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.     

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.     

Pregnancy: The effect of dose of mifepristone and gestation on the efficacy of medical abortion with mifepristone and misoprostol

Although it has been demonstrated that a combination of mifepristoneand a prostaglandin is an effective method of inducing abortionin early pregnancy, the optimum dose of the antigestogen isunknown. Women (n = 220) requesting abortion in early pregnancy(63 days amenorrhoea) were randomized to receive a single doseof either 600 or 200 mg mifepristone followed 48 h later bya single dose of 600 µg misoprostol by mouth. The percentageof women who had a complete abortion (93.6% confidence interval90.4–95.5%) was identical in the two groups. There wasno significant difference in the number of women who passedthe fetus within 4 h of receiving the prostaglandin (64 versus74%), the days of bleeding (14.6 ± 1.1 versus 15.3 ±0.9) nor in the onset of the next period (39.7 ± 1.3versus 36.7 ± 1.3) respectively between the groups receiving200 or 600 mg mifepristone. However, the complete abortion ratewas significantly higher in women 49 days compared to women50–63 days amenorrhoea (97.5 versus 89.1% respectively;P < 0.02). There was no difference in any of the other parametersat different weeks of gestation. We conclude: (i) that the recommendeddose of mifepristone could be reduced from 600 to 200 mg withoutloss of clinical efficacy, (ii) that the combination of mifepristoneand 600 µg misoprostol is a highly effective alternativeto vacuum aspiration for inducing abortion in women < 50days amenorrhoea and (iii) at gestation >56 days, this combinationmay result in too many incomplete abortions to be clinicallyacceptable.     

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: 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.     

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).     

Infertility: The desire for multiple births in couples with infertility problems contradicts present practice patterns

Paradoxically, the attitude of infertility patients towardsmultiple births has never been investigated. We therefore generateda survey by questionnaire, which was sent to 3800 consecutiveunselected couples with infertility problems: 582 responseswere received (15% response rate) and analysed. The percentagedistribution of the responses to 21 questions, addressing attitudestowards and knowledge about the risk of multiple gestations,was the main outcome. Worry about multiple births was expressed,independent of the number of multiples, although fear aboutmultiple conceptions was rejected by a large majority (64%).The risk of a twin birth was not strongly perceived, but theperception of risk increased with increasing numbers of multiples:triplets (50–62%), quadruplets or more (71–72%).A desire for the conception of twins was expressed by 67–90%of couples, a desire for the conception of triplets was equallyexpressed and rejected, and for a multiple gestation beyondtriplets was rejected by 73–82% of couples. Patients wereeducated about the risks of selective embryo reduction and respondedin a bimodal fashion to the option of utilizing this procedure,with equal numbers being willing to consider or reject it. Age,parity and length of infertility did not affect the couples‘worry or fear about multiples. The desire for twins and triplets,however, was correlated significantly with age (twins, P = 0.032;triplets, P = 0.03); there was no such correlation for largermultiples. The length of infertility was correlated with a positiveattitude towards multiples beyond triplets (P = 0.029) but wasnot correlated with a desire for twins or triplets. Prior paritydid not affect the attitude towards multiples at all. The lengthof infertility was also correlated significantly to an understandingof risk (twins, P = 0.034; triplets, P = 0.001; quadrupletsand more, P = 0.05), while age and parity was not. The considerationof selective embryo reduction as a treatment option was correlatedsignificantly with age (P = 0.0001), while the understandingof associated risks was evenly distributed, independent of patientcharacteristics. We conclude that infertility patients appearto be educated about the risks of multiple births. A strongdesire for multiple births, as long as this can be limited totriplets or less, increases with advancing female age. Increasinglength of infertility, however, increases the willingness formultiples beyond triplets. Increasing female age increases thereadiness to consider selective embryo reduction as a treatmentoption. Attitudes do not differ between couples with primaryand secondary infertility. Patient attitudes are thus not inagreement with the existing practice patterns in infertilitytherapy. A re-evaluation of some of these practice patternsmay therefore be indicated.     

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.     

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.     

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.     

