Concentration of interleukin-1{beta} correlates with prostaglandin E2 and F2{alpha} in human pre-ovulatory follicular fluid

To investigate the role(s) of interleukin-1 (IL-1) in humanovarian function, we measured the concentrations of IL–1,prostaglandins (PGs) and steroids in follicular fluid of 90stimulated ovaries, with reference to oocyte maturation. Concentrationsof IL-1 were significantly higher in the follicles from whichmature oocytes were recovered than in follicles from which oocytescould not be recovered (P < 0.05). IL-1 concentrations alsoincreased in association with oocyte maturation. Positive significantcorrelations were seen between IL-1 and prostaglandin E2 (PGE2)(r = 0.47, P < 0.001), and between IL-1 and prostaglandinF₂ (PGF₂) (r = 0.22, P < 0.05) in pre-ovulatory follicularfluid, but not between IL–1 and oestradiol, or betweenIL-1 and oestradiol, or between IL-1 and progesterone. 0Follicularfluid IL-1 might contribute to prostaglandin-induced oocytematuration and ovulation.     

Endometriosis and human infertility: a new investigation into the role of eutopic endometrium

BACKGROUND: Endometriosis is related to infertility even in the absenceof mechanical alterations of the reproductive tract. Even thoughthe pathogenesis of this phenomenon is still unclear, an impairedendometrial receptivity has been recently suggested. The aimof the present study was to investigate if endometriotic peritonealfluids (EPF) could interfere with endometrial stromal cell (ESC)decidualization and if tumor necrosis factor (TNF)- could beinvolved in the EPF effect. METHODS: Eutopic ESC were isolated from patients with or without endometriosis.ESC were treated with 17β-estradiol 10^–8 M and 6-methyl-17-hydroxyprogesteroneacetate2x10^–7 M for 16 days. In vitro decidualization was morphologicallyand biochemically assessed. We analysed whether ESC decidualizationcould be affected by EPF or peritoneal fluids from control patients(CPF), with or without soluble TNF- receptor 1 (sTNFR-1). RESULTS: Compared with ESC from control patients, eutopic ESC from patientswith endometriosis showed an impaired decidualization. Decidualizationof normal ESC was morphologically normal but biochemically abnormalin the presence of EPF, which was able to decrease the secretionof decidualization markers. sTNFR-1 was able to partially counteractthis effect. CONCLUSIONS: In endometriosis, the milieu surrounding the uterine cavitymay be involved in impaired eutopic ESC decidualization, partiallydue to increased peritoneal levels of TNF-.     

Embryonic resistance to tumour necrosis factor-{alpha} mediated cytotoxicity: novel mechanism underlying maternal immunological tolerance to the fetal allograft

The cytokine tumour necrosis factor- (TNF) has been postulatedto play an essential role in the cytotoxic activity of cell-mediatedimmunity against allogenic or tumour cells invading the host.Several tumour cell lines, however, are resistant to TNF mediatedcytotoxicity and respond paradoxically by cellular proliferationand by autocrine secretion of TNF. In view of the metastaticcharacter of the mammalian embryo, the aim of this study wasto assess the potential of murine embryos to secrete TNF invitro, to express TNF receptors and to resist TNF mediated cytotoxicityduring their in-vitro development to the blastocyst stage. Thepotential of human embryos to secrete TNF in vitro until theblastocyst stage was also investigated. From a total of 11 humanembryos, which were allowed to proceed to blastocyst formation,seven secreted TNF in the range of 2–117 pg/ml/24 h. Atotal of 123 C57BL/6J mouse embryos were studied of which 55%secreted TNF in the range of 1.25–3.95 mg/ml/24 h. Thepresence of high levels of exogenous TNF (10–300 IU) wasnot detrimental to the in-vitro development of murine embryos.Using immunohistochemical techniques, we were not able to detectthe presence of type I or II TNF receptors on the surface ofmurine embryos. Our findings suggest that human and C57BL/6Jmurine embryos have the potential to secrete TNF in vitro duringthe developmental stages leading to blastocyst formation. Inboth species, the presence of TNF in the culture medium didnot cause subsequent necrosis of the conceptus, suggesting thatmammalian embryos may be TNF resistant cell lines. The observedembryonic resistance to TNF may be explained by the absenceof TNF receptors by which the cytotoxic effect is usually mediated.It is suggested that embryonic resistance to physiological concentrationsof TNF released by effectors of the host's immune system, couldbe via a mechanism underlying the maternal immunological toleranceto the fetal allograft.     

