Comparison of human chorionic gonadotrophin in human urine and amniotic fluid as estimated by a polyclonal and by a specific monoclonal assay in very early pregnancy

Urinary human chorionic gonadotrophin (HCG) was estimated in42 samples from patients in very early pregnancy or shortlyafter early spontaneous abortion, in which initial testing showeda positive urinary HCG in the Hybritech Tandem ICON II monoclonal2 min test. There were no false positives with this test evenwhen the blue colour was difficult to distinguish from the whitebackground, i.e. considerably less than the calibration bluespot at 50 mlU/ml. The Serono polyclonal -HCG radioimmunoassay(RIA) detected urinary intact HCG (plus free -subunit) at concentrationsas low as 10 mlU/ml IS 61/6 for bioassay (the old standard),i.e. 22mlU/ml 1st IRP 75/537 for immunoassay (the new standard).The LKB-Pharmacia DELFIA monoclonal fluoroimmunoassay (FIA)detected urinary intact HCG, without free -subunit, to a minimumconcentration of 18 mlU/ml 75/537. The proportion of monoclonal/polyclonalwas 2–155%. In normal amniotic fluid at 16–20 weeksof pregnancy, HCG concentrations were 3180? 2270 (755–11000) mlU/ml with the FIA.     

Predictive value of human chorionic gonadotrophin in the outcome of early pregnancy after in-vitro fertilization and spontaneous conception

This prospective study analyses the value of the -subunit ofhuman chorionic gonadotrophin (-HCG) in 120 pregnancies obtainedafter in-vitro fertilization (IVF)-embryo transfer. Spontaneousconception cycles (n = 16) were also analysed allowing a comparisonbetween these two forms of conception. Of the 120 clinical pregnancies,48 started as single gestations and 50 started with two or moresacs. There were 14 clinical abortions and eight ectopic pregnancies.All subjects had blood samples taken under a fixed protocolon days 11, 14, 17, 20 and 23 after follicular aspiration. Weeklysamples were obtained thereafter until day 60 from ovum retrieval.Transvaginal ultrasounds were performed at weekly intervals,starting on day 23 after follicular aspiration. In spontaneousconception cycles blood samples were obtained daily, startingon the day of follicular rupture. In spontaneous conceptioncycles and in IVF– embryo transfer conceptions, the doublingtime (DT) of ;-HCG was 1.4 ± 0.3 and 1.6 ± 0.4days respectively. This difference was not significant. In multigesta–tions,the DT was 1.5 ± 0.3 days. The absolute values of -HCGin early spontaneous gestations were significantly higher thanin IVF—embryo transfer cycles, suggesting that the blastocystimplants with less cellular mass when initiated in vitro ascompared with the in-vivo condition. The early prediction ofectopic pregnancy and spontaneous clinical abortion was analysedby the -HCG profile as well as the absolute values in comparisonto normal pregnancies. Both parameters showed significant differencesas early as the interval between days 11 and 23 from follicularaspiration. This study provides a comprehensive approach tothe evaluation of the outcome of early gestation in terms ofthe predictability of single and multigestation, ectopic pregnancyand early abortion.     

Pregnancy: Oocyte donation to women of advanced reproductive age: pregnancy results and obstetrical outcomes in patients 45 years and older

