation.     

Infertility: Ovarian synchrony factor: a new ultrasound parameter in the prognosis of follicular rupture

The present study was carried out to present a new ultra-soundparameter used in stimulated cycles and called the ovarian synchronyfactor(OSF), which reflects the response of the total follicularcohort. It is calculated from the formula: OSF = (total no.of follicles 16 mm)/(total no. of follicles 10mm). OSF wasdetermined on the day of human chorionic gonadotrophin (HCG)administration (indicated when at least one follicle was 16mm)by measuring the widest follicular diameters with an ATL ultrasoundapparatus model Ultramark 4, with a 5.0 MHz vaginal transducer,in a total of 221 cycles stimulated for non-invasive assistedreproduction techniques (i.e. no oocyte retrieval). A new ultrasoundexamination was performed 56–60 h after HCG administrationto determine the possible presence of follicular rupture indicatedby the disappearance of the follicular image and/or a >5mmdecrease in the widest follicular diameter. The mean OSF inthe group of patients with rupture of at least one follicle(195 cycles) was mean ±SD, 0.57 ±0.25, as opposedto a mean ±SD of 0.39 ± 0.25 for the group withoutfollicular rupture (26 cycles). The Mann–Whitney testshowed that the OSF of the group with follicular rupture wassignificantly higher than that detected in the group withoutfollicular rupture (P <0.01). This information suggests thatovarian stimulation protocols should produce a synchronous follicularresponse (an OSF as close as possible to 1), i.e. that the follicularlots developing in both ovaries should not vary widely in size.This follicular homogeneity should facilitate the follicularrupture process.     

Fertilization and early embrology: The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signalduring early pregnancy in primates. CG has close structuraland functional similarities to pituitary luteinizing hormone(LH) which is regulated by gonadotrophin releasing hormone (GnRH).Tostudy the regulatory mechanism of CG secretion in primate embryos,we examined the production and timing of secretion of GnRH inperi-implantation embryos of the rhesus monkey. In-vivo fertilized/developedmorulae and early blastocysts, recovered from non-superovulated,naturally-bred rhesus monkeys by non-surgical uterine flushing,were cultured in vitro to hatched, attached and post-attachedblastocyst stages using a well-established culture system. Wemeasured GnRH and CG in media samples from cultured embryoswith a sensitive radioimmunoassay and bioassay, respectively.The secretion of GnRH (pg/ml; mean ± SEM) by embryos(n = 20) commenced from low levels (0.32 ± 0.05) duringthe pre-hatching blastocyst stage to 0.70 ± 0.08 at 6–12days and 1.30 ± 0.23 at 13 days of hatched blastocystattachment and proliferation of trophoblast cells. GnRH concentrationsin culture media obtained from embryos (n = 5) that failed tohatch and attach were mostly undetectable (0.1). Samples thatdid not contain detectable GnRH failed to show detectable CG.Immunocytochemical studies, using a specific monoclonal anti-GnRHantibody (HU4H) as well as polyclonal antisera (LR-1), revealedthat immunopositive GnRH cells were localized in pre-hatchingblastocysts (n = 4), in blastocysts (n = 2) after 5–10days of attachment and in monolayer cultures (n = 4) of well-establishedembryonic trophoblast cells. GnRH positive staining was seenonly in cytotrophoblasts but not in syncytiotrophoblasts. Similarly,cytotrophoblast, but not syncytiotrophoblast, cells of the rhesusplacenta were immunopositive. In controls, either in the absenceof antibody or in the presence of antibody pre-absorbed withGnRH, these cells failed to show stain. These observations indicate,for the first time, that an immunoreactive GnRH is producedand secreted by blastocysts during the peri-attachment periodand by embryo-derived cytotrophoblast cells in the rhesus monkey.