Implantation: Effect of platelet activating factor on embryonic development and implantation in the mouse

Platelet activating factor (PAF) was administered to femalemice in order to investigate its effect on ovulation rate andon oocyte quality including their in-vitro embryonic development,implantation and uterine receptivity. In experiment 1, 4-week-oldfemale mice were assigned to receive PAF or phosphate bufferedsaline for 4 consecutive days. On the second day of this treatment,pregnant mares' serum gonadotrophin was administered and humanchorionic gonadotrophin (HCG) 48 h later, after which copulationoccurred. Oocytes were collected on the following day and evaluated.The mean number of oocytes and zygotes (two pronuclear stageembryos) recovered from the PAF-treated group was not differentfrom the control group (31 versus 27), but the proportion ofzygotes was higher in PAF-treated group than in controls (83versus 68%, P 0.05, PAF versus controls). Although the rateof in-vitro first cleavage was not different in the two groups(82 versus 69% respectively), hatching was higher in the PAF-treatedgroup than control mice (99 versus 83%, P 0.01). In experiment2, the in-vitro developed blastocysts from experiment 1 weretransferred into the uterus of day 3 pseudopregnant PAF-treatedor control recipients. Three different combinations of intrauterinetransfer were performed; PAF embryo to control recipient (PAFC:n = 19), control embryo to PAF recipient (CPAF: n = 19), andcontrol embryo to control recipient (CC: n = 22). Implantationand abortion were assessed on day 19 post-transfer. The implantationrate of CPAF (23.7%) was lower than CC (31.1%, P 0.05), butwas not different from PAFC (31.2%). Further, CPAF showed ahigher abortion rate per embryo (29.6%) than PAFC (12.7%, P 0.05), but was not different from CC (24.4%). In the presentstudy, PAF administration enables females to produce oocyteswith a higher potential for fertilization, in-vitro developmentand implantation, but has a detrimental effect on uterine receptivityto embryos.     

Reactive oxygen species stimulate prostaglandin F2 alpha production in human endometrial stromal cells in vitro

BACKGROUND: The present study was undertaken to investigatethe effect of reactive oxygen species on prostaglandin F2 (PGF2)production by human endometrial stromal cells (ESC). METHODSAND RESULTS: Isolated ESC were incubated with hydrogen peroxide,which induces lipid peroxidation. Hydrogen peroxide increasedboth intracellular and medium concentrations of PGF2 (P <0.01). A time course study showed that hydrogen peroxide significantlyincreased PGF2 concentrations in the medium after 6 h incubation(P < 0.01), after which no further increase was observed.To study whether the increase in PGF2 production caused by hydrogenperoxide was mediated by cyclooxygenase, ESC were incubatedwith indomethacin (0.5 µg/ml), an inhibitor of cyclooxygenase,in the presence of hydrogen peroxide. Indomethacin significantlyblocked the increases in PGF2 production caused by hydrogenperoxide (P < 0.01). Hydrogen peroxide also increased PGF2production by decidualized ESC (P < 0.01), induced by theincubation with medroxyprogesterone acetate (10^–6 mol/l)and oestradiol (10^–8 mol/l). CONCLUSIONS: Reactive oxygenspecies stimulate PGF2 production in ESC, suggesting that theymight influence endometrial function by regulating PGF2 production.     

Immunology: Autoimmunity to spermatozoa, asymptomatic Chlamydia trachomatis genital tract infection and {gamma}{delta} T lymphocytes in seminal fluid from the male partners of couples with unexplained infertility

