Bassem Refaat

Multi-marker assessment of ovarian reserve predicts oocyte yield after ovulation induction

BACKGROUND Many hormone and ultrasound measurements have been assessed as possible markers of ovarian reserve and to identify potential poor responders to ovulation induction. The objective of this study is to determine whether multiple biomarkers measured in blood samples collected immediately before commencement of ovulation induction for IVF can predict the outcome of ovarian stimulation. METHODS We conducted a prospective observational study, including 356 unselected women undergoing ovulation induction/IVF at two centers. Anti-Müllerian hormone (AMH), inhibin B and FSH were measured before commencement of ovulation induction. The main outcome measures were the number of oocytes retrieved and pregnancy outcome. RESULTS Univariate analyses showed that age, FSH, inhibin B and AMH were significant predictors for poor oocyte yield. AMH presented the highest receiver operating characteristic area under the curve (ROC(AUC)) of 0.827 indicating a good discriminating potential for predicting poor ovarian response, followed by FSH with an ROC(AUC) of 0.721. In the multivariate analysis, the variables age, FSH and AMH remained significant and the resulting model provided a high ROC(AUC) of 0.819. Women with an ovarian reserve test of <0.3 have more than a 75% chance of having their treatment cycle canceled, but a value over 0.73 indicates a 38% chance of pregnancy. Number of oocytes and oocyte yield per unit FSH administered were correlated with log model for no pregnancy (r = -0.217, P < 0.001 and r = -0.367, P < 0.001, respectively) but had limited predictive value. CONCLUSIONS A derived estimate of ovarian reserve demonstrated superior ability for predicting oocyte yield after ovulation induction when compared with any single endocrine marker (AMH, inhibin B, FSH).

The expression of inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in ectopic pregnancy

OBJECTIVE To investigate the production of inducible nitric oxide synthase (iNOS) in the fallopian tube (FT) during the menstrual cycle and whether epithelia from FTs bearing an ectopic pregnancy differ from healthy tubes in iNOS expression. DESIGN Prospective study. SETTING Academic unit of reproductive and developmental medicine. PATIENT(S) Fallopian tubes from the different stages of the menstrual cycle (n=12), FTs bearing an ectopic pregnancy (n=15), and FTs from pseudopregnant women (n=6) were collected. INTERVENTION(S) In the pseudopregnant group, patients were injected with hCG in the days leading up to hysterectomy. Samples were processed for immunohistochemistry staining and quantitative reverse transcriptase polymerase chain reaction. MAIN OUTCOME MEASURE(S) To compare iNOS protein and messenger RNA expression between the different groups. RESULT(S) This is the first report on cyclicity in iNOS production by human fallopian tube during the menstrual cycle. The intensity of expression of iNOS was higher in the ectopic pregnancy group compared with the pseudopregnant group (P<0.05). CONCLUSION(S) The cyclicity in iNOS expression by the tube suggests its involvement in fertilization and early embryonic development. Pathologic generation of nitric oxide through increase iNOS production may decrease tubal ciliary beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in ectopic pregnancy.

The expression of MUC1 in human Fallopian tube during the menstrual cycle and in ectopic pregnancy

BACKGROUND Ectopic pregnancy is a major cause of maternal morbidity and mortality with increasing incidence worldwide. We have investigated whether epithelia from Fallopian tubes (FTs) bearing an ectopic pregnancy differ from normal tubes in expression of MUC1. METHODS Since it is not possible to collect FTs from women carrying a healthy pregnancy, we studied tissue collected at the time of hysterectomy for benign disease. Women were injected with hCG in the days leading up to hysterectomy, and pseudopregnancy confirmed by the presence of high serum progesterone levels and the decidualization of the endometrium. FTs from the different stages of the menstrual cycle (n = 24), tubes bearing an ectopic pregnancy (n = 15) and pseudo-pregnant tubes (n = 6) were collected and examined using immunohistochemistry and quantitative RT-PCR. RESULTS MUC1 was present at the apical surface of the tubal epithelial cells throughout the menstrual cycle, but intracellular localization was minimal in the follicular phase, increasing to a maximum in the luteal phase. MUC1, including the glycoform recognized by antibody 214D4, was found at the apical surface of tubal epithelium in both the ectopic and pseudo-pregnant groups and the intracellular expression was much stronger in the pseudo-pregnant group than in the ectopic group. The 214D4 epitope was absent from tubal tissue adjacent to ectopic implants. CONCLUSIONS The decrease in MUC1 expression and altered glycosylation in tubal epithelium from ectopic pregnancy may reflect an increase in receptivity.

