Progestogens and pregnancy loss.

Progestational agents are often prescribed to prevent pregnancy loss. Progestogens affect implantation, cytokine balance, natural killer cell activity, arachidonic acid release and myometrial contractility. Progestogens have therefore been used at all stages of pregnancy including luteal-phase support prior to pregnancy, threatened miscarriage, recurrent miscarriage, and to prevent preterm labor. In luteal support, a Cochrane review reported that progestogens were associated with a higher rate of live births or ongoing pregnancy in the progesterone group (odds ratio 1.77, 95% confidence interval (CI) 1.09-2.86). Evidence suggests that progestogens are also effective for treating threatened miscarriage. Again, in a Cochrane Database review, progestogens were associated with a reduced odds ratio of 0.53 (95% CI 0.35-0.79) when progestogens were used. In recurrent miscarriage, progestogens also seem to have a beneficial effect. A meta-analysis of progestational agents showed a 28% increase in the live birth rate (relative risk 0.72, 95% CI 0.53-0.97). For the last 30 years, progestogens have been used to prevent preterm labor. Recent meta-analyses also report beneficial effects. This review summarizes the literature and the author's experience using progestogens to prevent pregnancy loss.

Recurrent miscarriage and hCG supplementation: a review and metaanalysis.

Human chorionic gonadotropin (hCG) has been used to prevent subsequent miscarriages after previous recurrent miscarriages. In addition to the luteotrophic effects, hCG has uterine immune and autocrine actions. hCG also affects cytokine expression. A Cochrane database systematic review has indicated that hCG seems to prevent further miscarriages, (OR for miscarriage = 0.26, 95% CI 0.14-0.52). However, the trials in the Cochrane database were not matched for the number of miscarriages, 1°, 2° or 3° aborter status, maternal age, etc. and no account was made for chromosomally abnormal pregnancies. All of these impact on the subsequent prognosis and may confound the results. The previous trials in the literature all assessed urinary (u-hCG) rather than recombinant hCG (r-hCG), raising the question whether the effect on pregnancy outcome is due to hCG itself, or other urinary proteins present in u-hCG. A new trial is indicated in which r-hCG is compared to u-hCG and the most effective compared to placebo. Treatment and placebos arms should be stratified for the prognostic factors above and the results corrected for fetal chromosomal aberrations. Until such a trial is carried out, the use of hCG supplementation is empiric.

Sixth meeting of the European Forum on antiphospholipid antibodies. How to improve the understanding of the antiphospholipid syndrome?

The main objective of these meetings is to promote international collaboration in various clinical and research projects. This paper is the summary of the 2007 Ljubljana meeting, and offers an overview of the proposed projects. The technical and methodological details of the projects will be published on the forum's web site (http://www.med.ub.es/MIMMUN/FORUM/STUDIES.HTM).

Autoantibodies as predictors of pregnancy complications.

Certain autoantibodies which are found in autoimmune diseases including CTDs can impair fertility. Reproductive failure may present as pregnancy loss, either as miscarriage, intrauterine fetal death or stillbirth. There are also late obstetric complications such as intrauterine growth restriction, pre-eclampsia and pre-term birth. This review summarizes the possible influences of autoantibodies in reproductive failure, and particularly their predictive value (if available). The aPLs detectable by lupus anticoagulant, anti-cardiolipin or anti-beta2 glycoprotein I assays are associated with pregnancy loss and have a positive predictive value (PPV) of 75%. In spite of the general consensus on the management of pregnant aPL-positive women, few well-designed clinical trials have been reported and there is also insufficient data about the PPV of treatment. Anti-thyroid antibodies have been associated with pregnancy loss, and indeed have a PPV of 40%. However, no antibody is pathognomic for pregnancy loss. It may be more appropriate to assess a combination of antibodies rather than one antibody. However, a large meta-analysis of published trials is required in order to determine the prevalence of each particular autoantibody and different combinations of antibodies in different forms of reproductive failure.

Anti-phospholipid antibodies and infertility.