The relationship between an undetected, asymptomatic Chlamydiatrachomatis genital tract infection, the concentration of andb T cells in semen and sperm autoimmunity was examined in 48male partners of couples with unexplained infertility. ImmunoglobulinA (IgA) antibodies to C.trachomatis were detected in seminalfluids from 14 (29.2%) of the men. Only four of these were positivefor circulating anti-chlamydial IgA, suggesting that the stimulusfor antibody production was within the genital tract. In contrast,four men were positive for anti-chlamydial IgG in their semen;all were also seropositive for anti-chlamydial IgG. T lymphocytesbearing the and antigen receptors were present in every semensample. Men with seminal anti-chlamydial IgA, however, had significantly(P = 0.035) elevated semen T cell concentrations (median 3100cells/ml) than did men lacking this antibody (median 1400 cells/ml);concentrations of T cells were comparable in both groups. Genitaltract sperm autoimmunity, as shown by antibodies bound to motileejaculated spermatozoa, was detected in 13 (27.1%) men. Thepresence of these antibodies was associated with elevated concentrationsof both (median 4200 versus 700 cells/ml) and (median 5000versus 850 cells/ml) T cells (P = 0.0002 and 0.0001 respectively).Men with antisperm antibodies only in their serum had seminalT cell concentrations comparable with men testing negative forantisperm antibodies. Anti-chlamydial IgA was identified insemen from four of 10 men with IgA bound to their spermatozoaand in none of the men with only spermatozoabound IgG. Therewas no relationship between sperm quality and the occurrenceof seminal IgA antibodies to either C.trachomatis or spermatozoa.An asymptomatic C.trachomatis infection activates T cells withinthe male genital tract, which may lead to antisperm antibodyformation and immune-mediated infertility.     

Pregnancy: Cervical ripening with the cytokines interleukin 8, interleukin 1{beta} and tumour necrosis factor {alpha} in guinea-pigs

It has been suggested that the collagenolytic enzymes releasedfrom white blood cells which infiltrate the pregnant human uterinecervix at term are responsible for connective tissue changeswhich take place during the ripening process. Similarly, aninfiltration of inflammatory cells occurs in pregnant guinea-pigseither spontaneously at term or at preterm after treatment withthe antiprogestin onapristone. The objective of this study wasto evaluate the effects of the inflammatory cytokines interleukin8 (IL-8), interleukin 1 (IL-1), tumour necrosis factor (TNF-)and a combination of IL-1 and TNF- on cervical ripening in guinea-pigsduring advanced pregnancy. The cytokines were applied locally(intracervically) in a gel for 2 days and the effects were assessedon the third day by both extensibility measurements and morphologicalevaluation. IL-8 treatment on days 42 and 43 post coitum (p.c)and on days 48 and 49 p.c. (term: day 67± 3 p.c.) significantly(P < 0.05) increased cervical extensibility at both stagesof pregnancy. Although IL-1 treatment (days 42 and 43 p.c.)led to a slight increase in cervical extensibility, this effectwas not statistically significant. An electron microscope studyperformed on days 48 and 49 p.c. revealed a pronounced cervicalripening accompanied by the dissolution of collagen fibres,stromal oedema and the infiltration of polymorphonuclear leukocytesin all cytokine-treated groups. The morphological effects ofIL-8 and IL-1 were indistinguishable from those observed duringnormal cervical ripening at term. In contrast, TNF-, both aloneand in combination with IL-1, brought about a severe inflammatoryreaction with a massive infiltration of lymphocytes, marcophagesand polymorphonuclear leukocytes at the investigated dose. Weconclude that the local application of the inflammatory cytokinesIL-8, IL-1 and TNF- produces cervical ripening without inducinglabour in pregnant guinea-pigs; the morphological effects ofIL-8 and IL-1 being similar to the physiological cervical ripening.Our data support the view that cytokines, particularly IL-8,may play an important role during physiological, pathologicaland induced cervical ripening and could be clinically usefulas an adjunct to labour and delivery.     

Implantation: Tumour necrosis factor {alpha} inhibits in-vitro decidualization of human endometrial stromal cells

Interleukin-1 (IL-1) has been reported previously to inhibitthe in-vitro decidualization of human endometrial stromal cellsas assessed by progesterone-induced prolactin production andmorphological transformation. In this study we examined whetherother cytokines, such as tumour necrosis factor-(TNF), interferon-(IFN), IFN or granulocyte-macrophage colony-stimulating factor(GM-CSF), could affect the decidualization of human endometrialstromal cells in vitro. Of these cytokines, TNF significantlysuppressed prolactin production in a dose-dependent manner,with no apparent effect on cell number. The morphological transformationof endometrial stromal cells was also inhibited by TNF. TNFand IL-1 significantly suppressed cAMP-stimulated prolactinproduction by endometrial stromal cells. Neither the progesteroneconcentration in the supernatant of the endometrial stromalcell culture system nor intracellular calcium concentrationof the endometrial stromal cells were affected by the additionof TNF or IL-1. These results indicated that TNF and IL-1 suppressboth progesterone-induced and cAMP-mediated prolactin productionin endometrial stromal cells, and that this inhibition was notattributable to direct effects on progesterone metabolism orrelated to Ca2+-mediated signal transduction. These experimentssuggested that a local increase of TNF and IL-1 under certainpathological conditions in vivo may disturb blastocyst implantationand/or the maintenance of pregnancy by inhibiting the decidualizationof endometrial stromal cells.     