Role of Activins and Inducible Nitric Oxide in the Pathogenesis of Ectopic Pregnancy in Patients with or without Chlamydia trachomatis Infection

ABSTRACT Chlamydia trachomatis infection can lead to pelvic inflammatory disease, ectopic pregnancy (EP), infertility, and chronic pelvic pain in women. Activins and inducible nitric oxide synthase (iNOS) are produced by the human fallopian tube, and we speculate that tubal activins and iNOS may be involved in the immune response to C. trachomatis in humans and their pathological alteration may result in tubal pathology and the development of EP. Blood and fallopian tubes were collected from 14 women with EP. Sera were analyzed by enzyme-linked immunosorbent assay to detect antibodies against chlamydial heat shock protein 60 (chsp60) and the major outer membrane protein of C. trachomatis. Confirmation of C. trachomatis serology was made using the microimmunofluorescence test. The patients were classified into three groups according to their serological results, and immunohistochemistry and quantitative reverse transcription-PCR were performed to investigate the expression of candidate molecules by tubal epithelial cells among the three groups. This is the first study to show an increase in the expression of activin βA subunit, type II receptors, follistatin, and iNOS within the human fallopian tube of EP patients who were serologically positive for C. trachomatis. A similar expression profile was observed in the fallopian tubes with detectable antibodies only against chsp60. These results were shown at the mRNA and protein levels. We suggest that tubal activin A, its type II receptors, follistatin, and NO could be involved in the microbial-mediated immune response within the fallopian tube, and their pathological expression may lead to tubal damage and the development of EP.

Why does the fallopian tube fail in ectopic pregnancy? the role of activins, inducible nitric oxide synthase, and MUC1 in ectopic implantation

OBJECTIVE To investigate the role of activin-βA subunit, activin type II receptors, inducible nitric oxide synthase (iNOS), and MUC1 in the pathogenesis of ectopic pregnancy (EP) and their involvement in the determination of the implantation site. DESIGN Observational study. SETTING Academic unit of reproductive and developmental medicine. PATIENT(S) Four women at the luteal phase, three pseudopregnant women at the time of hysterectomy for benign disease, and 10 archived cases of EP. We collected 14 Fallopian tubes were collected from four women at the luteal phase and three pseudopregnant women at the time of hysterectomy for benign disease; specimens from implantation site, trophoblast and remote sites from the implantation site were collected from 10 archived cases of EP. INTERVENTION(S) Immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S) Comparison of the expression of candidate molecules between the different groups. RESULT(S) The expression of activin-βA subunit, activin type II receptors, and iNOS was statistically significantly increased and expression of MUC1 statistically significantly decreased in tubes bearing an EP. There was no statistically significant difference in the expression of the candidate molecules between the implantation and remote sites. Candidate molecules were also expressed in the trophoblast. CONCLUSION(S) The pathological expression of candidate molecules by tubes bearing an EP is not involved in the determination of implantation site. Additionally, candidate molecules may play a role in the regulation of trophoblast cells in vivo during early pregnancy.

The expression of activins, their type II receptors and follistatin in human Fallopian tube during the menstrual cycle and in pseudo-pregnancy

BACKGROUND The Fallopian tube (FT) is the site of fertilization and early embryonic development. We have previously reported the expression of activins, their receptors and follistatin by the FT. Here, our aim was to study the expression of tubal activins, their type II receptors and follistatin during the menstrual cycle and following exposure to hCG in vivo. METHOD A set of 30 FTs were collected from cycling women (n = 12) at different stages of the cycle (n = 4 in each stage) and pseudo-pregnant women (n = 3) at the time of hysterectomy for benign disease. The pseudo-pregnant women were injected with hCG in the days leading up to hysterectomy, and pseudo-pregnancy was confirmed by the persistence of amenorrhea, the presence of corpus luteum and decidualization of the endometrium. FT specimens were examined using immunohistochemistry and quantitative RT-PCR. RESULTS The expression of activin βA- and βB-subunits, activin type IIA and IIB receptors, and follistatin varied throughout the menstrual cycle, being lowest in the follicular phase and highest in the luteal phase. These results were demonstrated at the mRNA and protein level by quantitative RT-PCR and immunohistochemistry (P< 0.05). HCG injection rescued the expression of the candidate molecules from falling to the follicular stage levels but the expression remained lower than in the luteal phase. CONCLUSIONS We suggest that activins play a role in tubal physiology and early embryonic development. Additionally, exposure of the tubal epithelium to hCG modulates the expression of tubal activins.

