Discrimination between chronological and ovarian age in infertile women aged 35 years and older: predicting pregnancy using basal follicle stimulating hormone, age and number of ovulation induction/intra-uterine insemination cycles

A marked decline in fertility rates has been demonstrated inwomen >35 years of age. We have previously demonstrated theimportance of basal follicle stimulating hormone (FSH) concentrationsplus chronological age to predict pregnancies in women aged40 years undergoing ovula-tion induction therapy. The purposeof the current study was to extend our previous study and determinethe impact of age, basal FSH concentrations and ovulation induction/intra-uterine insemination (IUI) treatment cycles on pregnancyrates in infertile women aged 35 years. This prospective observationalstudy was performed at a tertiary university fertility centre.Assessments of basal hormonal status and ovulation inductionprotocols were performed. The main outcome measured was clinicalpregnancies. A total of 770 treatment cycles in 179 women aged35 years were analysed. The impact of basal FSH concentrationson treatment outcomes could be bifurcated into a favourablegroup (FSH 23 mlU/ml) and a poor prognosis group (FSH 24 mlU/ml).A multivariate logistic regression model was generated whichaccurately predicted pregnancies. There was a high degree ofcorrelation between predicted pregnancies and observed pregnancies(r = 0.86). We conclude that age, number of treatment cyclesand the interaction term basal FSH x age are useful and significantpredictors of pregnancies in patients aged 35 years undergoingovulation induction/IUl therapy.     

Does free androgen index predict subsequent pregnancy outcome in women with recurrent miscarriage?

BACKGROUND: Several studies have investigated plasma androgen levels inwomen with recurrent miscarriage (RM) with conflicting resultson whether an association between hyperandrogenaemia and RMexists. However, none of these studies included sensitive androgenmeasurements using a large data set. We therefore investigatedthe free androgen index (FAI) in a large number of women withRM in order to ascertain whether hyperandrogenaemia is a predictorof subsequent pregnancy outcome. METHODS: We studied 571 women who attended the Recurrent MiscarriageClinic in Sheffield and presented with 3 consecutive miscarriages.Serum levels of total testosterone and sex hormone-binding globulinwere measured in the early follicular phase and FAI was thendeduced. RESULTS: The prevalence of hyperandrogenaemia in RM was 11% and in asubsequent pregnancy, the miscarriage rate was significantlyhigher in the raised FAI group (miscarriage rates of 68% and40% for FAI > 5 and FAI 5 respectively, P = 0.002). CONCLUSIONS: An elevated FAI appears to be a prognostic factor for a subsequentmiscarriage in women with RM and is a more significant predictorof subsequent miscarriage than an advanced maternal age (40years) or a high number (6) of previous miscarriages in thisstudy.     

Ovulation induction combined with intrauterine insemination in women 40 years of age and older: is it worthwhile?

The use of ovulation induction combined with intrauterine insemination(IUI) as a treatment for subfertility in women with patent Fallopiantubes has increased in recent years. Little is known regardingthe efficacy of this treatment in women aged40 years. We reviewedour data in our ovulation induction with IUI programme for 168consecutive patients aged 40 years undergoing a total of 469cycles of treatment. Either sequential clomiphene citrate andhuman menopausal gonadotrophins or daily gonadotrophins wereutilized along with timed IUI insemination. In 402 completedcycles, 28 clinical pregnancies occurred. The pregnancy lossrate was 34.4%. The overall ongoing/viable pregnancy rates perinitiated and completed cycles were 4.47 and 5.22% respectively.No viable pregnancies occurred in 136 cycles in women aged 43years. The ongoing/viable cycle fecundity rates for women aged40, 41, and 42 years were 9.6, 5.2, and 2.4% percycle respectively.When utilized in women aged40 years, ovulation induction withIUI is most likely to result in successful pregnancy in women40–42 years of age. Women43 years should consider otheralternatives such as adoption or egg donation.     

Doubling the human menopausal gonadotrophin dose in the course of an in-vitro fertilization treatment cycle in low responders: a randomized study

The effect of doubling the human menopausal gonadotrophin (HMG)dose in the same treatment cycle in which the ovarian responseafter 5 days of ovarian stimulation with 225 IU/day is ‘low’,has been evaluated in a prospective randomized study. Forty-sixpatients met the ultrasound and oestradiol criteria for enrolmentin the study, one patient participated twice. In 22 patientstreatment was continued with 225 IU HMG/day and in 25 patientsthe HMG dose was increased to 450 IU/day. No effect of doublingthe HMG dose was found on the length of the ovarian stimulation,peak oestradiol values, number of follicles 11 and 14 mm indiameter respectively on ultrasound on the day of HCG administration,number of cancelled cycles, number of oocytes at follicularpuncture and the number of patients with 3 oocytes at retrieval.It is concluded that doubling the HMG dose in the course ofan IVF treatment cycle is not effective in enhancing ovarianresponse in low responders. This is in accordance with currenttheories on follicular growth, which state that follicular recruitmentoccurs only in the late luteal and early follicular phase ofthe menstrual cycle.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Ovary: Immunolocalization of transforming growth factor-{beta}1 and transforming growth factor-{beta}2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate thecellular distribution of transforming growth factor-₁ (TGF₁)and transforming growth factor ₂ (TGF₂) at different stagesof follicle development in the prepubertal mouse ovary underthe following conditions: (i) after pregnant mare's serum gonadotrophin(PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin(HCG) treatment; (iii) after PMSG and HCG treatment plus mating.In the immature ovary, TGFF₁ and TGF₂ immunoreactivities arelocalized in theca and granulosa cells and in oocytes. AfterPMSG treatment, TGF₁ and TGF₂ immunoreactivities are localizedin granulosa cells; in addition, TGF₂ staining is noted in thematrix surrounding antral cells. Staining for both TGF₁ andTGF₂ drops in the theca but persists in the oocyte. PMSG plusHCG treatment results in a significant increase in TGF₁ andTGF₂ immunoreactivity in the theca and in the maintenance ofTGF₁ staining in both basal granulosa cells and cumulus cellswhereas TGF₂ immunoreactivity is essentially localized in thematrix surrounding cumulus cells. Staining for TGF₁ and TGF₂persists in the oocyte. Following PMSG plus HCG treatment andmating, TGF₁ immunoreactivity is localized in the luteal cellsof corpora lutea and TGF₂ shows a similar localization pattern.This study provides evidence that TGF₁ and TGF₂ peptides areexpressed in specific cell types during induced follicular maturationin the mouse ovary.     

Molecular aspects of implantation: Human trophoblast adhesion to matrix proteins: inhibition and signal transduction

At the time of implantation, the extracellular matrix proteinslaminin and fibronectin are abundant in the decidua and aredistributed pericellularly around each individual stromal cell.First trimester human trophoblast expresses both laminin andfibronectin receptors, specifically the ₁₁, ₅₁, ₆₁ and ₆₄ integrinheterodimers. In this study we have demonstrated that in-vitroadhesion of first trimester human trophoblast to purified extracellularmatrix proteins and to purified decidual stromal cell monolayerscan be inhibited by monoclonal antibodies directed against appropriateintegrin subunits and by synthetic peptides containing an arginine-glycine-asparticacid sequence. Monoclonal antibodies (mAbs) to the ₅ and ₁ integrinsubunits and a synthetic peptide significantly inhibited adhesionto fibronectin. Binding of trophoblast to laminin was blockedwith mAbs to the ₆ and ₁ but not ₁ and ₄ integrin subunits.Similarly, integrin-mediated adhesion to monolayers of decidualstromal cells could be blocked with mAbs to the ₅, ₆, ₁ and₄ integrin subunits. Integrin-mediated signal transduction innormal and malignant trophoblast was investigated by Westernblotting. A 115 kDa protein was the major tyrosine phosphorylatedprotein detected in trophoblast after binding to laminin orfibronectin. The profile of tyrosine phosphorylated proteinsdiffered for malignant trophoblast.     

Genetics: Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and {Delta}F508 mutations

W1282X (W) and F508 () are the two most common mutations ofthe cystic fibrosis Israeli population. Patients who are homozygotes(WW and ) as well as compound heterozygotes (W) present a severephenotype of the disease. In the present study, we have developeda polymerase chain reaction (PCR)-based method for the detectionof both mutations simultaneously in a single blastomere. Unfertilizedhuman oocytes and single polyspermic blastomeres were subjectedto a two-round PCR amplification: a first round of multiplexPCR followed by a second round of nested PCR, done seperatelyat each locus. Clear signals at both loci were obtained in 51%(47/65) of oocytes and 69% (24/35) of blastomeres. The genotypeof the single cell analysed was determined by endonuclease digestionof the W products and by heteroduplex formation of the F products.This diagnostic system will allow the identification of affectedembryos (WW, , W) as well as phenotypically normal carriers(W++), and therefore may be used for cystic fibrosis preimplantationdiagnosis in families who carry either or both mutations     

Relationship between tumour necrosis factor {alpha} and sex steroid concentrations in the follicular fluid of women with immunological infertility

Experimental evidence suggests a tight relationship betweencytokines and the reproductive system. Tumour necrosis factor (TNF), a cytokine produced by activated macrophages and mesenchymalcells, seems to participate in the control of fertility. Therefore,the present study was undertaken to evaluate the concentrationsof TNF in the follicular fluid of female patients with immunologicalinfertility, as well as the possible role of this cytokine infollicular development. Concentrations of TNF, 17β-oestradiol,progesterone, androstenedione and testosterone were measuredin the follicular fluid of patients with immunological infertilityand patients with a tubal factor of infertility, who servedas a control group. Patients with immunological infertilityhad significantly higher concentrations of TNF in their follicularfluid compared to the control group. In contrast, oestradiolconcentrations were significantly lower in the former group.The intrafollicular concentrations of the other steroids measureddid not differ significantly between the two groups. The fertilizationrate of ova from follicles included in the study was significantlylower in patients with immunological infertility compared tocontrol subjects (19.1 and 57.1% respectively). In conclusion,this study shows that patients with infertility of immunologicalorigin have increased follicular fluid concentrations of TNFand lower oestradiol concentrations. We speculate that elevatedTNF concentrations in the human follicle may negatively influenceboth ovulation- and fertilization-related events.     

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.     

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.     

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.