Integrins are not involved in the process of human sperm-oolemmal fusion

BACKGROUND: We investigated whether integrins are required forthe human sperm–oocyte binding and fusion processes. METHODS:The expression of several integrin subunits at the human oocyteplasma membrane was investigated using immunofluorescence microscopy,and the functional role of integrins expressed at the humanoocyte surface in sperm–oocyte interaction was studiedusing a zona-free human oocyte binding and fusion assay. A totalof 144 unfertilized oocytes were stained with anti-integrinantibodies and 147 zona-free unfertilized oocytes were inseminatedin the presence of various anti-integrin antibodies that wereexpressed in oocyte plasma membrane. RESULTS: The antibodiesof six integrin subunits (₂, ₃, ₅, ₆, _V, _M) and six integrinsubunits (₁, ₂, ₃, ₄, ₅, ₆) were bound to the surface of fixedunfertilized oocytes. In contrast, the presence of ₁ and ₄ subunitscould not be verified. The human sperm–oocyte bindingwas only partially inhibited by blocking antibodies of ₂, ₃,₅, ₆, _V, _M, ₁, ₂ and ₃ with a maximum of 55% inhibition, butantibodies of ₄, ₅ and ₆ showed no effect on sperm–oolemmalbinding. A similar reduction of the number of fused sperm wasobserved. However, the ratio of fused sperm to total sperm (boundand fused) was not impaired by all integrin antibodies, suggestingthat integrins had no role in the sperm–oolemmal fusionprocess. CONCLUSIONS: These results suggest that one of thebinding mechanisms can be inhibited by integrin antibodies butthat this mechanism does not play an essential role in the humansperm–oolemmal binding and fusion processes. The othermechanisms, insensitive to integrins, may involve both bindingand fusion processes in human oocytes.     

Integrins and adhesion molecules: A positive correlation between expression of {beta}1-integrin cell adhesion molecules and fertilizing ability of human spermatozoa in vitro

The purpose of this study was to investigate firstly whether(1-integrin cell adhesion molecules are expressed by human spermatozoa,and secondly whether there is any relationship between the expressionof 1-integrin cell adhesion molecules and the fertilizing abilityof human spermatozoa in vitro. A total of 50 semen samples wereexamined. The samples were obtained from the male partners ofcouples undergoing in-vitro fertilization (IVF) for either unexplained,tubal or male factor infertility. A panel of six monoclonalantibodies against 1-integrin cell adhesion molecules and immunohistochemicaltechniques were used to identify the presence of these moleculeson the spermatozoa. The percentage of spermatozoa showing strongimmunolabelling with each monoclonal antibody was assessed ineach sample. The relationship between these results and theaetiology of infertility and incidence of fertilization wasexamined. 1-Integrins, and primarily the ones with 4-, 5- and6-chains, were expressed by human spermatozoa. Compared withsemen samples from unexplained or male factor infertility patients,samples from tubal infertility patients had a significantlyhigher (P < 0.05) percentage of spermatozoa expressing adhesionmolecules. There was a positive correlation between the expressionof 4, 5 and a6 adhesion molecules and the fertilizing abilityof spermatozoa. The positive correlation between the presenceof certain (1-integrin cell adhesion molecules and the fertilizingability of human spermatozoa suggests that integrins may beputative determinants in egg-sperm recognition and interaction.     

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.     