Thymoquinone subdues tumor growth and potentiates the chemopreventive effect of 5-fluorouracil on the early stages of colorectal carcinogenesis in rats

Colorectal cancer (CRC) is one of the most prevalent cancers and has a high mortality rate. Insensitivity and the limited therapeutic efficacy of its standard chemotherapeutic drug, 5-fluorouracil (5-FU), represents an important challenge in CRC treatment. The robust antitumor properties of thymoquinone (TQ), the main bioactive constituent of Nigella sativa, have recently been demonstrated on different cancers. We investigated whether TQ could potentiate the chemopreventive effect of 5-FU to eradicate the early stages of CRC and elucidated its underlying mechanisms. An intermediate-term (15 weeks) model of colorectal tumorigenesis was induced in male Wistar rats by azoxymethane (AOM), and the animals were randomly and equally divided into five groups: control, AOM, AOM/5-FU, AOM/TQ, and AOM/5-FU/TQ. TQ (35 mg/kg/d; 3 d/wk) was given during the seventh and 15th weeks post-AOM injection, while 5-FU was given during the ninth and tenth weeks (12 mg/kg/d for 4 days; then 6 mg/kg every other day for another four doses). At week 15, the resected colons were subjected to macroscopic, histopathological, molecular, and immunohistochemical examinations. Interestingly, 5-FU/TQ combination therapy resulted in a more significant reduction on AOM-induced colorectal tumors and large aberrant crypts foci than treatment with the individual drugs. Mechanistically, 5-FU and TQ remarkably cooperated to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and TBRAS and upregulate the expression of anti-tumorigenesis DKK-1, CDNK-1A, TGF-β1, TGF-βRII, Smad4, and GPx. Overall, our findings present the first report describing the in vivo enhancement effect of combined TQ and 5-FU against early stages of CRC; however, further studies are required to determine the value of this combination therapy in an advanced long-term model of CRC and also to realize its clinical potential.

Role of activins in embryo implantation and diagnosis of ectopic pregnancy: a review

Embryo implantation is a major prerequisite for the successful establishment of pregnancy. Ectopic implantation outside the intrauterine cavity and the development of ectopic pregnancy (EP) is a major cause of maternal morbidity and occasionally mortality during the first trimester. EP may be induced by failure of tubal transport and/or increased tubal receptivity. Activins, their type II receptors and follistatin have been localised in the human endometrial and tubal epithelium and they are major regulators of endometrial and tubal physiology during the menstrual cycle. Pathological expression of activins and their binding protein, follistatin, was observed in tissue and serum samples collected from EP. Several studies with different designs investigated the diagnostic value of a single measurement of serum activin-A in the differentiation between normal intrauterine and failing early pregnancy and the results are controversial. Nevertheless, the diagnostic value of activins in EP, including the other activin isoforms (activin-B and –AB) and follistatin, merits further research. This review appraises the data to date researching the role of activins in the establishment of normal pregnancy and, pathogenesis and diagnosis of tubal EP.

Serum Activins and Follistatin during the Treatment of Chronic Hepatitis C Genotypes 1 and 4 and Their Correlations with Viral Load and Liver Enzymes: A Preliminary Report

Aims. To measure the effect of pegylated interferon-α therapy on serum activin-A, activin-B, and follistatin and their correlation with viral load and liver fibrosis in chronic hepatitis C (CHC). Methods. This study was cross-sectional and sera were collected from 165 participants classified into 7 groups: 40 healthy negative control, 33 treatment naïve patients as positive control, 19 patients at week 4, 22 at week 12, and 19 at week 24 of treatment initiation and 21 responders and 11 nonresponders at the end of 48-week treatment protocol. Serum candidate proteins were measured using ELISA and liver fibrosis was assessed by AST platelet ratio index (APRI). Results. CHC significantly increased activins and decreased follistatin compared to negative control (P < 0.05). Activin-A and follistatin levels returned to the levels of negative control group at weeks 4, 12, and 24 following treatment initiation and were significantly different from positive control (P < 0.05). Both proteins were significantly different between responders and nonresponders. Activin-A correlated positively and significantly with the viral load and APRI. Conclusion. CHC modulates serum activin-A and follistatin and they appear to be influenced by pegylated interferon-α therapy. Further studies are needed to explore the role of activins in CHC.

The prevalence of Chlamydia trachomatis and Mycoplasma genitalium tubal infections and their effects on the expression of IL-6 and leukaemia inhibitory factor in Fallopian tubes with and without an ectopic pregnancy.

This was a prospective case–control study that measured the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG) by an IVD CE multiplex PCR kit in fresh Fallopian tubes (FT) obtained from 96 ectopic pregnancies (EP) and 61 controls in the midluteal phase of the cycle. We later measured the expression profile of IL-6, leukaemia inhibitory factor (LIF) and their signalling molecules, in respect to the type and number of infections, by immunohistochemistry, ELISA and quantitative RT-PCR. The frequencies of CT, and MG mono- and co-infections were significantly higher in EP. IL-6, LIF, their receptors and intracellular mediators were significantly up-regulated at the gene and protein levels in positive compared with negative FTs within each group (P < 0.05). EP tubal samples with co-infections showed the highest significant expression of the candidate cytokines by all techniques (P < 0.05). CT and MG are frequent in EP and up-regulate the tubal expression of IL-6, LIF and their signalling molecules. Both cytokines could be involved in the tubal immune response against bacterial infections, as well as the pathogenesis of EP. Further studies are needed to explore the roles of IL-6 family in infection-induced tubal inflammation and EP.