FLOW CYTOMETRIC DETECTION OF SUBHAPLOID NUCLEI IN HUMAN SPERM AS A MEASURE OF DNA FRAGMENTATION AND APOPTOSIS.

The use of assisted reproductive technologies (ARTs) is increasing worldwide. In order to predict the rate of pregnancy after ART the DNA fragmentation index (DFI) of ejaculated spermatocytes may be a better marker than conventional semen quality parameters. Spermatocytes with fragmented DNA are associated with apoptotic stages and are characterized by a low DNA content. The subhaploid nuclei of DNA-damaged spermatocytes can be easily detected by flow cytometry. We here analyzed the percentage of subhaploid nuclei of semen samples from 163 patients aged 26 to 74 years who consulted one of the ten centres for reproductive medicine which routinely send sperm samples to our laboratory in order to determine special sperm parameters. The percentage of subhaploid nuclei indicating the DFI of spermatocytes did not correlate with age and sperm volume, but inversely correlated with sperm concentration and the percentage of motile spermatocytes. This is in concordance with previous studies which demonstrated that DNA damage of spermatozoa correlates with conventional semen quality parameters. Since DNA-damaged spermatocytes are associated with an impaired outcome of assisted conception technologies, this method could help to monitor sperm quality of subfertile men after measures to increase sperm quality and to improve selection criteria of cryopreserved sperm samples in assisted reproduction medicine.

Anti-Mullerian hormone confirms the novel classification of female functional androgenization including polycystic ovary syndrome.

OBJECTIVE: Functional androgenization (FA) can be divided into five groups corresponding to the predominant organ pathology as recently shown by our group: functional cutaneous androgenization (FCA, skin) and FA syndrome (FAS) I (ovary, lean individual), II (adrenal gland), III (ovary, fat tissue, pancreas, and hyperinsulinemia), and IV (residual FA dysfunctions). Group-specific clusters are based on primary variables such as LH, testosterone, DHEAS, sex hormone-binding globulin (SHBG), body mass index (BMI), glucose, insulin, and enlarged polyfollicular ovaries. Because anti-Müllerian hormone (AMH) positively correlates with the antral follicle count, its relevance as an additional primary variable for classifying FA was investigated. DESIGN: In this study, 178 patients with FA were consecutively enrolled and classified into the five FA groups as described earlier and 30 women with regular menstrual cycles served as control. METHODS: Primary variables and serum AMH were analyzed in the early follicular phase. RESULTS: FA patients showed significantly elevated AMH levels (11.1±6.7 ng/ml) versus control (3.0±2.0 ng/ml; P<.0001). AMH was significantly increased in groups FAS I (15.6±5.8 ng/ml) and FAS III (11.6±6.6 ng/ml) compared with groups FCA (7.0±3.8 ng/ml), FAS II (5.05±3.0 ng/ml), and FAS IV (6.9±4.6 ng/ml) and correlated positively (P<.0001) with LH (r=0.538) and testosterone (r=0.368). In regression and multivariate analyses, AMH was not dependent on SHBG, DHEAS, BMI, glucose, or insulin. In receiver operating characteristic analysis, 9.21 ng/ml AMH showed 90% specificity with 71.2% sensitivity for the diagnosis of the two ovarian FA groups, FAS I and III. CONCLUSION: AMH confirms the novel stratification system and constitutes a useful primary variable in the algorithm of FA classification.

Expression patterns of CRH, CRH receptors, and CRH binding protein in human gestational tissue at term.

Recent research suggests a significant role for placental corticotropin-releasing hormone (CRH) in controlling human parturition. This paper describes the expression of CRH, CRH receptors 1 and 2, and CRH binding protein (CRH-BP) in gestational tissue in late pregnancy. Placenta, myometrium, decidua, and fetal membranes were collected after uncomplicated pregnancies at term caesarian section before the onset of labour. The localisation and mRNA expression of CRH, CRH receptors, and CRH-BP were studied by immunohistochemistry and reverse transcription (RT)-PCR. CRH receptors were detected in placenta, myometrium, decidua, and fetal membranes. We demonstrated for the first time the presence of CRH receptors on resident macrophages and on endothelial cells. CRH receptor 1 mRNA was detected in all tissues investigated by RT-PCR, whereas CRH receptor 2 mRNA was restricted to myometrium and decidua. CRH mRNA was widely expressed in all tissue under study. Novel findings are also presented on the expression of CRH-BP in the myometrium. This widespread expression of the CRH system in gestational tissue suggests a paracrine role for CRH in the birth process (e.g. effects on macrophages and endothelial cells).

Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women.

Nitric oxide (NO) is a potent relaxant of smooth muscle and possibly plays a role in maintaining uterine quiescence during pregnancy. Clinical studies have shown beneficial effects of the stable NO donor glyceryl trinitrate (GTN) for the inhibition of pathological myometrial contractility that occurs in preterm labor or dysmenorrhea. Since there are contradictory results regarding the mediation of the relaxing effect of NO, the myometrial prostaglandin synthesis during superfusion with NO donors was studied. Human myometrial strips obtained either at term Cesarean sections before the onset of labor or after hysterectomies in premenopausal women were studied in a superfusion system. After the manifestation of spontaneous contractions, GTN was added in low doses comparable with in vivo levels (0.4-40 nM) and the effect on myometrial activity, intracellular cGMP and prostaglandin production was analyzed. Additionally, the effect of sodium nitroprusside (SNP)--which releases NO spontaneously--was compared with that of GTN. GTN caused a significant decrease in the contraction frequency of myometrial strips from both pregnant and non-pregnant women similar to that of SNP. There was no significant change in the myometrial synthesis of PGI2, PGF2 alpha and PGE2, whereas the intracellular cGMP content was increased. In conclusion, GTN showed a significant inhibitory effect on human myometrium in vitro in very low doses and therefore represents an interesting therapeutic alternative for the treatment of preterm labor and dysmenorrhea. GTN in low doses did not alter the prostaglandin synthesis of human myometrium.

Low density lipoprotein (LDL) subfractions during pregnancy: accumulation of buoyant LDL with advancing gestation.

Pregnancy is accompanied by changes in the maternal lipoprotein metabolism that may serve to satisfy the nutritional demands of the fetus. In this study lipoprotein metabolism was investigated in 23 women during normal pregnancy in the first, second, and third trimesters and in 15 healthy nonpregnant women with regular menstrual cycles. Lipid and apolipoprotein concentrations were measured in total plasma, very low density, intermediate density, low density (LDL), and high density lipoproteins, and in each of six LDL subfractions. During early pregnancy, triglycerides, and dense LDL were higher than in the nonpregnant state. With advancing gestation, triglycerides increased and the distribution of apolipoprotein B-100-containing lipoproteins became increasingly dominated by the accumulation of very low density and intermediate density lipoproteins and buoyant, triglyceride-rich LDL. This is the first study that investigates LDL subfractions in pregnancy using a method that strictly separates LDL subfractions by virtue of density. The accumulation of buoyant, triglyceride-rich lipoproteins may be related to the down-regulation of maternal lipase activities by placental hormones. As a consequence, the metabolic changes of late pregnancy may result in an increased flux of lipoprotein-derived lipids to the placenta, which, with advancing gestation, increasingly expresses receptors with a high affinity for triglyceride-rich lipoproteins.

Formation of proinflammatory cytokines in human term myometrium is stimulated by lipopolysaccharide but not by corticotropin-releasing hormone.

Human term myometrium is poorly characterized as a source of proinflammatory mediators involved in parturition. We have investigated the basal expression of cytokines in myometrium, as well as the effects of CRH and lipopolysaccharide (LPS) on cytokine release. Explants from term myometrium were challenged with CRH or LPS (1 microg/mL each) in short-term tissue culture. Interleukin (IL)-1beta++, IL-6, IL-8, and tumor necrosis factor (TNF)alpha concentrations in the medium were quantified by enzyme immunoassay. The major cytokines released after 24 h were IL-6 and IL-8. All cytokines investigated were stimulated significantly by LPS (P: < 0. 05) but not by CRH. Messenger RNA levels of these cytokines were investigated by RT-PCR. IL-1beta+ and IL-6 messenger RNA were present in preterm and term myometrium before and during labor, whereas IL-8 and TNFalpha were expressed only by myometrium in active labor. Furthermore, myometrial CRH receptors and macrophages were characterized immunohistochemically. We conclude that human term myometrium is a site of production of proinflammatory cytokines and is involved in the inflammation-like reactions mediating the birth process. Cytokine release in term myometrium seems not to be under control of CRH.

Altered lipid metabolism in preeclampsia and HELLP syndrome: links to enhanced platelet reactivity and fetal growth.