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 embryology: The protective action of polyvinylpyrrolidone-Percoll during the cryopreservation of mouse 2-cell embryos and its effect on subsequent developmental potential post-thaw in vitro and in vivo

The effects of cryopreservation, in media containing (FS3 +) or omitting (FS3) polyvinylpyrrolidone (PVP) in the form ofPercoll (PVP-Percoll), on the survival of 2-cell mouse embryoswas studied. Survival and zona pellucida disruption post-thaw,growth (assessed by in-vitro culture until the blastocyst stage)and development in vivo (assessed by implantation and livingfetus rates and the birth of live progeny) were all investigated.Initial post-thaw survival showed no statistically significantdifference (P 0.05) between FS3+ (91.1 ± 9.8%) and FS3(84.5 ± 6.6%). However, there was a statistically significant(P 0.05) reduction in the incidence of zona damage when thefreezing solution contained PVP-Percoll compared to the control(3.6 ± 1.0 and 8.7 ± 0.6% respectively) and astatistically significant (P 0.05) greater number of embryosdeveloping in vitro to the blastocyst stage (84.8 ±7.1and 72.3 ± 6.1% respectively). The rates of implantationwere not significantly different: 72.2 ± 7.0% for FS3+and 51.2 ± 30.7% for the non-frozen control group. Thepercentage of live fetuses was also similar between the experimentaland control groups: 27.4 ± 10.6 and 24.3 ± 113%respectively. We conclude that the presence of polymers canprotect embryos against cryoinjury and that PVP in the formof PVP-Percoll provides a non-toxic alternative to PVP in itsnative form, during the cryopreservation of mouse 2-cell embryos.     

Endometriosis: Leuprolide in a 3-monthly versus a monthly depot formulation for the treatment of symptomatic endometriosis: a pilot study

An open-label randomized pilot study was conducted to evaluatethe efficacy and acceptability of 6 months treatment with leuprolidein a 3-monthly versus a monthly i.m. depot injection for therelief of chronic pelvic pain in women with endometriosis. Atotal of 30 women aged 18-38 years were allocated to the 3-monthlydepot arm (n = 15) or to the monthly depot arm (n = 15) afterlaparoscopic diagnosis of pelvic endometriosis. Mean (SD) deepdys-pareunia scores according to a 0–3 point verbal ratingscale decreased from 1.8 (0.9) at baseline to 1.3 (0.7) at theend of treatment in the 3-monthly depot group and from 2.1 (1.2)to 1–3 (0.7) in the monthly depot group. Correspondingvalues in non-menstrual pain scores fell from 2.1 (0.6) to 1.1(03), and from 2.1 (0.8) to 1.2 (0.4) respectively, withoutstatistically significant differences between the groups. Serumluteinizing hormone (LH) and 17-oestradiol concentrations weresignificantly suppressed at 12 and 24 weeks compared with baselinevalues, without differences between the groups. The monthlydepot caused a slightly more marked inhibition of serum folliclestimulating hormone (FSH) levels with respect to the 3-monthlypreparation. Mean (SD) endometriosis scores at baseline andat 6-month follow-up laparoscopy were respectively 32.8 (25.1)and 12.2 (9.3) in the 3-monthly depot group and 29.0 (22.7)and 13.1 (15.3) in the monthly depot group (paired Mest, P 0.05). Mean percentage decrease in lumbar spine bone mineraldensity was 5.2% in the former and 4.9% in the latter subjects.In the 3-monthly depot group, 13 women graded the tolerabilityof their treatment schedule as ‘good’ compared withseven in the monthly depot group (² = 5.40, P = 0.02).     

