Frank D. Yelian

Effects of tumor necrosis factor-alpha on human trophoblast cell adhesion and motility

PROBLEM: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor‐alpha (TNF‐α) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF‐α may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF‐α on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion.

The prevalence of ureaplasma urealyticum, mycoplasma hominis, chlamydia trachomatis and neisseria gonorrhoeae infections, and the rubella status of patients undergoing an initial infertility evaluation

PurposeTo determine the prevalence of positive test for Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) infections, and their corresponding Rubella status when undergoing workup for infertility.MethodsRetrospective chart review to determine infection status for UU, MH, CT, and NG as determined by cervical swab, as well as the serum Rubella antibody titer.ResultsA total of 46 patients of the patients reviewed were positive for UU (20.1%), three patients were positive for MH (1.3%), five patients were positive for CT (2.2%) and one patient was positive for NG (0.4%). Rubella immunity was confirmed in 90.3% of patients.ConclusionApproximately one quarter of women presenting to an infertility clinic seeking to conceive were found to have a positive test for UU, MH, CT or NG infection. Additionally, almost 10% of the patients were Rubella non-immune at the time of presentation for infertility evaluation.

Ethanol-induced expression of cytokines in a first-trimester trophoblast cell line.

OBJECTIVES Altered cytokine expression at the fetoplacental interface may be a potential mechanism for the development of fetal immune dysfunction in children with fetal alcohol syndrome. This study was conducted to determine whether first-trimester trophoblasts respond to ethanol exposure by the induction of specific cytokines. STUDY DESIGN HTR-8/SVneo trophoblast cells were cultured in vitro in the presence of either ethanol (0.5% [vol/vol]), lipopolysaccharide (1 microg/mL), or ethanol and lipopolysaccharide. Expression of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 was examined by Northern analysis and enzyme-linked immunosorbent assay. RESULTS Culture in the presence of ethanol, lipopolysaccharide, or lipopolysaccharide and ethanol resulted in the increased transcription and secretion of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 at significantly greater levels (P < .01) than control cultures. CONCLUSIONS Human first-trimester trophoblasts express high levels of cytokines when cultured in the presence of ethanol. Trophoblasts may therefore be an important exogenous source of cytokines for the fetus, and altered cytokine levels during early gestation may have an adverse effect on the development of the fetal immune system.

Repair kinetics of amnion cells after microsurgical injury.

OBJECTIVE There is a paucity of data regarding healing of fetal membranes. We assessed the repairing ability of a microsurgically-injured monolayer of amnion-derived cells compared to an endothelial cell line. MATERIALS AND METHODS Amnion-derived cells (FL cells) were grown to confluence in 10% fetal calf serum (FCS). A microsurgical incision was performed under an inverted microscope. The area of each incision was measured at times 0, 6, 12 and 24 h using MCID M4 image analysis software. The experiments were repeated using 1% FCS. The observations were also made replacing the media at 6 and 12 h. Umbilical-cord-derived endothelial cells were used for comparison. The rate of repair of the incision was measured using three methods: (a) absolute closure rate (ACR): (A0-A1)/T, where A0 is the original incisional area, and A1 is the new area at interval T: (b) relative percentage rate (RPR): (A0-A1)/A0/T x 100, and (c) healing rate (HR): (A0-A1)/A0 x 100. RESULTS Amnion cells were capable of repairing the microsurgical defect. The ACR decreased over time, and was higher with larger defects. A lower concentration of FCS nor the addition of fresh media altered the repair process significantly. Endothelial cells were significantly faster than amnion cells or amnion-supplemented cells with endothelial media. The RPR remained relatively constant for all groups, suggesting Gompertzian kinetics. CONCLUSIONS Amnion cells are capable of repairing a surgical defect at a relatively constant percent rate, but are slower than an endothelial cell line. The decreased rate of closure over time may be due to relative contact inhibition. Further studies will concentrate on the molecular and cellular mechanisms involved in this process.

Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study

BACKGROUND Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy. METHODS Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells. RESULTS Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value. CONCLUSIONS This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

Endometrial Thickness Dynamics and Morphologic Characteristics During Pituitary Downregulation With Antagonists in Assisted Reproductive Technology Cycles

Objective. The purpose of this study was to evaluate whether the dynamics of endometrial stripe thickness during gonadotropin‐releasing hormone (GnRH) antagonist pituitary downregulation in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles are related to implantation and pregnancy outcomes. Methods. This retrospective cohort study evaluated 115 conventional IVF/ICSI cycles. All patients underwent ovarian stimulation with gonadotropins and the GnRH antagonist ganirelix acetate. The endometrial stripe was measured transvaginally daily from the day of initial GnRH antagonist administration to the day of the human chorionic gonadotropin (hCG) trigger and then transabdominally on the day of embryo transfer. We created 5 categories (0–4) of endometrial thickness variation, considering significant a daily variation of 1.5 mm. Our aim was to predict whether the endometrial thickness dynamics or morphologic characteristics were related to the duration of ovarian stimulation, duration of ganirelix use, or estradiol levels during ovarian stimulation and whether they would influence implantation and pregnancy rates. Results. No relationship was found between the duration of ovarian stimulation, duration of ganirelix use, and estradiol level (expressed as the area under the curve), and endometrial thickness dynamics or morphologic characteristics. Despite a thinner endometrial thickness in 37% of the cycles on the day of the hCG trigger compared with the beginning of GnRH antagonist stimulation, there was no correlation between endometrial dynamics and pregnancy outcomes. There was, instead, a positive relationship between a trilaminar endometrial morphologic pattern with a positive pregnancy test result, successful implantation, and ongoing pregnancy (P < .05). Conclusions. Despite a net decrease in thickness in almost 50% of cases, endometrial dynamics did not correlate with pregnancy outcomes. Conversely, a trilaminar endometrial morphologic pattern on the day of embryo transfer was positively related to pregnancy outcomes.

Adrenomedullin Levels in Normal and Preeclamptic Pregnancy at Term

Objective: To describe maternal plasma levels of adrenomedullin (AM), a hypotensive and natriuretic peptide, in normal and preeclamptic women at term. Study design: Maternal plasma AM levels were determined in 13 preeclamptic and 15 normotensive primigravidas by radioimmunoassay. Plasma samples were obtained with the patients in the lateral recumbent position before the administration of any medications. Results: Women with preeclampsia had significantly elevated AM levels when compared with normotensive controls (42.3 ± 10.5 pg/mL versus 16.9 ± 3.1 pg/mL, P < .011). Conclusion: In this pilot study, AM levels were significantly increased at term in preeclamptic women.

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration *

Objective To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. Design and Methods Human semen samples were treated with 1 to 1,000 µ M cocaine hydrochloride for up to 2hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. Results After a short exposure (15minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. Conclusion Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Successful pregnancies with uterine leiomyomas and myomectomy at the time of caesarean section

Objective To ascertain the impact of uterine leiomyomas on pregnancy outcome, and to determine the effectiveness of myomectomy at the time of caesarean delivery. Methods A retrospective study was conducted on pregnant women with uterine leiomyomas. Clinical information including the course of the pregnancy, mode of delivery, pathology findings, and postpartum course were extracted from medical records and analysed for statistical significance. Results There were 50 pregnancies associated with uterine leiomyomas. During pregnancy, the leiomyomas grew >2 cm in 46% of cases. Only 6% were affected by symptoms of red degeneration. There were 2% mid trimester inductions of labour, 6% vaginal deliveries, and 92% caesarean sections. Of the 46 caesarean sections, 47% were due to obstetrical factors and 89% of patients underwent myomectomy at the time of caesarean delivery. The mean blood loss from myomectomy at the time of caesarean section was 260 ml (200–700 ml), and 5% of patients who underwent myomectomy were transfused. Conclusions It is possible to carry a pregnancy successfully to term when the pregnancy is complicated by uterine leiomyomas. When caesarean delivery is needed, myomectomy can be performed at the time of caesarean section routinely without significant complications.

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion.