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.     

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.     

Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome

BACKGROUND: The sperm chromatin structure assay (SCSA) has beensuggested as a predictor of fertility in vivo as well as invitro. The available data however, have been based on limitednumbers of treatments. We aimed to define the clinical roleof SCSA in assisted reproduction. METHODS: A total of 998 cycles[387 intrauterine insemination (IUI), 388 IVF and 223 ICSI]from 637 couples were included. SCSA results were expressedas DNA fragmentation index (DFI) and high DNA stainable (HDS)cell fractions. Outcome parameters were biochemical pregnancy(BP), clinical pregnancy (CP) and delivery (D). RESULTS: ForIUI, the odds ratios (ORs) for BP, CP and D were significantlylower for couples with DFI >30% as compared with those withDFI 30%. No statistical difference between the outcomes of ICSIversus IVF in the group with DFI 30% was seen. In the DFI >30%group, the results of ICSI were significantly better than thoseof IVF. CONCLUSIONS: DFI can be used as an independent predictorof fertility in couples undergoing IUI. As a result, we proposethat all infertile men should be tested with SCSA as a supplementto the standard semen analysis. When DFI exceeds 30%, ICSI shouldbe the method of choice.     

Acrosin activity in human spermatozoa in relation to semen quality and in-vitro fertilization

Acrosin, a sperm proteinase released during acrosomal exocytosis,facilitates penetration through the oocyte vestments. The purposeof this investigation was to determine if a correlation existsbetween the acrosin activity of ejaculated human spermatozoa,before motility enrichment techniques, and in-vitro fertilization(IVF) success using selected (glass wool or swim-up) spermatozoa.Since all the oocytes were retrieved from women receiving exogenoushormonal stimulation and a mixed population of mature and immatureoocytes were encountered, only cases with 50% mature oocyteswere analysed. Under these conditions, the acrosin activitywas significantly greater (P < 0.01) in the ejaculates inwhich spermatozoa ultimately fertilized <70% of the matureoocytes, than in the ejaculates in which spermatozoa ultimatelyfertilized 70% of the mature oocytes. Furthermore, a strongcorrelation (r = 0.962, P = 0.0001) was detected between pre-IVFacrosin activity and subsequent high (70%) IVF success. Acrosinactivity from normozoo-spermic and oligoasthenozoospermic menwas also compared and was significantly (P < 0.01) higherfor the normozoo-spermic group. These data suggest that measurementof acrosin activity may be a valuable clinical laboratory assayfor assessing the sperm fertilizing potential and that low acrosinactivity is associated with abnormal semen characteristics.     

Andrology: Insulin-like growth factor-I and {alpha}2-macroglobulin in seminal plasma correlate with semen quality

Insulin-like growth factor-I (IGF-I) and ₂-macroglobulin (₂-M)are believed to be involved in the development of germ cells.IGF-I is mainly controlled by concentrations of human growthhormone (HGH), influences cell proliferation and differentiationand its action is mediated by insulin-like growth factor-bindingproteins (IGFBP), placental protein 14 (PP14) and prostate-specificantigen (PSA). ₂-M acts as a broad spectrum proteinase inhibitorand a binding protein for many cytokines and hormones, e.g.inhibin and activin. This study was designed to identify concentrationsof these molecules in seminal plasma in normal semen samplesof healthy men, correlations with semen quality, the relationshipof IGF-I and ₂-M with factors affecting male fertility, andwhether vasectomy influences the concentrations of these molecules.Concentrations of IGF-I and₂-M in human seminal plasma wererelated to semen quality, basal concentrations of HGH, testosterone,IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 inseminal plasma and to serum concentrations of luteinizing hormone(LH) and follicle stimulating hormone (FSH). Commercially availableassays were used to analyse 69 semen samples of various qualityand 11 post-vasectomy samples. IGF-I concentrations in seminalplasma were significantly correlated with the percentage ofmorphologically normal spermatozoa (r = 0.748, P = 0.00001)and sperm concentration (r = 0301, P = 0.011), but negativelycorrelated with serum FSH (r = –0.506, P = 0.00006) andPSA in seminal plasma (r = –0388, P = 0.0009). Total ₂-Mwas significantly correlated with sperm count (r = 0.423, P= 0.0005), percentage of progressively motile spermatozoa (r= 0.444, P = 0.00019), quality of motility (sperm motile efficiency,r = 802, P = 0.00001) and straight line velocity (r = 0.411,P = 0.0013). Correlation between the sperm concentration andHGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomyreduced the concentration of total ₂-M (P = 0.00008) and HGH(P = 0.0068) in the seminal plasma; IGF-I was also reduced aftervasectomy when the total ejaculate amount was considered. ThusIGF-I and ₂-M are significant for the germ cell development:IGF-I hi the maturation of spermatozoa and ₂-M in progressivemotility.     

Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding

BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) isa common cause of failure of fertilization in vitro. This studywas to determine if DSZPB is caused by defective pathways upstreamof protein kinase A (PKA) and C (PKC), or reduced protein tyrosinephosphorylation (TP). METHODS: Infertile men with DSZPB and either normal sperm morphology(NSM) 14% (n = 15) or 5% (n = 15) were studied. Sperm–ZPbinding test was performed by incubation of motile sperm withoocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP,PKA activator) or phorbol myristate acetate (PMA, PKC activator).TP of capacitated sperm in medium was assessed by immunofluorescencewith an anti-phosphotyrosine monoclonal antibody. RESULTS: For normal sperm with normal sperm–ZP binding, both PMAand dbcAMP significantly enhanced sperm–ZP binding ina dose–response manner. Only dbcAMP, but not PMA, significantlyincreased TP of capacitated sperm. In DSZPB men with severeteratozoospermia (NSM 5%), neither PMA nor dbcAMP enhancedsperm–ZP binding, despite dbcAMP significantly increasingthe TP of capacitated sperm for all samples. In contrast, forDSZPB with NSM 14%, PMA caused significantly increased spermbinding up to normal levels (40 sperm bound/ZP) in five men,and dbcAMP had a similar result in two men. Again TP was significantlyenhanced only by dbcAMP, but not by PMA. CONCLUSIONS: There is defective signalling in pathways upstream of PKC andPKA in some men with DSZPB and normal semen analysis. Stimulationof TP by dbcAMP does not enhance sperm–ZP binding capacityin DSZPB men with low TP, regardless of sperm morphology.     