Normal pregnancy is a physiological condition of balanced hypercoagulability. However, in preeclamptic pregnancies, the coagulation and fibrinolytic cascades are highly activated, accompanied by pathological blood rheology and endothelial dysfunction. This may result in disseminated intravascular coagulation (DIC). Atherosclerosis research showed that lipids may interfere with coagulation and cause endothelial dysfunction. Therefore, we analyzed the lipoprotein distribution and platelet counts in uncomplicated preeclamptic and HELLP syndrome pregnancies. In addition, a correlation between the fetal circulation determined by Doppler velocimetry and the maternal lipid metabolism was investigated. Fasting serum was collected from 24 women in the third trimester of uncomplicated pregnancies, 9 women with severe preeclampsia, and 6 women with HELLP syndrome. Cholesterol (CH), triglycerides (TGs), and apolipoproteins were analyzed in serum and in very-low-density (VLDL), intermediate-density (IDL), low-density (LDL), and high-density (HDL) lipoproteins separated by ultra-centrifugation. Compared with normal pregnancies, TGs in serum, VLDL, IDL, LDL, and HDL were significantly increased in preeclampsia; no difference in CH concentrations was observed. During HELLP syndrome, IDL-TGs were increased compared with normal pregnancies. There was no clear correlation between fetal hemodynamics and maternal lipid metabolism, but there was a significant negative correlation between maternal platelet counts and serum TG levels. Because TG-rich particles may play an important role in thrombin generation and may induce platelet aggregation, the observed changes in lipoprotein metabolism in preeclampsia and HELLP syndrome may contribute to the coagulopathy seen in these conditions.

Cyclooxygenase-1 and -2 in human placenta and placental bed after normal and pre-eclamptic pregnancies.

In pre-eclampsia, the ratio of prostacyclin:thromboxane production rate is decreased favouring the vasoconstrictive thromboxane. One of the rate-limiting steps in prostaglandin synthesis is cyclooxygenase (COX) activity. Therefore, we investigated the expression of COX-1 and COX-2 in human placenta and placental bed. Tissue specimens from the 29th to 40th week of pregnancy were obtained from Caesarean sections after uncomplicated and pre-eclamptic pregnancies before the onset of labour. COX-1 and COX-2 were localized immunohistochemically with the identification of positive cells by double immunofluorescence staining. The protein and mRNA levels were analysed by immunoblotting and quantitative reverse transcriptase-polymerase chain reaction. Expression of both COX-1 and COX-2 could be observed in placenta and placental bed. COX-1-like immunoreactivity was observed in most cell types with strongest staining in macrophages. Only macrophages, endothelium, vascular leiomyocytes and fibroblasts stained positively for COX-2. In placenta, COX-1 and -2 expression was unchanged after pre-eclampsia. In placental bed, protein and mRNA levels of COX-1 were increased in the pre-eclamptic group (P < 0.05), whereas COX-2 expression did not differ significantly from normal pregnancies. An increased expression of COX-1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed. A therapy with drugs inhibiting COX-1 might be beneficial in this condition.

Distribution of the A and B forms of the progesterone receptor messenger ribonucleic acid and protein in uterine leiomyomata and adjacent myometrium.

The two forms of the progesterone receptor, PR-A and PR-B, are independently regulated at the transcriptional level, and show distinct responses to progesterone antagonists. We were interested in possible differences in the PR-A to PR-B ratio between uterine myometrium and leiomyomata (fibroid), that might influence the response of fibroids to progesterone agonists and antagonists, and thus have consequences for the treatment of this condition. Fibroid and adjacent normal myometrium were obtained from 11 women undergoing hysterectomy. Immunohistochemistry using a monoclonal antibody which recognizes both PR-A and PR-B showed exclusively nuclear staining, and this was stronger in the leiomyomata than in adjacent myometrium. An antibody specific for PR-B gave fainter staining of both tissues. Western blotting confirmed a higher concentration of PR in leiomyomata than myometrium in eight out of 11 cases. In all cases both forms were present, with a consistent dominance of PR-A over PR-B. However an RNase protection assay showed that there was no difference between the concentrations of mRNA encoding PR-A and PR-B, or between the mRNA concentrations in leiomyomata and normal myometrium. We conclude that the observed differences between the levels of immunoreactive PR in leiomyomata and myometrium may result from post-translational control, and support the use of progesterone antagonists in the treatment of leiomyomata.

Isolation of macrophages (Hofbauer cells) from human term placenta and their prostaglandin E2 and thromboxane production.