We analysed the results of oocyte donation to women of advancedreproductive age (45 years old) and followed their pregnanciesthrough to delivery in order to assess obstetrical outcomes.Patients (n = 162) aged 45–59 years (mean ± SD;47.3 ± 3.4 years) underwent 218 consecutive attemptsto achieve pregnancy. Oocytes (16.2 ± 7.2 per retrieval)were provided by donors 35 years old. Cleaving embryos (8.2± 4.8 zygotes/couple) were transferred trans-cervically(4.5 ± 1.1 per embryo transfer) to recipients prescribedoral micronized oestradiol and intramuscular progesterone. Followingoocyte aspiration there were six instances of non-fertilization(2.8%) and 212 embryo transfers. A total of 103 pregnancieswas established for an overall pregnancy rate (PR) of 48.6%,which included 17 preclinical pregnancies, 12 spontaneous abortions,and 74 delivered pregnancies (clinical PR 40.6%; delivered PR34.9%). Multiple gestations were frequent (n = 29; 39.2% ofpregnancies) and included 20 twins, seven triplets, and twoquadruplets. Two of the triplet and both of the quadruplet pregnanciesunderwent selective reduction to twins. Antenatal complicationsoccurred in 28 women (37.8% of deliveries) and included pretermlabour (n = 9), gestational hypertension (n = 8), gestationaldiabetes (n = 6), carpel tunnel syndrome (n = 2), pre-eclampsia(n = 2), HELLP syndrome (n = 2), and fetal growth retardation(n = 2). 48 (64.8%) deliveries were by Caesa-rean section. Thegestational age at delivery for singletons was 383 ±1.3 weeks (range 35–41 weeks), with birth weight 3218± 513 g (range 1870–4775 g); twins 35.9 ±2.0 weeks (range 32–39 weeks), birth weight 2558 ±497 g (range 1700-3450 g); and triplets 33.5 ± 0.7 weeks(range 32-34 weeks), birth weight 1775 ± 190 g (range1550-2100 g). Neonatal complications (4.6% of babies born) includedgrowth retardation (n = 2), trisomy 21 (n = 1), ventricularseptal defect (n = 1), and small bowel obstruction (n = 1).There were no maternal or neonatal deaths. We conclude thatoocyte donation to women of advanced reproductive age is highlysuccessful in establishing pregnancy. However, despite carefulantenatal screening, obstetrical complications are common, oftensecondary to multiple gestation.     

Is luteal function maintained by factors other than chorionic gonadotrophin in early pregnancy?

Women with ectopic pregnancy (n = 14) and early embryonic arrest(‘blighted ovum’) (n = 9) were studied 16 days afterconception, at a time when they were asymptomatic and serumconcentrations of -human chorionic gonadotrophin (HCG) werein the normal range and increasing at an apparently normal rate.Serum progesterone and oestradiol concentrations were comparedwith those from normal women matched for gestational age andserum -HCG concentration whose singleton intra-uterine pregnanciesproceeded normally beyond 20 weeks. Mean serum progesteroneconcentrations were significantly lower in the women with ectopicpregnancies than in matched controls (P < 0.002); however,there was no difference in the serum progesterone concentrationsbetween women with blighted ova and matched controls. Statisticallysignificant differences were not seen in serum oestradiol concentrationsbetween either group and matched controls. Similarly there wasno difference in serum progesterone or oestradiol concentrationsin 20 women who conceived ectopic pregnancies and 20 women conceivingblighted ovum pregnancies and their matched intra-uterine controlswhen conception followed ovarian stimulation. The low serumprogesterone concentrations seen in ectopic pregnancy suggestthat there is a specific and selective deficiency in progesteronesynthesis, which implies that factors other than HCG may influenceluteal function.     

The role of a single free {beta}-human chorionic gonadotrophin measurement in the diagnosis of early pregnancy failure and the prognosis of fetal viability

This prospective controlled study investigated the concentrationsof free -human chorionic gonadotrophin (HCG) subunit in 554women with a singleton intrauterine or tubal pregnancy. Theypresented with vaginal bleeding and/or abdominal pain in thefirst 18 weeks of pregnancy. The control group comprised 156women with musculo-skeletal pain and no vaginal bleeding. Theirpregnancies continued to term. The study group comprised 398women (141 threatened-continuing pregnancies, 37 threatened-miscarriages,185 non-continuing pregnancies and 35 tubal pregnancies). Free-HCG concentrations were significantly lower in the non-continuing,threatened-miscarriage and tubal pregnancy groups [mean 4.62,6.50 and 4.27 ng/ml respectively; 95% confidence interval (CI)3.75–-5.69, 4.46–9.48 and 2.92–6.2 respectively]than in the control and threatened-continuing groups (mean 41.61and 48.22 ng/ml respectively; 95% CI 34.53–50.13 and 42.03–55.32respectively) (P < 0.001 in all cases). A cut-off value at20 ng/ml was found to differentiate between the ‘viable’(control and threatened-continuing) and the ‘abnormal’(non-continuing, threatened-miscarriage and tubal) pregnancies,with 88.3% sensitivity and 82.6% positive predictive value.An excellent diagnostic and prognostic usability of free HCGwas confirmed by a receiver operating characteristic curve plotIn conclusion, a single serum free -HCG measurement taken inearly pregnancy is valuable in the immediate diagnosis of earlypregnancy failure and the long-term prognosis of viability.     