Antiphospholipid syndrome (APS) or the presence of antiphospholipid antibodies (aPL), usually presents as pregnancy loss. However, aPL have also been reported to affect implantation, placentation, and early embryonic development. The binding of aPl to beta2GP1 may lead to breakdown of the phospholipid adhesion molecules between different elements of trophoblast. As aPL affect placental growth and function, aPl may prevent implantation presenting as infertility. Lupus anticoagulant and anticardiolipin antibody have been implicated in the prothrombotic effects of APS. Antibodies to other phospholipids such as anti-phosphatidylserine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl Inositol etc. may be more relevant in infertility. Their role remains to be clarified. There is theoretical evidence from animal models and clinical infertility practice that aPL has a role in infertility. However, a large-scale meta-analysis has failed to confirm the association. To determine whether infertility or even pregnancy loss is associated with aPL, it is necessary to know that the embryo is chromosomally normal. Pregestational diagnosis has shown that up to 60% of embryos may be chromosomally aneuploid in failed in vitro fertilization (IVF); hence, may confound our understanding concerning the association between aPL and infertility, failed IVF or even pregnancy loss.

Cytokines in recurrent miscarriage.

Cytokines act at all stages of pregnancy from implantation to parturition. This review examines their relevance in recurrent miscarriage. However, recurrent miscarriage may be due to an inherently abnormal embryo (e.g., chromosomal abberations) or maternal factors (e.g., uterine anomalies or antiphospholipid antibodies). In the former, cytokines are not causative, but may be part of the mechanism of abortion. In the antiphospholipid syndrome, cytokines such as TNFalpha and IL-6 may be responsible for the associated thrombosis. Hence, an appropriate cytokine milieu could be responsible for whether the antibodies are pathogenic or merely an epiphenomenon. Natural killer cells seem to have a key role in immunosurveillance of the invading trophoblast. However, if activated by TNFalpha, natural killer cells may induce apoptosis in the trophoblast possibly leading to miscarriage. This action is inhibited by TGFbeta. Early ultrasound scanning and embryoscopy have revealed structural anomalies in karyotypically normal embryos which have terminated in first trimester missed abortion. Teratogens such as cyclophosphamide cause fetal demise by excessive apoptosis. Excessive apoptosis may be mediated by TNFalpha, TGFbeta and other cytokines. GM-CSF has been reported to prevent teratogenesis in laboratory animals. Both immunomodulation and hormonal support (progesterone or hCG supplements) have been used to improve the live birth rate in recurrently aborting women. Each may modulate the balance between various cytokines. Although neither hormonal support or immunopotentiation have been proven to be beneficial, the results and the role of cytokines themselves can only be assessed in trials of karyotypically normal embryos.

Autoantibody panel screening in recurrent miscarriages.

PROBLEM: Antiphospholipid autoantibodies (aPL), antithyroid antibodies and anti-extractable nuclear antigens (anti-ENA) have all been reported to be associated with recurrent miscarriages (RM) and infertility. However, this association remained controversial. MATERIALS AND METHODS: Fifty-eight women with impaired fertility (38 women with RM and 20 women with infertility, but no miscarriages) and 28 control parous women were screened for seven autoantibodies [antithyroglobulin (aTG), antithyroid peroxidase (aTPO), anticardiolipin (aCL), antiphosphatidyl-serine (aPS), antiprothrombin antibodies (aPT), anti-beta 2 glycoprotein 1 (abeta2GP1), and anti-ENA]. There was no evidence for autoimmune diseases in the patients or the control. The analysis was also performed with several panels of autoantibodies, each of which contained two or more autoantibodies. RESULTS: Anti-TPO was the only antibody to be associated with RM (P = 0.01). A significant association was found between RM, and autoantibodies in the 'aTG + aTPO + anti-ENA' or 'aTG + aTPO' panels. The 'aTG + aTPO + anti-ENA' panel was also associated with RM when the analysis was performed only on 17 women who had secondary infertility: 10 from the 38 women with RM, and seven from the 20 women with infertility and no miscarriages. A significant association (P < 0.001) was also apparent between anti-CL and anti-PS and infertility compared with the 28 control women. CONCLUSIONS: RM was associated with autoantibodies to aTPO and the combined panel of aTPO, aTG and anti-ENA, but not with aPL. aPL were associated with infertility.

ART in recurrent miscarriage: preimplantation genetic diagnosis/screening or surrogacy?

