Modulation of Cytokine Production by the Dydrogesterone Metabolite Dihydrodydrogesterone.

PROBLEM: Previous studies have shown that dydrogesterone, the orally administered progestogen, upregulates the production of Th2 cytokines and downregulates Th1 cytokine production. This study was designed to ascertain whether dihydrodydrogesterone (DHD), the major metabolite of dydrogesterone, is similarly capable of modulating cytokine production by peripheral blood mononuclear cells (PBMC) from women with a history of unexplained recurrent miscarriage. METHOD OF STUDY: Mitogen-stimulated PBMC from women with unexplained recurrent miscarriage were exposed to progesterone or dydrogesterone or DHD, and the levels of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines were estimated by ELISA. To ascertain whether DHD mediates its effects via the progesterone receptor, RU486, a progesterone agonist, was added to cultures along with mitogen and DHD. RESULTS: The metabolite DHD, like its parent molecule dydrogesterone, suppresses the production of the pro-inflammatory cytokines IFN- γ and TNF-α and upregulates the production of the anti-inflammatory cytokine IL-4. The progesterone antagonist RU486 reverses the effect of DHD, suggesting that this molecule mediates its cytokine-modulating effect via the progesterone receptor. CONCLUSION: Dihydrodydrogesterone retains the immunomodulatory effects of the progestogen dydrogesterone by bringing about a shift in cytokine production profiles that might be conducive to the success of pregnancy.

Molecular basis for the effects of zinc deficiency on spermatogenesis: An experimental study in the Sprague-dawley rat model.

INTRODUCTION: The objective of this study is to investigate the molecular mechanisms underlying the effects of zinc deficiency on spermatogenesis in the Sprague-Dawley (SD) rat. MATERIALS AND METHODS: Three groups of eight adult male SD rats were maintained for 4 weeks on a normal diet as control, zinc deficient diet and zinc deficient diet with zinc supplementation of 28 mg zinc/kg body weight respectively. Using standard techniques, the following parameters were compared between the three groups of experimental animals at the end of 4 weeks: (a) Serum zinc, magnesium (Mg), copper (Cu), selenium (Se) and cadmium (Cd), (b) serum sex hormones, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX), (c) interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), Bcl-2, Bax and caspase-3 expression in the testes, (d) assessment of apoptosis of testicular cells using electron microscopy and (e) testicular volume and histology using the orchidometer and Johnsen score, respectively. RESULTS: The zinc deficient group showed a reduction of testicular volume, serum concentrations of Zn, Cu, Se, Mg, SOD, GPX, IL-4, Bcl-2 and testosterone (P < 0.05), as well as increased levels of serum Cd, MDA and tissue TNF-α, Bax, caspase-3 and apoptosis of the germ cells (P < 0.05) compared with control and zinc supplementation groups. CONCLUSION: Zinc deficiency is associated with impaired spermatogenesis because of reduced testosterone production, increased oxidative stress and apoptosis. These findings suggest that zinc has a role in male reproduction.

Changes in the diameter and valve closure time of leg veins in primigravida women during pregnancy.

OBJECTIVE: The aim of this study was to monitor the changes that develop in leg veins of primigravida women during pregnancy. METHODS: Sixty primigravida women volunteered to undergo clinical evaluation and duplex ultrasound examination of both lower limb veins to monitor changes in vein diameter and valve closure time (VCT) during pregnancy and 3 months postpartum by duplex ultrasound. A total of four readings were taken for each subject, one reading for each trimester and the last reading at 3 months postpartum. RESULTS: The mean (± standard deviation) age of participants was 26.82 ± 2.47 years; 39 limbs (32.5%) and 65 limbs (54.2%) developed C1-C3 venous changes during the second and third trimesters, respectively. Three months post partum, 36 limbs (30%) continued to have C1-C2 changes. Only four limbs in four subjects developed varicose veins along the great saphenous vein, and their VCT was more than 1 second. These subjects were found to have a family history of varicose veins. Duplex examinations showed that there was a gradual increase in the diameter and VCT from the second trimester through the third trimester of pregnancy in all examined venous segments. These changes were statistically significant by Friedman and related-samples Wilcoxon signed rank tests within the same legs (P = .001) but not between legs in the same subject (P > .05), even with adjustment for body mass index (P = .001-.049). CONCLUSIONS: In primigravida women, lower limb veins showed gradual increase in vein diameter and in VCT starting from the second trimester. These changes reverted to baseline in most cases 3 months after delivery.

Intrauterine growth restriction: cytokine profiles of trophoblast antigen-stimulated maternal lymphocytes.