Pregnancy: Proliferative activity in ectopic trophoblastic tissue

Clinical observations have shown that tubal pregnancies developindividually different biological activities such as differentgrowth rates, levels of beta human chorionic gonadotrophin (-HCG),or rates of tubal wall destruction. In the present study, weevaluated the proliferative activity of ectopic cytotrophoblastictissue using immunocytochemistry with antibodies to Ki-67 (cloneMIB-1). The rates of proliferation obtained were related tothe maternal serum -HCG values. Reference data were obtainedfrom placentas of intact intrauterine pregnancies c(group I,n = 14). The proliferative activity of this tissue was comparedto that of cytotrophoblastic tissue of tubal pregnancies (groupII, n = 27). Ki-67-immunostained as well as non-stained cytotrophoblasticnuclei of the villi and the trophoblastic columns were countedseparately, and results were expressed as percentage of positivecells. Serum -HCG values were determined twice, 48 h and immediatelybefore operation. The cytotrophoblastic cells of intact intrauterinepregnancies (group I) showed uniform and high proliferativeactivities (80% on average in villi, 84% on average in columns).The average Ki-67 proliferation rate was significantly lower(P < 0.001) in trophoblastic tissue of tubal pregnancies(group II; 42% on average in villi, 61% on average in columns).Within the group of tubal pregnancies, higher intragroup differenceswere observed. The number of Ki-67-labelled cells was independentof the absolute preoperative serum -HCG values in both groups,yet they were clearly related to the relative increase of P-HCGin maternal serum. At higher proliferation rates, there wasa significant, growing increase of -HCG values (P < 0.01).We have found immunohistochemical evidence to support the previousclinical speculations that tubal pregnancies develop more heterogeneouslyand more slowly than intact intrauterine pregnancies. The developmentof the -HCG concentrations may be taken as an indirect parameter,reflecting proliferative activity of the trophoblast.     

Pregnancy: Adverse effect of a homogeneous hyperechogenic endometrial sonographic pattern, despite adequate endometrial thickness on pregnancy rates following in-vitro fertilization

We have previously presented data to show that in patients whohad in-vitro fertilization (IVF)—embryo transfer usingovarian stimulation involving the luteal phase leuprolide acetate—humanmenopausal gonadotrophin (HMG) regimen, poor pregnancy resultsensued if either the endometrial thickness was < 10 mm ora homogeneous hyperechogenic sonograpic pattern was presentimmediately prior to taking a human chorionic gonadotrophin(HCG) injection. There were only 15 cases with this hyperechogenictype endometrium (and no pregnancies). The purpose of the presentstudy was to evaluate the influence of a hyperechogenic endometriumwhen the endometrial thickess was 10 mm, in a more extensiveseries, in women having IVF—embryo transfer using thesame ovarian stimulation regimen. A total of 273 consecutivecycles, where endometrial thickness was 10 mm, were evaluated(not including the 85 cycles previously reported). Of 22 patientswith the hyperechogenic pattern, one achieved a chemical pregnancy(-HCG >500 mIU/ml) and none achieved clinical pregnancies(ultrasound confirmation). In contrast, 67 of 251 (26.7%) patientsconceived with other echo patterns (x² analysis = 5.9, df =1, P = 0.01). These data thus confirm, in a larger series, thenegative influence of this type of echo pattern on subsequentpregnancy rates following the luteal phase leuprolide acetate—HMGovarian stimulation regimen.     