Placental macrophages (Hofbauer cells) are located close to trophoblast cells and fetal capillaries, which makes them ideal candidates for involvement in regulatory processes within the villous core. Their production of various cytokines and prostaglandin (PG) synthesizing enzymes has previously been shown immunohistochemically. Hofbauer cells were isolated from human placenta after term deliveries by Ficoll and Percoll gradient centrifugation. Remaining trophoblast cells were removed with anti-epidermal growth factor (EGF)-receptor-coated Dynabeads followed by differential adherence. The identity of isolated cells was investigated by immunohistochemistry with anti-CD68, which showed that >90% cells were positive. After a 36 h recovery period in either 20% O2 or 5% O2, fresh medium was applied and PGE2 and thromboxane (TXA2) production analysed by enzyme immunoassay at 4, 8, and 24 h. PGE2 and TXA2 were both produced by placental macrophages with PGE2 synthesis being predominant. Concentrations of both could be stimulated by lipopolysaccharide with maximum effect after 24 h. Culture in low oxygen caused decreased PGE2 concentrations, whereas TXA2 production remained unchanged. In conclusion, the presented isolation protocol allows further study of Hofbauer cell function. This study also presents novel findings regarding the prostaglandin production of term Hofbauer cells under normal and hypoxic conditions.

Expression of prostacyclin and thromboxane synthases in placenta and placental bed after pre-eclamptic pregnancies.

Prostacyclin and thromboxane are potent antagonistic regulators of vascular tone and platelet aggregation. In pre-eclampsia, the ratio of their metabolites is decreased. Little is known about the local regulation of intrauterine prostacyclin and thromboxane production in this condition. Placenta and placental bed biopsies were obtained from uncomplicated and pre-eclamptic pregnancies. Prostacyclin synthase (PCS) and thromboxane synthase (TXS) and their mRNA's were localized by immunohistochemistry using monoclonal antibodies and in situ hybridization. Protein and mRNA levels were quantified by immunoblot and RNase protection assay. PCS-like immunoreactivity was found in endothelial cells and leiomyocytes, whereas fetal and maternal macrophages showed positive staining for TXS. Their mRNA was localized to trophoblast and endothelium, and TXS mRNA could also be detected in macrophages. Quantitative analysis showed no significant difference in intrauterine protein or mRNA expression after pre-eclampsia. The prostacyclin and thromboxane production seems to be compartmentalized within the uteroplacental unit. The expression of their synthesizing enzymes might be regulated post-transcriptionally. Additional regulation of prostaglandin production could be metabolically or on the substrate level and requires further elucidation.

Arachidonate metabolism in human placenta, fetal membranes, decidua and myometrium: lipoxygenase and cytochrome P450 metabolites as main products in HPLC profiles.

Eicosanoids play a key role in pregnancy maintenance and parturition. We investigated the metabolism of arachidonic acid (AA) in short-term tissue cultures of placenta, fetal membranes, decidua and myometrium. Tissues were obtained from caesarean sections before the onset of labour after uncomplicated pregnancies. The released metabolites were analysed by high performance liquid chromatography (HPLC) and specific immunoassays. In radiotracer experiments tissues were labelled with [3H]-AA and metabolites released after incubation with calcium ionophore A23187 were profiled by HPLC. Decidua was more active in metabolizing AA (turnover 34 per cent) than myometrium (28 per cent), placenta (21 per cent) and fetal membranes (17 per cent). Main product in placenta, decidua and myometrium was 12-hydroxyeicosatetraeinoic (12-HETE) (decidua: 19 per cent of released radioactivity, myometrium 14 per cent, placenta 7 per cent). Fetal membranes formed 5-HETE as main product. Another major metabolite in placenta, fetal membranes and decidua was characterized by HPLC as 5(6)-epoxyeicosatrienoic acid. Only myometrium released appreciable amounts of prostaglandins in form of 6-keto-prostaglandin F1 alpha. In non-radioactive experiments formation of eicosanoids from endogenous AA was investigated by HPLC (fluorescence- and UV-detection) and immunoassays. These experiments confirmed the high production of 12-HETE and the low formation of prostaglandins. Our results suggest that the biological role of AA-metabolites, other than prostaglandins, have as yet been underestimated.

[Prostaglandins and labor onset]. / Prostaglandine und Geburtsbeginn.

Prostaglandins and lipoxygenase metabolites of arachidonic acid (Eicosanoids) are crucial paracrine regulators of labor. There are many informations about in vitro production and the physiological or pathophysiological role and clinical importance of these substances. However, the all decisive mechanism of the involvement of eicosanoids in birth process is still unknown. In this review we describe the present knowledge about endocrine, paracrine and autocrine regulations of uterine contractions.

Immunohistochemical localization of thromboxane synthase in human intrauterine tissues.