Expression of melatoninergic receptors in human placental choriocarcinoma cell lines

BACKGROUND: Melatonin crosses the placenta and enters the fetalcirculation. Moreover, experimental data suggest a possibleinfluence of melatonin on placental function and fetal developmentin humans. To date, the expression and role of melatonin receptorsin human placenta choriocarcinoma cell lines and in human termplacental tissues remain to be elucidated. METHODS AND RESULTS:Results from RT–PCR, western blotting and confocal microscopydemonstrated that the MT1, MT2 and ROR1 melatonin receptorsare expressed in the human term placental tissues and in choriocarcinomacell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbentassay showed that 6-chloromelatonin (a melatonin agonist) inhibits,in a dose-dependent manner, forskolin-stimulated hCG- secretionin JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but hadno effect on basal human chorionic gonadotrophin (hCG-) levels.This effect of 6-chloromelatonin on forskolin-stimulated HCG-secretion was abolished by pertussis toxin (PTX), suggestingthat melatonin regulates hCG- production by an action involvingan inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatoninstimulated basal hCG- secretion (P < 0.001). CONCLUSION:These results demonstrate, for the first time, the expressionof melatonin receptors in human term placental tissues and inchoriocarcinoma cells and suggest a possible paracrine/autocrinefunction for melatonin in human placenta.     

Integrins and adhesion mlecules: Cell adhesion molecules on the oocyte and preimplantation human embryo

The presence of cell adhesion molecules on human oocytes, earlyembryos, and pre-hatched blastocysts was examined by indirectimmunofluorescence and compared to the distribution found onfirst trimester villous placenta with the same antibodies. Sixintegrin subunits (3, V, 1, 3, 4, 5) were observed consistentlythroughout preimplantation development. Evidence was also obtainedfor the presence of integrin subunits 2, 4, L, 2, and 7 on asmall number of oocytes. A more restricted developmental analysisof E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that thesecell adhesion molecules are also present on oocytes and earlyembryos. L-selectin was detected on oocytes but was not foundon 8-cell embryos. The oocyte and early blastomeres have complexsurfaces in which the integrin and CAM families are represented.     

Female age predicts embryonic implantation after ICSI: a case-controlled study

From 1 October 1991 until 31 December 1993, 1270 cycles forintracytoplasmic sperm injection were performed. Of these, 71(5.6%) were carried out in women 40 years of age. The semencharacteristics in couples40 years of age or <40 years weresimilar. The mean male age for the older group of women was47.1 years (range 34–67) versus 35 years (range 25–71)for the younger group of women (P <0.001). The mean femaleage was 41.9 years (range 40–-47) and 31.8 years (range23–39). The numbers of cumulus—oocyte complexesand metaphase-II oocytes were significantly lower in women 40years of age (P < 0.001). The mean numbers of replaced embryoswere respectively 2.3 (133/59) in women 40 years of age and2.5 (160/63) in women <40 years of age. The delivery rateper retrieval and per transfer was significantly lower in women40 years of age (P < 0.05). The delivery rates per retrievaland per transfer were respectively 7% (5/71) and 8.5% (5/59)in the older group of women versus22.5% (16/71) and 25.4% (16/63)in the younger group. Female age is the predictive factor forembryonic implanatation.     

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).     

Endocrinology: Prostaglandin F2{alpha} stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells

Human ovarian follicular fluid contains a number of insulin-likegrowth factor binding proteins (IGFBP) of which IGFBP-3 is themost abundant. IGFBP-3 synthesis is growth hormone-regulated.We studied the effect of prostaglandin F₂ (PGF₂) on IGFBP-3secretion by cultured human granulosa-luteal cells from follicularaspirates of women participating in an in-vitro fertilizationprogramme. The IGFBP-3 concentration was measured using a specificmonoclonal immunofluorimetric assay. Contrary to a previousreport on unstimulated follicles, this study demonstrated apositive correlation between follicular fluid IGFBP-3 concentrationand follicular size. PGF2a was found to stimulate in a dose-dependentfashion the secretion of IGFBP-3. Significant (p < 0.05)effects were found at PGF₂ concentrations of 10^–8, 10^–7and 10^–6 M. Because IGFBP-3 inhibits progesterone productionstimulated by insulin-like growth factor (IGF)-I, the PGF₂-inducedstimulation of IGFBP-3 production may be one of the mechanismswhereby PGF₂ exerts its luteolytic effect via the IGF system     

Endocrinology: Substrate dependency of C19 conjugates in hirsute hyperandrogenic women and the influence of adrenal androgen