Implantation: Impact of trophoblast penetration through the basal membrane on the efficacy of drug therapy in tubal pregnancies

Concentrations of -human chorionic gonadotrophin (HCG) of 2500IU/l are generally considered to be maximal for successful drugtherapy of tubal pregnancies [instillation of prostaglandin-F₂(PGF₂) or hyperosmolar glucose]. The purpose of our study wasto ascertain if there was an association between the significantlyhigher failure rates above this threshold value and the histologicallydetermined anatomopathological substratum. We therefore evaluatedthe impact of trophoblast penetration through the basal membraneof the Fallopian tube on the efficacy of drug therapy. Pre-operativeserum -HCG concentrations were compared with the histologicallydetermined trophoblast penetration, distinguishing between ectopicpregnancies with intra-luminal growths up to the myosalpinx,and those with extra-luminal growths going beyond the basalmembrane and penetrating the myosalpinx. Basic data were obtainedfrom a group of patients who received primary surgical treatmentbut it had never been the intention for them to receive drugtherapy (independently of their initial -HCG values; group I,n = 43). These reference data were compared with the findingsin preparations from another group of patients obtained duringsecondary surgical intervention, performed to achieve finalcure of tubal pregnancy after failure of primary PGF₂ instillation(group II, n = 30). Group I patients showed a significantlyhigher rate of intra-luminal trophoblast growths (P = 0.0001)at -HCG values <2500 IU/l; above this threshold value, extra-luminalspread was found significantly more often (P = 0.0001). In histologicalpreparations from group II, however, the number of extra-luminalgrowths was significantly higher even at low -HCG values (P= 0.007); at values above the threshold level, the distributionsin the two groups were similar. These results suggest that drugtherapy of tubal pregnancy becomes inefficient in tubal pregnanciesas soon as the trophoblast penetrates the basal membrane ofthe Fallopian tube.     

Evaluation of {beta}-endorphin and interleukin-6 in seminal plasma of patients with certain andrological diseases

Human semen contains large amounts of opioid peptides and cytokines.We have measured the concentrations of interleukin (IL)-6 in140 semen samples and of -endorphinin 77 semen samples. Themedian concentration of endorphinin seminal plasma from normozoospermicmen(n = 23) was 154.7 pg/ml (10th—90th percentiles, 42.0—774.6),and there was no significant difference in the -endorphin concentrationamong normozoospermic, oligozoospermic (n= 28), asthenozoospermic(n= 15), azoospermic(n= 4) and post-vasectomy (n= 7) samples.There was no correlation between -endorphin concentration andsperm characteristics, nor with blood hormones. Endorphinconcentration was lower in cases with immunelogical infertility,as revealed by a positive direct mixedantiglobulin reactiontest (n = 12) ( > 0.01), than inmatched controls. The medianconcentration of IL-6 insamples with normal sperm concentration,motility andmorphology with or without white blood cells (n=39) was 26.1 pg/ml (10th–90th percentiles, 7.3–172.3),and there was no significant difference in the IL-6 concentrationamong normozoospermic, oligozoospermic (n= 46),asthenozoospermic(n= 32), azoospermic (n= 13) and post-vasectomy (n= 10) samples.The IL-6 concentration was significantly higher in cases ofvaricocele (n= 22)without white blood cells in semen (P <0.001) than in matched controls without varicocele (n= 23).In addition, the IL-6 concentration was elevated (P < 0.0001)in cases with accessory sex gland inflammation (n= 40). IL-6concentration was positively correlated with white blood cellsin semen (n= 60, r = 0.59, P < 0.0001), but there was nocorrelation with -endorphin concentration. The IL-6 concentrationchosen to differentiate between cases with and without accessorygland inflammation was 45.3 pg/ml, with a specificity of 80.6%and a sensitivity of 92.5%. It is concluded that -endorphinin seminal plasma playsan immune suppressive role, and thatincreased IL-6 concentration may be related to testicular dysfunctionincases with varicocele. Furthermore, IL-6 is an accurate markerof accessory sex gland inflammation.     