Intrauterine growth restriction (IUGR) is an important perinatal syndrome that poses several serious short- and long-term effects. We studied cytokine production by maternal peripheral blood lymphocytes stimulated by trophoblast antigens. 36 women with a diagnosis of IUGR and 22 healthy women with normal fetal growth were inducted. Peripheral blood mononuclear cells were stimulated with trophoblast antigens and levels of the proinflammatory cytokines IL-6, IL-8, IL-12, IL-23, IFNγ, and TNFα and the anti-inflammatory cytokines IL-4, IL-10, and IL-13 were measured in culture supernatants by ELISA. IL-8 was produced at higher levels by blood cells of the IUGR group than normal pregnant women, while IL-13 was produced at lower levels. IL-8, IFNγ, and TNFα were higher in IUGR with placental insufficiency than in normal pregnancy. IL-12 levels were higher and IL-10 levels were lower in IUGR with placental insufficiency than in IUGR without placental insufficiency. We suggest that a stronger pro-inflammatory bias exists in IUGR as compared to normal pregnancy and in IUGR with placental insufficiency when compared to IUGR without placental insufficiency. Several ratios of proinflammatory to anti-inflammatory cytokines also support the existence of an inflammatory bias in IUGR.

Ex-vivo oocyte retrieval for fertility preservation.

OBJECTIVE: To report a novel fertility preservation strategy in a woman with recurrent serous borderline ovarian tumor in the conserved ovary involving ex-vivo retrieval of in vivo matured oocytes and subsequent embryo cryopreservation. DESIGN: Case report. SETTING: Tertiary infertility care unit. PATIENT(S): A 27-year-old woman presented for follow-up visit with a history of borderline serous adenocarcinoma treated conservatively with left oophorectomy and fertility-sparing laparoscopic staging. Ultrasound scan revealed a recurrent disease in the right ovary. INTERVENTION(S): Ex-vivo retrieval of mature oocytes after ovarian stimulation. MAIN OUTCOME MEASURE(S): Fertility preservation. RESULT(S): The patient underwent ovarian stimulation followed by a laparotomy and oophorectomy on the day of oocyte retrieval. A puncture of the follicles was performed in the operating theatre with a maximum ischemia time of 14 minutes. Eleven mature oocytes were aspirating, resulting in seven zygotes for cryopreservation. CONCLUSION(S): Mature oocytes can be successfully retrieved ex-vivo from the oophorectomy specimen after a controlled ovarian hyperstimulation (COH) protocol. This method provides a possible strategy for fertility preservation in patients with recurrent ovarian cancer without the risk of cancer cells spillage associated with the standard transvaginal oocyte retrieval.

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).

Lithium protects against toxic effects of cadmium in the rat testes.

PURPOSE: To investigate the protective effect of Lithium against the toxic effect of Cadmium in the rat testes. METHODS: Twenty four adult male Sprague-Dawley rats were treated with four different regimens: Cadmium only, Cadmium and lithium, lithium only and controls. Rats were sacrificed after 6 weeks and testicular levels of pro-inflammatory cytokine (IL-4), anti-inflammatory cytokine (TNF-α), Pro-apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) were measured by ELISA while serum levels of FSH, LH, Prolactin and Testosterone were measured using the Vidas parametric system. Antioxidant status (MDA, SOD) was also assessed in serum. Histopathological changes of testes were examined using light and electron microscopy. Immunohistochemical staining for Bax, Bcl-2 and Caspase 3 were performed. RESULTS: Treatment with lithium was associated with significant reduction in the toxic effects of Cadmium as shown by reduced testicular levels of TNF-α, serum levels of Malondialdehyde and testicular level of Bax, and increased levels of IL-4, Zn-Cu SOD, Bcl-2 and Testosterone. Testicular histopathology showed that Cadmium produced an extensive germ cells apoptosis and the addition of lithium in Cadmium-treated rats significantly reduced cadmium-induced testicular damage. CONCLUSION(S): Lithium has a protective effect against cadmium-induced testicular apoptosis in the rat.

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.

Role of activins and inducible nitric oxide in the pathogenesis of ectopic pregnancy in patients with or without Chlamydia trachomatis infection.

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 betaA 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.

Redirection of cytokine production by lymphocytes from women with pre-term delivery by dydrogesterone.