Recently, assisted reproductive techniques have been used to prevent further miscarriages in women with recurrent miscarriage. One approach uses either screening or diagnosis of embryonic chromosomes prior to embryo replacement [preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD)]. The second approach involves surrogacy. However, PGS/PGD assumes that the embryo is chromosomally abnormal, and that the mother should receive a chromosomally normal embryo. Surrogacy assumes that the embryo is normal and that the maternal environment needs to be substituted. This article examines the place of both techniques in different types of recurrent miscarriage, and tries to give guidelines as to which technique is preferable depending on the likelihood of an embryonic chromosome aberration. In repeated fetal aneuploidy or in the older patient, PGS or PGD are preferable. However, with high numbers of miscarriages, or in autoimmune pregnancy loss, surrogacy is preferable. In the light of recent work, it is uncertain which treatment mode is indicated in balanced parental chromosome aberrations. In conclusion, both techniques have a place, but probably only in those patients with a poor prognosis in whom assisted reproductive techniques will be shown to improve the subsequent live birth rate above the spontaneous rate.

NQO2 gene is associated with clozapine-induced agranulocytosis.

Clozapine is a dibenzodiazepine neuroleptic with atypical pharmacological and clinical profiles. Treatment with this drug may be complicated with agranulocytosis (AGR). It is likely that defective oxidative mechanism may be the cause of AGR. A candidate gene, dihydronicotinamide riboside (NRH) quinone oxidoreductase 2 (NQO2), which is involved in detoxification of drugs, was selected. This gene has been mapped to the short arm of chromosome six. The gene was studied by single-strand conformation polymorphism analysis and direct sequencing in 98 schizophrenic patients that were treated with clozapine. Eighteen of these patients developed AGR. Ten polymorphisms in the coding regions, in intron 1, and in the promoter region were found, two of which were novel. Comparisons of the polymorphisms in the first intron in AGR patients and controls suggested that this site might be connected with AGR. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that the level of NQO2 mRNA is low in AGR patients compared with the control group. Such a reduction in message suggests that the NQO2 gene may be involved in the development of clozapine-induced AGR.

Treatment of pregnant patients with antiphospholipid syndrome.

Antiphospholipid Syndrome (APS) has been widely recognized as a risk factor for the recurrence of both thrombosis and pregnancy losses; however the optimal treatment of patients is debatable. The aim of this paper was to establish a consensus among experts on the treatment of APS in pregnancy. A questionnaire that described possible different clinical situations was sent to the International Advisory Board of the 10th International Congress on Antiphospholipid Antibodies. Sixteen experts from different medical branches and different geographic areas sent their replies. The consensus was that treatment for APS pregnant patients should be low molecular weight heparin (LMWH) and low dose aspirin (LDA). The dosage, and frequency of LMWH depends on different situations, including the body weight and past history. Patients with previous thromboses usually receive two injections per day. Warfarin can also be used from 14 to 34 weeks, for patients with previous stroke or severe arterial thromboses. The use of intravenous immunoglobulin (IVIG) seems to be restricted to patients with pregnancy losses despite conventional treatment. The experts usually advised barrier methods of contraception, intrauterine device (if the patient is not taking corticosteroids) or progestins. Oral contraception with oestrogens was usually avoided.

Hepatic infarctions during pregnancy are associated with the antiphospholipid syndrome and in addition with complete or incomplete HELLP syndrome.

Antiphospholipid antibody syndrome (APS) is associated with adverse pregnancy outcomes and maternal complications including thrombotic events and early pre-eclampsia. HELLP syndrome (Hemolysis, Elevated Liver enzymes, Low Platelets) represents a unique form in the spectrum of pre-eclampsia. This report describes four patients with pregnancy-associated hepatic infarctions. All four had APS and HELLP syndrome, which was complete in one patient and incomplete in three patients, with elevated liver enzymes in all, and either thrombocytopenia or hemolysis in two. In the literature, we found descriptions of an additional 24 patients who had 26 pregnancies with concomitant hepatic infarction. Of the total 28 patients, anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LAC) were assessed in 16 patients, out of whom 15 were found to be positive. Hepatic infartction during pregnancy was associated almost always with APS, with HELLP (2/3 complete, 1/3 incomplete), and only in one-third of the pregnancies with pre-eclampsia (PE).

Thromboprophylaxis improves the live birth rate in women with consecutive recurrent miscarriages and hereditary thrombophilia.