Time-dependent effects of transforming growth factor {alpha} on aromatase activity in human granulosa cells

Transforming growth factor a (TGF) is implicated as a paracrinegrowth factor in the regulation of human granulosa cell function.To investigate this further, we have examined the actions ofTGF on the basal and folliclestimulating hormone (FSH)-stimulatedaromatase activity of human granulosa cells to determine howthis growth factor influences oestrogen biosynthesis in thefollicle. Granulosa cells from women having in-vitro fertilizationduring untreated or gonadotrophin-stimulated cycles were culturedfor 1–6 days in the presence or absence of FSH or TGFat a range of doses. Aromatase activity, expressed as oestradiolproduction, was determined after culture during a 3 h test period.After 2 days, TGF (1–300 ng/ml) decreased basal and FSH-stimulatedaromatase activity in a dose-dependent manner (ED50 = 3 ng/ml).In contrast, after 4 days, TGF enhanced both basal and FSH-stimulatedaromatase activity. Repeated experiments revealed a consistentpattern of inhibition on day 2, which was more marked in thepresence of FSH (reduction by 30.6 ± 9.1%, mean ±SEM; n = 14; P < 0.01), and stimulation on day 4 in boththe absence (increased by 61.4 ± 20.6%, mean ±SEM; n = 6; P < 0.05) and presence of FSH (increased by 36.0± 15.2%, mean ± SEM; n = 8; P < 0.05). Theresults provide further evidence that TGF is a paracrine factorin the control of oestrogen biosynthesis, but the actions canbe either inhibitory or stimulatory depending on the durationof exposure.     

Pregnancy: Placental isoferritin patterns during normal first trimester and tubal gestations

Placental isoferritin (PLF) has been shown to be involved inthe down-regulation of the maternal immune system during pregnancy.In a prospective study, serum PLF concentrations were measuredin 33 pregnant women with singleton, normal, ongoing first trimestergestations and compared with those of 22 women with tubal gestations.Diagnoses were based on endocrinological, sonographic, intra-operativeand histopathological criteria. Venous blood was obtained fromboth groups for PLF determination before evacuation of the pregnancyproducts. B-Human chorionic gonadotrophin (HCG), 17B-oestradioland progesterone were determined at surgery for the tubal pregnancypatients. The mean ± SD PLF concentrations were 18 ±14, 25.4 ± 42.3 IU/ml among normal and tubal gestationsrespectively. Significant differences between normal and tubalpregnancies were found (P < 0.05). Based on PLF measurements,sensitivity (67%) and specificity (33%) values were found tobe similar for the normal and ectopic pregnancies. No correlationwas found between the other measured pregnancy hormones andPLF for the tubal pregnancy group. Low PLF concentrations amongpathological gestations may reflect abnormal trophoblastic activity.The simultaneous assessment of PLF and -HCG concentrations whichprobably originate from different trophoblastic cells, is recommendedfor better diagnosis and monitoring of first trimester placentalactivity.     

Endocrinology: Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin

A recently described two-site enzyme immunoassay incorporatinga pre-assay oxidation step was validated and used to measureserum concentrations of dimeric inhibin in five normally cyclingwomen and in 13 women undergoing gonadotrophin therapy. Recombinanthuman inhibin A (standard) gave an assay response curve whichwas parallel to those for human serum samples and recovery ofexogenous inhibin added to serum samples before assay was quantittive(109±8%, n=11). During the normal menstrual cycle dimericinhibin concentration increased from 9.0±2.0 pg/ml duringthe early follicular phase to reach a mid-cycle peak of 55.3±11.1pg/ml coincident with the pre-ovulatory gonadotrophin surge.After falling to 27.9 ± 5.7 pg/ml 1 day after the luteinizinghormone surge, inhibin then rose in parallel with serum progesteroneto reach a peak value of 115.6 ± 19.3 pg/ml during themid-luteal phase, before falling to 14.1±4.9 pg/ml bythe onset of next menses. During the follicular phase, dimericinhibin concentrations were closely correlated with those ofserum oestradiol (r,= 0.69; P< 0.001), whereas during theluteal phase they were most closely correlated with serum progesteroneconcentrations (r = 0.73; P < 0.001). Daily treatment withhuman meno-pausal gonadotrophin promoted a progressive increasein serum dimeric inhibin concentration which increased 20-foldin 6 days. In the same period total-inhibin (measured by radioimmunoassay)increased 5-fold, while serum oestradiol increased 30-fold.Although the assay cross-reacted with dimeric inhibin formsof molecular masses in the range 200–30 kDa, chromatographyof superovulatory human serum revealed that the fully processed 30 kDa form is the predominant circulating form, although aproportion of this (30%) is reversibly associated with serumbinding protein(s).     

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.     