Serum C₁₉ conjugates, specifically 3-androstanediol glucuronide(3G), reflect peripheral androgen action through the actionof 5-reductase activity. The origin of 5-reduced C₁₉ conjugateshas been controversial and it has been suggested that they arederived primarily from adrenal androgens. We examined concentrationsof 3G, 3-androstanediol sulphate (3S), androsterone glucuronide(AoG) and androsterone sulphate (AoS) in 40 hirsute hyperandrogenicwomen. These patients were divided into four groups based uponindividual, combined or normal concentrations of the adrenalandrogens dehydroepiandrosterone (DHEAS) and 11-hydroxy-androstenedione.Testosterone, unbound testosterone and androstenedione weresimilar in these groups. Serum 3G was equally high in all groupsand was correlated significantly with hirsutism, while the otherconjugates were not. Androsterone glucuronide was raised inall groups but was higher in patients with raised DHEAS. Serum3S was raised in all groups and was higher where both adrenalandrogens were raised. Serum AoS was highly correlated withDHEAS. Serum 3G was correlated with unbound testosterone andandrostenedione but not with the adrenal androgens. The glucuronideconjugates were correlated with one another as were the sulphateconjugates but glucuronides and sulphates were not correlated.These data confirm ovarian and adrenal dependency of C₁₉ conjugates.Serum 3G appears to reflect hirsutism most accurately and isleast dependent on adrenal androgens in patients with mixedhyperandrogenism.     

Diagnosing and preventing inherited disease: Detection of sickle gene by coelocentesis in early pregnancy: a new approach to prenatal diagnosis of single gene disorders

Coelomic fluid, placental tissue and maternal blood were collectedat 7–10 weeks gestation from each of 58 women undergoingelective termination of pregnancy for psychological indications.In all samples, a 364 bp fragment of the human -globin genespanning positions –23 to 341 was amplified. The restrictionendonuclease Ddel was used to detect the sickle mutation whichabolishes its restriction site. -Globin DNA was successfullyamplified from all samples. In 53 cases a normal maternal -globingenotype was detected. In three out of five cases, where thematernal haemoglobin phenotype was HbAS, heterozygosity forthe sickle mutation was demonstrated on analysis of coelomicfluid. In the remaining two cases a normal -globin genotypewas observed. Three further coelomic fluid samples were foundto be heterozygous for the sickle mutation. In these instancesthe maternal haemoglobin phenotype was normal, indicating paternaltransmission of the sickle gene. The results of the presentstudy have established that the diagnosis of sickle cell anaemia,and potentially other human single gene disorders, is feasibleby coelocentesis.     

Interleukin-1{alpha} and -{beta} modulation of luteinized human granulosa cell oestrogen and progesterone biosynthesis

This study was designed to test the hypothesis that inter-leukin-1(IL-1) and directly affect progesterone, and oestradiol productionin cultures of purified human granulosa cells. Luteinized granulosacells were obtained from women during in-vitro fertilizationcycles. Granulosa cells with and without associated white bloodcells were cultured in the presence of IL-1 and IL-1 (0.5–50ng/ml) for 48 h. Media were changed at 24 h intervals and assayedfor progesterone and oestradiol. In separate experiments, granulosacell viability was assessed with the tetrazolium salt reductionassay, haemocytometer cell counts, and Trypan blue dye exclusion.Our results indicate that progesterone synthesis by basal andhuman chorionic gonadotrophin (HCG)-stimulated granulosa cellsco-cultured with white blood cells was inhibited by 5.0 ng/mlof IL-1 and IL-1 at 48 h of culture. In the presence of whiteblood cells, granulosa cell oestradiol synthesis was inhibitedby IL-1 but not IL-1. Oestradiol was inhibited after both 24and 48 h of culture and was maximally affected by 5.0 ng/mlof IL-1. In contrast, basal and HCG-stimulated oestradiol productionby granulosa cells cultured free of white blood cells was inhibitedonly by IL-1. IL-1 at 5.0 ng/ml produced maximal inhibitionof basal oestradiol (57%) and HCG-stimulated oestradiol (41%)production at 48 h of culture. Gonadal steroid inhibition byIL-1 and IL-1 was not mediated through cytotoxic or antiproliferativeeffects on granulosa cells. Specificity of the granulosa cellresponse to IL-1 and IL-1 was demonstrated by abrogation ofsteroid inhibition with anti-IL-1 and IL-1 neutralizing antibodies.In conclusion, IL-1 directly inhibited the production of oestradiolby human ovarian granulosa cells. IL-1 and IL-1 also exertedindirect effects on steroid production via white blood cellsthat are usually present in granulosa cell cultures if stepsare not taken to remove them. These data support the hypothesisthat cytokines play an important role in intra-ovarian regulationof steroid biosynthesis.