PROBLEM: To study the ability of dydrogesterone to modulate the production of pro-inflammatory and anti-inflammatory cytokines by lymphocytes from women undergoing pre-term delivery (PTD). METHOD OF STUDY: Peripheral blood mononuclear cells (PBMC) from 18 subjects undergoing PTD were stimulated with the mitogen phytohemagglutinin in the presence and absence of progesterone and dydrogesterone. The levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 in culture supernatants were then estimated by enzyme-linked immunoabsorbant assay. Cytokine production in the presence and absence of progesterone and dydrogesterone were compared. RESULTS: The exposure of PBMC to dydrogesterone resulted in a significant inhibition in the production of the pro-inflammatory cytokines IFN-gamma and TNF-alpha and a significant increase in the levels of the anti-inflammatory cytokine IL-4, resulting in a substantial shift in the ratio of Th1/Th2 cytokines. CONCLUSION: Dydrogesterone induces a shift in cytokine bias, by inhibiting pro-inflammatory cytokine production and increasing anti-inflammatory cytokine production.

Insulin resistance in women with recurrent spontaneous miscarriage of unknown aetiology.

OBJECTIVE: The objective of this study was to explore the role of insulin resistance in women with recurrent spontaneous miscarriage (RSM). Patients with > or =3 consecutive spontaneous miscarriages were classified as having RSM. SUBJECTS AND METHODS: Thirty-five non-pregnant women presenting with RSM in our specialized RSM clinic constituted the study population, while 30 non-pregnant, parous, fertile women without RSM served as controls. The study and control patients were matched for age, ethnicity and body mass index (BMI). Blood was extracted from these patients for fasting blood glucose and fasting insulin simultaneously, and a battery of investigations including ultrasonography was performed on all the patients with RSM. RESULTS: There was no significant difference in the mean fasting glucose for the study and control patients (5.5 +/- 0.7 vs. 5.3 +/- 0.6 mmol/dl) and in the mean fasting insulin (15.0 +/- 5.1 and 12.9 +/- 2.9 mU/l). There was a significant difference between the mean parity of the study and control patients (0.7 +/- 0.7 vs. 1.7 +/- 0.5, p < 0.0001) and also the mean number of miscarriages (4.2 +/- 1.2 vs. 0.1 +/- 0.4, p < 0.0001). There was, however, no significant difference in the insulin resistance of both groups as calculated using the Homeostasis Model Assessment and the fasting glucose insulin ratio of <4.5. CONCLUSION: Insulin resistance was not significantly associated with RSM in our study. Further studies may be needed.

The effect of obesity on the outcome of infertility management in women with polycystic ovary syndrome.

INTRODUCTION: Obesity has become a worldwide epidemic with ever increasing incidence and public health problems in both developing and developed countries. OBJECTIVE: The objective of the study is to investigate the incidence of obesity among patients with polycystic ovarian syndrome attending infertility clinic and the effect on treatment outcome. METHODOLOGY: Two hundred and seventy women with polycystic ovarian syndrome attending the infertility clinic were evaluated clinically, biochemically, and laparoscopically. They were stratified according to their body mass index (BMI) as follows: normal weight: 18-24; overweight: 25-29, obese:30-34, and grossly obese: > or = 35. Therapy included induction of ovulation with clomiphene citrate and gonadotrophins. The patients were followed up through during induction of ovulation and pregnancy. RESULTS: There were ethnic differences in mean BMI. Significantly more obese women had oligomenorrhoea (p<0.01) and anovulation (p<0.01) than women with normal weight. Obesity adversely affected the outcome of ovulation induction with clomiphene citrate and gonadotrophins; 79% of women with BMI 18-24 ovulated at 6 months compared to 15.3% in those with BMI 30-34 (p<0.001) and 11.8% in women with BMI > or = 35 (p<0.001). The pregnancy rate and outcome were also adversely affected by obesity. CONCLUSION: Obesity has a negative impact on the outcome of treatment of infertility. Weight reduction programme should be an essential component of infertility management.

Measurement of inhibin A and inhibin pro-alphaC in early human pregnancy and their role in the prediction of pregnancy outcome in patients with recurrent pregnancy loss.