The effect of thromboprophylaxis with low molecular weight heparin (LMWH), on the subsequent live birth rate, in thrombophilic women with recurrent miscarriage has not been sufficiently assessed. The present study is a cohort study undertaken to assess the effect of enoxaparin on the subsequent live birth rate in women with hereditary thrombophila. Eighty-five patients with three or more consecutive pregnancy losses and a hereditary thrombophilia subsequently conceived. Thirty-seven were treated with daily subcutaneous injections of enoxaparin 40 mg and 48 were not treated. The outcome of the subsequent pregnancy was assessed in both groups of patients in terms of live births or repeat miscarriage. Forty-seven of the 85 patients were subsequently delivered, 38 have miscarried. Twenty-six of the 37 pregnancies in treated patients (70.2%) resulted in live births, compared with 21 of 48 (43.8%) in untreated patients (P < 0.02, OR 3.03, 95% CI 1.12-8.36). The beneficial effect was seen mainly in primary aborters, i.e. women with no previous live births (P < 0.008, OR 9.75, 95% CI 1.59-52.48). This benefit was also found in patients with a poor prognosis for a live birth (five or more miscarriages), where the live birth rate was increased from 18.2% to 61.6%. However, the benefit was not statistically significant, probably due to the small number of patients. If the beneficial effects of enoxaparin are confirmed by additional studies, thromboprophylaxis can be recommended for patients with hereditary thrombophilia and recurrent pregnancy loss.

Pregnancy outcome after laparoscopy or laparotomy in pregnancy.

STUDY OBJECTIVE: To assess obstetric performance and fetal outcomes after laparoscopy or laparotomy performed during pregnancy. DESIGN: Nationwide, multicenter, retrospective chart review (Canadian Task Force classification II-2). SETTING: Seventeen hospitals throughout Israel: 12 university or university-affiliated hospitals and 5 general hospitals. PATIENTS: Three hundred eighty-nine pregnant women. INTERVENTION: Laparoscopy or laparotomy for various indications. MEASUREMENTS AND MAIN RESULTS: Of 192 laparoscopies performed, 141 were during the first, 46 during the second, and 5 during the third trimester; respective figures for 197 laparotomies were 63, 110, and 24. No intraoperative complications were reported for either procedure. Six and 25 women had complications after laparoscopy and laparotomy, respectively. There was no significant difference in abortion rates between groups. Mean gestational age at delivery and mean birthweight were comparable between groups. No significant difference was found in frequency of fetal anomalies between groups or when compared with the Israel register of anomalies. CONCLUSION: Operative laparoscopy seems to be as safe as laparotomy in pregnancy.

TNF-alpha in pregnancy loss and embryo maldevelopment: a mediator of detrimental stimuli or a protector of the fetoplacental unit?

PURPOSE: Tumor necrosis factor alpha (TNF-alpha), a multifunctional cytokine, has been identified in the ovary, oviduct, uterus, and placenta, and is expressed in embryonic tissues. For many years TNF-alpha was mainly considered to be a cytokine involved in triggering immunological pregnancy loss and as a mediator of various embryopathic stresses. However, data collected during the last decade has characterized TNF-alpha not only as a powerful activator of apoptotic, but also antiapoptotic signaling cascades, as well as revealed its regulatory role in cell proliferation. This review summarizes and conceptualizes the studies addressing TNF-alpha-activated intracellular signaling and the possible functional role of TNF-alpha in embryonic development. METHODS: Studies addressing the role of TNF-alpha in intercellular signaling, in vivo studies addressing the functional role TNF-alpha in spontaneous and induced pregnancy loss, and studies addressing the role of TNF-alpha in fetal malformations were reviewed. Comparative studies in TNF-alpha knockout and TNF-alpha positive mice were performed to evaluate embryonic death, structural anomalies in fetuses, the degree of apoptosis and cell proliferation, and the activity of molecules such as caspases 3 and 8, the NF-kappaB, (RelA), IkappaBalpha in some target embryonic organs shortly after exposure to embryopathic stresses. RESULTS: It is proposed that the possible essential function of TNF-alpha may be to prevent the birth of offspring with structural anomalies. CONCLUSIONS: TNF-alpha will boost death signaling to kill the embryo if initial events (damages) triggered by detrimental stimuli may culminate in structural anomalies, and stimulate protective mechanisms if the repair of these damages may prevent maldevelopment.

Cytokine expression in the uterus of mice with pregnancy loss: effect of maternal immunopotentiation with GM-CSF.