Uterine tissues are known to be able to synthesize thromboxane A2 (TXA2), but there is little information about the nature of cells actually responsible for its production. In this study human placenta, fetal membranes, umbilical cord and pregnant myometrium were investigated immunohistochemically. The avidin-biotin method for a monoclonal antibody against human thromboxane synthase (Tü 300) was applied on frozen tissue sections. In placenta, fetal membranes and umbilical cord, staining was positive for Hofbauer cells and fibroblasts. Further, in sections of placenta, capillary endothelium showed antigenicity for TX synthase. Leiomyocytes in the umbilical cord vessels contained the enzyme as well. Preparations of pregnant myometrium were shown to express TX synthase in leiomyocytes, endothelial cells and connective tissue cells. Amnion, trophoblast and decidua did not possess antigenicity for this enzyme. Since TXA2 plays an important role for the regulation of vascular tone and aggregation of platelets and may stimulate myometrial contractions during parturition, the abundance of TX synthase in pregnancy-specific tissues confirms previous in vivo and in vitro observations. Further, TXA2 synthesized by Hofbauer cells may be involved in immunological reactions during pregnancy, and the number and level of activation of Hofbauer cells may be closely related to the initiation of labour. Thromboxane production by the endothelium lining the fetal vessels points to its regulatory role for the blood flow in the fetoplacental unit.

Eicosanoid production by frozen tissue in vitro is markedly changed.

The influence of storing deep frozen human tissue specimens on their eicosanoid production in tissue culture was evaluated. Pieces of placenta and fetal membranes (n = 6) were divided and part processed immediately after obtaining the tissues, part after being kept at -20 degrees C for some days. Eicosanoid production was studied in tissue culture with incubation in oxygenated Hank's balanced salt solution (HBSS) at 37 degrees C for 1 h. Prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane A2 (TXA2) and leukotriene B4 (LTB4) production were significantly different in the tissues frozen prior to the incubation. This should be considered when interpreting eicosanoid synthesis in tissue cultures for their significance in vivo.

Eicosanoid production by intrauterine tissues before and after labor in short-term tissue culture.

Prostanoid production by intrauterine tissues from pregnant and non-pregnant women has been studied intensively over the last decade. Little is known about the lipoxygenase metabolites of arachidonic acid (AA). The production of prostaglandins and HETEs by pregnancy specific human tissues was investigated in a short-term culture system. Tissue samples were obtained after uncomplicated pregnancies from placenta, fetal membranes and decidua of deliveries before (n = 6) and after the onset of labor (n = 8) and incubated for 1 hour in oxygenated HBSS. In the supernatant, PGE2, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were measured with RIA and 15-, 12- and 5-HETE with HPLC and UV-detection. The main AA-metabolite in all tissue incubations was 12-HETE. Decidua produced 12 to 28 times more prostaglandins than placenta and fetal membranes with 6-keto-PGF1 alpha as the main metabolite. The main cyclooxygenase derivative measured from placenta and fetal membrane incubations was TXB2. After labor, fetal membranes showed an increase in total prostaglandin (significant for PGE2) and a decrease in HETE synthesis. The physiologic significance of 12-HETE in reproduction is still poorly understood, but a shift in AA metabolism from HETEs to prostaglandins may be involved in the initiation of labor. Furthermore, these results point to different roles of the tissue compartments within the pregnant uterus for the parturition process.

The role of eicosanoids in reproduction.

The central role of eicosanoids in reproduction was studied in areas of important clinical interest. First, their involvement in pregnancy-induced hypertension was investigated. Urine of normotensive and hypertensive pregnant women was analysed for 6-keto-PGF1 alpha, TXB2 and PGE2 by HPLC/RIA. PGE2 and 6-keto-PGF1 alpha excretion was markedly reduced in the preeclamptic subgroup of hypertensive patients during the last two trimesters. A reduced urinary excretion of 6-keto-PGF1 alpha, TXB2 and PGE2 was also found in a hypertension animal model (rat). Further, tissue cultures of human placentas, deciduas and fetal membranes from hypertensive pregnancies displayed a reduced prostaglandin production. Secondly, in the same in-vitro model the central role of PGE2 of fetal membrane origin for the beginning or parturition was shown. Thirdly, concerning endometrial function, the enhancement of PGF2 alpha and PGE2 formation in secretory endometrial cells by estradiol-17 beta and progesterone was documented. Fourthly, lipoxygenase product content in peritoneal fluid of endometriotic patients did not differ from controls.