Immunology: Anti-endometrial lymphocytotoxicity and natural killer cell activity in baboons (Papio anubis and Papio cynocephalus) with endometriosis

This study was performed to test the hypothesis that anti-endometrial,lymphocyte-mediated cytotoxicity and natural killer (NK) activityare reduced in baboons with endometriosis when compared to animalswith a normal pelvis. Lymphocyte-mediated cytotoxicity was determinedin 28 baboons (15 with endometriosis, 13 with normal pelvis)and NK cell activity was evaluated in 42 baboons (31 with endometriosis,11 with normal pelvis). Anti-endometrial lymphocyte-mediatedcytotoxicity was determined by a 20 h assay with effector-targetratios of 50: 1 and 25: 1. The NK activity (K562 cell line astarget) was simultaneously measured in all animals during a4 h assay with effector:target ratios of 200: 1, 100: 1, 50:1, 25: 1, 12: 1, 6: 1 and 3: 1. Statistical analysis was performedusing analysis of variance, paired rank, Mann—Whitney,Kruskal—Wallis and Fisher exact tests where appropriate.Lymphocyte-mediated cytotoxicity was significantly lower (P< 0.025) in baboons with endometriosis (mean 5.9 ±8.7 %, median 0%, range 0–26%) than in animals with anormal pelvis (mean 22.9 ± 23.0 %, median 7%, range 0–78%). This difference could be explained by the absence of cytotoxicityin baboons with moderate to severe endometriosis, probably dueto high spontaneous release of ⁵¹Cr from labelled target cells.When stricter criteria were used and only animals with a labellingindex (maximal/spontaneous release) of 1.7 were analysed (n= 11), the anti-endometrial cytotoxicity was comparable betweenbaboons with and without endometriosis. NK cell activity wasalso comparable in primates with and without endometriosis.In conclusion, no difference in lymphocyte-mediated cytotoxicityand NK cell activity was observed between baboons with and withoutendometriosis.     

Fertilization and early embryology: Embryo quality and pregnancy potential of fresh compared with frozen embryos--is freezing detrimental to high quality embryos?

To determine the effect of cryopreservation on embryo qualityand the pregnancy potential of embryos, donated oocytes fromthe same donor (n = 24) were randomly allocated, with subsequenttransfer to two or more different ovum recipients resultingin at least one fresh and one frozen embryo transfer cycle fromthe same cohort of oocytes. Endometrial receptivity was controlledin all ovum recipients, and male factor patients were excluded.The number of embryos transferred, mean embryo grade transferred,number of high quality embryos (grade 2.5, grade 1 being best)transferred and embryo implantation and live birth rates arereported. Significantly more embryos (4.4 ± 1.2 versus3.3 ± 1.2, P < 0.00003) of higher quality (1.9 ±0.5 versus 2.1 ± 0.5, P < 0.013) and of a more advancedcell stage (3.0 ± 0.6 versus 2.6 ± 0.7, P <0.019) were transferred fresh than after cryopreservation respectively.Implantation rates/embryo [19/151 (12.6%) and 9/111 (8.1%)]and live birth rates/transfer [11/42 (26.2%) and 6/45 (13.3%)],from fresh and frozen transfers respectively, were not significantlydifferent despite the larger number of high quality embryostransferred fresh. Embryo cryopreservation adversely affectsembryo quality, but does not have detrimental effects on theimplantation or pregnancy potential of high quality embryos.Because of the loss of embryos during freeze — thawingduring frozen embryo cycles, every effort should be made toattempt a fresh transfer.     

Endocrinology: Randomized trial of misoprostol and cervagem in combination with a reduced dose of mifepristone for induction of abortion

Mifepristone (600 mg) in combination with a prostaglandin hasbeen demonstrated to be a safe, acceptable alternative to vacuumaspiration for induction of abortion in the first 9 weeks ofpregnancy. However, the efficacy and side-effects of differentprostaglandins used in combination with mifepristone have notbeen assessed in a randomized trial. In this study, 800 womenseeking an abortion at gestational age 63 days amenorrhoea wererandomized to receive either 0.5 mg gemeprost by vaginal pessary(group I) or 600 µg misoprostol (group II) by mouth –48h after taking 200 mg mifepristone by mouth. The side-effectsand number of complete abortions were used as measures of efficacy.There was no significant difference in the rate of completeabortion between group I [96.7%; 95% confidence interval (CI)94.9–98.5%, n = 391] and group II (94.6%; 95% CI 92.3–96.9,n = 386). It was not possible to assess the outcome with certaintyin the remaining 23 women. However, there were significantlymore ongoing pregnancies in the women who received misoprostolthan in those who received gemeprost (nine versus one, P <0.01) and in eight of these 10 women the gestation was >49days. Fewer women in group II required analgesia than in groupI (48 versus 60%, P < 0.001) although the number requestingopiate was similar in each group (6.9 versus 5.2%, P > 0.4).The incidence of nausea and vomiting after misoprostol (47.8and 21.9% respectively) was higher (P < 0.001) than aftergemeprost (33.9 and 12% respectively). The incidence of infectionand heavy bleeding was low in both groups (<2%) and onlyone woman required blood transfusion. We conclude that the recommendeddose of mifepristone and gemeprost can be reduced without impairingclinical efficacy in pregnancies up to 63 days amenorrhoea.Misoprostol is a safe alternative prostaglandin but has a higherincidence of ongoing pregnancies especially at gestation after49 days amenorrhoea.     