OBJECTIVE: To examine the temporal relationship among inhibin A, beta-hCG, and pro-alphaC in early pregnancy and to determine whether the measurement of these hormones has any role in prediction of pregnancy outcome in patients with recurrent spontaneous miscarriage. DESIGN: Prospective descriptive study. SETTING: A tertiary referral center for recurrent miscarriage. PATIENT(S): Thirty-six pregnant women with previous history of recurrent spontaneous pregnancy loss. INTERVENTION(S): Serial blood samples were collected prospectively at 6, 8, 10, and 12 weeks of gestation and were analyzed for inhibin A and inhibin pro-alphac using a two-site enzyme-linked immunosorbent assay as well as for beta-hCG using the microparticle enzyme immunoassay. MAIN OUTCOME MEASURE(S): Serum levels of inhibin A, inhibin pro-alphac, and beta-hCG. RESULT(S): The patients were allocated to two groups according to the pregnancy outcome: group 1 consisted of patients whose pregnancy continued beyond 20 weeks (control group); and group 2 consisted of patients who spontaneously aborted (aborted group). There was a significant difference in inhibin A concentrations between the control and aborted groups at 8, 10, and 12 weeks' gestation. Significant differences in beta-hCG concentrations between the two groups is evident only at 10 weeks' gestation. There were no significant differences in inhibin pro-alphac concentrations between the two groups at any gestational age. Assessment of the trend in the control group over the study period showed a significant increase in inhibin A and beta-hCG but not inhibin pro-alphac levels. CONCLUSION(S): Low serum levels of inhibin A at early gestational age in pregnancies destined to miscarry suggest a role for this glycoprotein as a marker for early pregnancy viability. Its measurement at the time of the first pregnancy test might be able to predict pregnancy outcome.

Differential expression of T-helper cytokines in the peritoneal fluid of women with normal ovarian cycle compared with women with chronic anovulation.

BACKGROUND: Estrogen and progesterone immunoregulate the genital environment by expression of cytokines and growth factors. OBJECTIVE: To investigate the pattern of expression of T-helper cytokines during the ovarian cycle compared with women with chronic anovulation resistant to clomiphene citrate. HYPOTHESIS: Expression of T-helper cytokines in women with chronic anovulation may be different from the pattern in women with a normal ovarian cycle. METHODS: We evaluated 31 infertile women having laparoscopy for evaluation of tubal patency and evidence of ovulation in two groups during (a) the luteal phase (17 women) and (b) the follicular phase (14 women). A third group was composed of 14 women with polycystic ovarian syndrome, but they were resistant to clomiphene citrate for induction of ovulation and had laparoscopic ovarian cautery. Peritoneal fluid was collected during laparoscopy. Estimation of T-helper cytokine interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, IL-4 and IL-6 in serum, peritoneal fluid and culture of the peritoneal mononuclear cells was performed by ELISA. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, estradiol and progesterone were evaluated by the Vidas Parametric System. RESULTS: The LH : FSH ratio was significantly higher in the women with polycystic ovaries than in the ovulatory groups. IL-2 and IFN-gamma were more highly expressed in the follicular phase but the T-helper 2 cytokines IL-4 and IL-6 predominated in the luteal phase, serum, peritoneal fluid and culture of the peritoneal mononuclear cells. From the follicular to the mid-luteal phase, IL-6 increased three to fivefold in the serum and peritoneal fluid, but there was low expression with anovulation. CONCLUSIONS: The peritoneal fluid levels of IL-4 and IL-6 are higher in the luteal phase. Low IL-6 levels in chronic anovulation may be a marker of resistance to clomiphene citrate.

The aetiology and pattern of recurrent pregnancy loss.

The aims of this study were to determine the aetiological factors and the pattern of recurrent pregnancy loss in Kuwait. Ninety consecutive patients attending the special recurrent miscarriage clinic were studied prospectively. A comprehensive history of all previous miscarriages and pregnancies, past medical and gynaecological events were established. A physical examination was performed. Extensive investigations were performed. Pregnancies which occurred during the study were monitored carefully. The mean age of the patients was 30.46+/-6.04 years. The patients were subdivided into the groups of secondary (57%) and primary (43%) recurrent miscarriages. Eighty-five per cent of all previous miscarriages occurred in the first trimester. The main aetiological factors were uterine anomaly 2.2%, chromosome anomaly (parental) 2.2%, PCOS, infections and other miscellaneous factors 21.1%, positive antiphospholipid antibodies 33.3% and unexplained in 35.6%. The overall live birth rate was 82% and maternal morbidity was low. Positive antiphospholipid antibodies are the most frequently associated cause of recurrent pregnancy loss in Kuwait.

Successful transvaginal ultrasound-guided ablation of a cervical pregnancy in a patient with simultaneous intrauterine pregnancy after in vitro fertilization and embryo transfer.

Heterotopic pregnancy, or simultaneous intrauterine and extrauterine gestation, is a relatively rare condition. However, induced ovulation and assisted reproductive technologies have markedly increased the incidence of this condition. In this article, a case of heterotopic pregnancy after in vitro fertilization and embryo transfer is presented in which the viable cervical pregnancy was treated by transvaginal ultrasound-guided puncture and injection of potassium chloride in conjunction with methotrexate at week 6 of gestation. At week 12 of gestation, the intrauterine gestation was viable and complete resorption of the cervical pregnancy had occurred. At week 30 of gestation, a healthy baby was delivered by Caesarian section after prelabour rupture of membranes.