Subcutaneous low dose leuprolide acetate depot versus leuprolide acetate for women undergoing ovarian stimulation for in-vitro fertilization

A total of 100 women undergoing ovarian stimulation with gonadotrophin-releasinghormone agonist (GnRHa) and a human menopausal gonadotrophin(HMG) for in-vitro fertilization (IVF) participated in thisrandomized comparative study. Leuprolide acetate at a dose of0.5 mg/day s.c. (n = 52, group I), or low-dose leuprolide acetatedepot at a dose of 1.88 nig s.c. (n = 48, group II), was startedon days 21–23 of the cycle. Stimulation with 225 IU/dayHMG was started after pituitary desensitization had been achieved.The luteal phase was supported by human chorionic gonadotrophin(HCG) i.m. injection. There were nostatistical differences inbaseline oestradiol (24.5 ± 4.8 versus 21.9 ±4.5 pg/ml) and follicle stimulating hormone (FSH) concentrations(3.9 ± 1.9 versus 3.2 $ 1.8 mlU/ml), and concentrationson the day of HCG administration of oestradiol (1657 ±245 versus 1512$165 pg/ml), luteinizing hormone (LH; 6.2 ±4.8 versus 5.6 ± 4.3 mlU/ml) and FSH (10.6 ± 2.8versus 10.8 ± 3.6 mIU/ml). There were also no statisticaldifferences in the HMG dosage (26.8 ± 1.8 versus 28.5± 1.5), the number of oocytes retrieved (7.6 ±3.0 versus 8.1 ± 4.3), the number of oocytes fertilized(5.3 ± 2.1 versus 5.6 ± 3.0) and the number ofembryos transferred (3.5 ± 1.3 versus 3.4 ± 1.6).There was no evidence of a premature LH surge in either group,but two patients appeared to have a poor response in the leuprolideacetate group (group I). There were 11 pregnancies (21.2%) afterthe use of leuprolide acetate and 12 pregnancies (25.0%) inthose given leuprolide acetate depot; no statistical differenceexisted between these two groups. Thus, an s.c. low-dose leuprolideacetate depot injection may offer a useful alternative for pituitarysuppression in ovarian stimulation for IVF.     

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.     

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.     

Endocrinology: Ovarian hyperstimulation syndrome: pre-ovulatory serum concentrations of interleukin-6, interleukin-1 receptor antagonist and tumour necrosis factor-{alpha} cannot predict its occurrence

The pathogenesis of the ovarian hyperstimulation syndrome (OHSS)is poorly understood. Since significant elevations in cytokinesare found in 01155, our objective was to conduct a prospectivecase-controlled study to assess if preovulatory cytokine serumconcentrations can predict its occurrence. The study group wasselected from in-vitro fertilization patients who subsequentlydeveloped severe OHSS, along with a matched group who did notdevelop this complication (n = 20), and a healthy normal controlgroup (n = 10). Interleukin-6 (IL-6), interleukin-1 receptorantagonist (IL-1RA) and tumour necrosis factor- (TNF) measurementswere performed with sensitive immune-assays and confirmed withbioassays. Serum IL-6 (mean concentration ± SEM: 4.38± 0.36 pg/ml), IL-1RA (829 ± 292 pg/ml) and TNF(15.5 ± 132 pg/ml) concentrations did not show differencesthroughout the normal menstrual cycle group. Cytokine variabilityand pre-ovulatory values were similar in OHSS compared to controlledovarian hyperstiinulation (COH) patients. However, average follicularphase serum 1L-6 concentrations were higher in OHSS (8.71 ±0.41 pg/ml) and COH (7.66 ± 0.38 pg/ml) patients thanin normally menstruating women (4.34 ± 0.99 pg/ml) (P< 0.0001). Pre-ovulatory serum 1L-6 concentrations were alsohigher in OHSS (9 ± 0.94 pg/ml) and COH (73 ±0.97 pg/ml) patients than in controls (4.57 ± 1.1 pg/ml)(P < 0.01 and P < 0.04 respectively). IL-1RA and TNF concentrationswere comparable in all the groups. This study suggests thatcytokine measurements cannot be used to predict the occurrenceof OHSS prior to the administration of human chorionic gonadotrophin.     

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.     

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.