It is believed that failure of the maternal immune system to actively support embryonic development, through production of the appropriate cytokine network, might be responsible for embryonic death. Thus, the aim of this study was to evaluate the possible involvement of cytokines such as tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta2 (TGF-beta2), which are crucial for normal embryonic development, in the early stages of mechanisms that mediate induced pregnancy loss. The early stages of the resorption process induced by lipopolysaccharide (LPS) were characterized by blood accumulation in the vicinity of the embryo, preceding any visible embryonic damage. At that time, immunohistochemical analysis revealed an increased expression of TNF-alpha in the primary and secondary decidua, which was reduced as the resorption process was completed. In contrast, TGF-beta2 expression was decreased in the primary and secondary decidua, as well as in the glandular epithelium, at all the times assessed. Maternal immunopotentiation with granulocyte macrophage-colony stimulating factor (GM-CSF), which controls maternal immune activities supporting normal embryonic development, decreased the resorption rate in LPS-treated mice while normalizing the expression of TNF-alpha and TGF-beta2 in the uterus of these animals throughout the ongoing resorption process. These results indicate a possible role for maternal immunopotentiation with GM-CSF in the mechanisms mediating the early stages of pregnancy loss, possibly via modulation of TNF-alpha and TGF-beta2 activity.

Apoptosis in the uterus of mice with pregnancy loss.

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis-regulating gene products p53 and bcl-2. METHOD OF STUDY: Pregnancy loss was induced by LPS or CP on days 9 or 12 of pregnancy, respectively. LPS- or CP-associated apoptosis was assessed by the TdT mediated dUTP-biotin nick end labeling (TUNEL) method as well as by DNA fragmentation analysis, while p53 or bcl-2 expression was evaluated by immunohistochemistry. RESULTS: Lipopolysaccharide treatment initiated a resorption process that was accompanied by the appearance of apoptotic cells in the uterus, which increased in number by 24 hr after treatment. Induction of pregnancy loss with CP resulted in the appearance of some apoptotic cells in the uterus, reaching a peak at 72 hr after treatment. DNA fragmentation analysis revealed a DNA ladder at 24 hr after LPS as well as 72 hr after CP treatment. Immunohistochemical analysis demonstrated a continuous p53 expression in the uterus of LPS- or CP-treated mice, which was somewhat elevated at the peak of the apoptotic process. On the other hand, bcl-2 expression in LPS-treated mice could be reciprocally correlated with the apoptotic process, appearing only at its initiation or completion, while in CP-treated mice it was continuously expressed except for some elevation at the completion of the apoptotic process. CONCLUSIONS: Our results suggest a possible role for the apoptotic process in mechanisms mediating pregnancy loss and indicate an involvement of p53 and bcl-2 in its regulation.

The role of pro- and anti-apoptotic molecular interactions in embryonic maldevelopment.

PROBLEM: Pregnancy loss and the occurrence of inborn structural anomalies are often preceded by excessive apoptosis in targeted embryonic and extraembryonic tissues. Apoptogenic stimuli activate both death and survival, signaling cascades consisting of molecules acting as activators and effectors, or negative regulators of apoptosis. The interplay between these cascades determines whether the cell which is exposed to an apoptogenic stimulus dies or survives. This review summarizes the functioning of pro- and anti-apoptotic molecules in embryos responding to various teratogens. The effect of potentiation of the maternal immune system on these molecules is also discussed. METHODS OF STUDY: The data on the functioning of various pro- and anti-apoptotic molecules in embryos exposed to various developmental toxicants, and embryos developing in a diabetic environment are reviewed. Techniques such as the TUNEL method, DNA fragmentation assay, electromobility shift assay (EMSA), fluorometric assay, immunohistochemistry, Western blot, In situ hybridization, have been used in our studies to detect apoptosis, and evaluate the functioning of molecules such as TNFalpha, caspases, NF-kappaB and IkappaB, p53, and bcl-2 in different embryonic and extraembryonic tissues. RESULTS: Our and other data summarized in this review have demonstrated that the doses of developmental toxicants required to induce pregnancy loss and gross structural anomalies induce excessive apoptosis shortly after treatment. Depending on the intensity and type of targeted tissues, this apoptosis was accompanied by alterations in the activity of the molecules which act as activators and effectors (e.g. caspase 3, caspase 8, caspase 2, p53) or negative regulators (bcl-2, NF-kappaB) of apoptosis. Maternal immunopotentiation, which decreases the level of induced and spontaneous pregnancy loss and the incidence and severity of teratogen-induced structural anomalies has been shown to modulate the expression of these molecules both in embryonic tissues and at the feto-maternal interface. CONCLUSIONS: The data presented in this review suggest that molecules such as TNFalpha, caspase 3, caspase 8, NF-kappaB, p53 and bcl-2, which are involved in the regulation of apoptosis, may also be involved in determining the sensitivity of the embryo to developmental toxicants. Maternal immunopotentiation may modulate the functioning of these molecules.