Pregnancy: Oocyte donation in menopausal women

Oocyte donation was performed by in-vitro fertilization andembryo transfer in 82 women. The average age of the patientswas 48 years (range 26–60), and 71 were over 40 yearsold, with an average follicle stimulating hormone (FSH) valueof 74±14 IU and oestradiol concentration of 30 ±9 pg/ml. Overall 32 clinical pregnancies were achieved, of which21 reached term, three are ongoing, and eight were lost (sixmiscarriages, one abruptio placentae, and one severe gestationalproteinuria hypertension in the second trimester). The 21 termpregnancies produced 26 newborns; one patient who had had threeembryos replaced delivered triplets and three patients had twins.Before the embryo transfer was performed, the endometrium thicknesswas accurately determined by vaginal ultrasound probe. The highestnumber of implanted embryos was reached when the endometriumthickness was 9–12 mm.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

Andrology: Nuclear decondensation of sperm head and failure at in-vitro fertilization: an ultrastructural study

The problem of unexplained male infertility was investigatedby electron microscopic study of spermatozoa from 51 males.The subjects were subdivided as follows: group A (n = 25) normalfertile males (controls), group B (n = 13) successful in-vitrofertilization (IVF) cases (fertilization rate >50%), groupC (n = 13) failed IVF cases. All subjects included in groupsB and C had a 6–12 year history of childlessness and IVFwas employed when other methods of assisted reproduction failed.The study of spermatozoa in fertile males (controls) was carriedout to establish baseline ultrastructural abnormalities. Inall 51 cases, an average of 330 (280–800) sperm headsand 660 (330–1190) sperm tails were studied. Decondensationof nuclear chromatin was observed in 70 ± 15% (mean ±SD) of spermatozoa in failed IVF cases, 16 ± 5% in successfulIVF cases and 7 ± 3% in controls. These results werefound to be statistically significant (P > 0.001). The meanvalue for motility of spermatozoa in all three groups was withinaccepted limits of normality. It is concluded that decondensationof nuclear chromatin seen by electron microscopy is one of themost important causes of male infertility. It is advocated thatelectron microscopic examination of semen should be carriedout in all cases of longstanding, unexplained male infertilitybefore embarking upon IVF programmes.     

Cytological studies of human zygotes exhibiting developmental arrest

Developmental arrest of 111 ( 5%) fertilized ova which had formedtwo pronuclei was observed during a 5-year period of an in-vitrofertilization programme. At least 30 zygotes demonstrated visiblepronuclei at 44–66 h after insemination, and 42 zygotesfragmented. Of the 107 prepared zygotes, 97 were informativeand revealed that developmental arrest occurred at differentstages of the cell cycle: from interphase (n = 48), to transitioninterphase-prophase (n = 20), to prophase (n = 11), and to metaphase(n = 13). The latter 13 zygotes were characterized by chromosomesets as follows: haploid (n = 2), diploid (n = 6), triploid(n = 1) and tetraploid (n = 4). Another five zygotes demonstrateddifferent numbers of metaphase chromosomes (between 10 and 40),as well as prematurely condensed chromosomes (PCC) as a resultof marked asynchrony in pronuclear morphogenesis. A total of18 zygotes exhibited asynchrony in the morphology of the twopronuclei. It is concluded that abnormal chromosome sets andpronuclear asynchrony might be causes for early developmentalarrest.