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.     

Andrology: An indiscriminate use of pentoxifylline does not improve in-vitro fertilization in poor fertilizers

In order to enhance fertilization in vitro, pentoxifylline (PTX)was used in couples showing low fertilization rates in previousin-vitro fertilization (IVF) attempts for the treatment of malefactorinfertility. Sibling oocytes were inseminated at random withspermatozoa prepared with or without PTX. After selection byPercoll gradient, sperm samples were divided into two equalaliquots. One aliquot was incubated in Earle's medium containing3.6 mM PTX (treatment group), the other aliquot was incubatedwith PTX-free Earle's medium (control group). After 30 min,both suspensions were washed twice. Sperm parameters after preparationdid not differ between treatment and control samples, and nordid the mean fertilization rates, which were 49.3 and 42.6%respectively. Cleavage characteristics and morphological qualityof the embryos were not significantly different between thetreatment and control groups. The results of this study demonstratethat indiscriminate use of PTX in an IVF programme increasesneither fertilization rate nor embryo transfer rate in poorfertilizers. More prospective research is needed to evaluatethe role of PTX in IVF in couples selected according to theeffect of the compound on sperm function, e.g. hyperactivationand acrosome reaction.     

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).     

Andrology: Analysis of the extent to which sperm movement can predict the results of ionophore-enhanced functional assays of the acrosome reaction and sperm-oocyte fusion

This study has examined the extent to which the informationgenerated by ionophore-enhanced bioassays of the acrosome reactionand sperm-oocyte fusion might be predicted from the computer-aidedanalysis of sperm motility Strong correlations (r 0.7) wereobserved between specific components of sperm movement in semenand the potential for A23187-induced sperm-oocyte fusion, generatinga stepwise regression coefficient of R = 0.663 on the basisof two criteria, percentage progressive motility and amplitudeof sperm lateral head displacement (ALH). The movement characteristicsof the spermatozoa recovered from the Percoll gradients gavean even higher R value of 0.838 on the basis of four variables(percentage rapid, average path velocity, straightness and ALH).In contrast, the ability of human spermatozoa to undergo acrosomereaction in response to A23187 exhibited a limited correlationwith sperm movement, whether these measurements were made inthe original semen sample or following Percoll purification(R 0.4). These results have diagnostic implications, sincesperm-oocyte fusion and the acrosome reaction clearly differin their relative dependence on sperm motility. In practicalterms, it should be noted that the computer-aided analysis ofsperm movement was shown to provide up to 70% of the informationgenerated by the more laboured assessment of sperm-oocyte fusion.     

Ovary and ovulation: Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles

To study the relationship between follicular atresia, apoptosis,and nitric oxide (NO) generation in follicular development,steroidogenesis, NO levels in follicular fluid and apoptosiswere analysed in the various sized follicles of women receivingovarian stimulation with human meno-pausal gonadotrophin (HMG)—humanchorionic gonado-trophin (HCG) treatments for in-vitro fertilization(IVF)-embryo transfer. The follicles were divided into threegroups by diameter: large follicle, 18 mm; medium follicle,12 and 15 mm; small follicle, 10 mm. Follicular fluid was obtainedfrom 20 women 34 h after HCG administration, and the concentrationsof oestradiol, progesterone and testosterone, and nitrite, nitrate,arginine and citrulline were measured. Granulosa cells obtainedfrom each group of follicular fluid were stained with Hoechstdye, and nuclear morphology was examined by a fluorescence microscopy.Oestradiol and progesterone concentrations in large follicleswere significantly (P < 0.01) higher than those in mediumor small follicles, and testosterone concentrations in smallfollicles were significantly (P < 0.01) higher than thosein large follicles. There were no significant differences inthe concentrations of nitrite, nitrate, arginine and citrullineamong three groups. The percentage of apoptotic cells with nuclearfragmentation was significantly (P < 0.01) higher in smallfollicles than in large follicles. The present results suggestedthat small follicles with poor response to HMG may undergo atresiathrough apoptosis. No significant difference in the follicularNO level between large and small follicles led us to speculateon a different responsiveness to NO in these two types of follicles.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Calcium signalling in human oocytes and embryos: two-store model revival

Since the first observations showing that sperm-induced activationof mouse oocytes is mediated by sustained oscillations of intracellularfree calcium concentration (Cuthbertson and Cobbold, 1985),similar patterns of sperm-induced calcium signals have beenobserved in various other mammalian species (Jones, 1998; review).Sperm-induced calcium oscillations were also observed in humanoocytes, both after conventional IVF (Taylor et al., 1993) andafter ICSI (Tesarik et al., 1994; Tesarik and Sousa, 1994). The rapid expansion of clinical applications of ICSI in theearly-to-mid 1990s raised considerable concern about the safetyof this technique, which prompted research into the differencesin the form of sperm-induced calcium signals generated in conventionally-fertilizedand ICSI-treated oocytes. It was