Growth factor expression by human oviduct and buffalo rat liver coculture cells.

OBJECTIVE: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth. DESIGN: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors. SETTING: Academic tertiary care hospital. PATIENT(S): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts. INTERVENTION(S): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5\u27 end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography. RESULT(S): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF. CONCLUSION(S): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development

Increased incidence of aberrant morphological phenotypes in human embryogenesis--an association with endometriosis.

PURPOSE: After noticing specific aberrant forms of development in preimplantation embryos from women with endometriosis, we embarked upon this study in order to examine the frequency of these events as compared with controls. METHODS: A total of 235 embryos representing 30 women undergoing 56 cycles of in vitro fertilization were retrospectively analyzed on videotape and placed into categories based upon the incidence of specific morphological criteria found during observations made on the first and second days following oocyte retrieval. RESULTS: Several of the aberrant nuclear and cytoplasmic events were found to be statistically increased in the group of embryos from women with endometriosis. CONCLUSIONS: The incidence of defined aberrant phenotypes in embryos from women with laparoscopically documented endometriosis may allow us to identify specific embryotoxic events with reference to endometriosis and other diagnoses

Use of hyaluronan in the selection of sperm for intracytoplasmic sperm injection (ICSI): Significant improvement in clinical outcomes-multicenter, double-blinded and randomized controlled trial

Study Question Does the selection of sperm for ICSI based on their ability to bind to hyaluronan improve the clinical pregnancy rates (CPR) (primary end-point), implantation (IR) and pregnancy loss rates (PLR)? Summary Answer In couples where ≤65% of sperm bound hyaluronan, the selection of hyaluronan-bound (HB) sperm for ICSI led to a statistically significant reduction in PLR.WHAT IS KNOWN AND WHAT THIS PAPER ADDSHB sperm demonstrate enhanced developmental parameters which have been associated with successful fertilization and embryogenesis. Sperm selected for ICSI using a liquid source of hyaluronan achieved an improvement in IR. A pilot study by the primary author demonstrated that the use of HB sperm in ICSI was associated with improved CPR. The current study represents the single largest prospective, multicenter, double-blinded and randomized controlled trial to evaluate the use of hyaluronan in the selection of sperm for ICSI.DESIGNUsing the hyaluronan binding assay, an HB score was determined for the fresh or initial (I-HB) and processed or final semen specimen (F-HB). Patients were classified as \u3e65% or ≤65% I-HB and stratified accordingly. Patients with I-HB scores ≤65% were randomized into control and HB selection (HYAL) groups whereas patients with I-HB \u3e65% were randomized to non-participatory (NP), control or HYAL groups, in a ratio of 2:1:1. The NP group was included in the \u3e65% study arm to balance the higher prevalence of patients with I-HB scores \u3e65%. In the control group, oocytes received sperm selected via the conventional assessment of motility and morphology. In the HYAL group, HB sperm meeting the same visual criteria were selected for injection. Patient participants and clinical care providers were blinded to group assignment.PARTICIPANTS AND SETTINGEight hundred two couples treated with ICSI in 10 private and hospital-based IVF programs were enrolled in this study. Of the 484 patients stratified to the I-HB \u3e 65% arm, 115 participants were randomized to the control group, 122 participants were randomized to the HYAL group and 247 participants were randomized to the NP group. Of the 318 patients stratified to the I-HB ≤ 65% arm, 164 participants were randomized to the control group and 154 participants were randomized to the HYAL group. Main Results and the Role of Chance HYAL patients with an F-HB score ≤65% demonstrated an IR of 37.4% compared with 30.7% for control [n = 63, 58, P \u3e 0.05, (95% CI of the difference-7.7 to 21.3)]. In addition, the CPR associated with patients randomized to the HYAL group was 50.8% when compared with 37.9% for those randomized to the control group (n = 63, 58, P \u3e 0.05). The 12.9% difference was associated with a risk ratio (RR) of 1.340 (RR 95% CI 0.89-2.0). HYAL patients with I-HB and F-HB scores ≤65% revealed a statistically significant reduction in their PLR (I-HB: 3.3 versus 15.1%, n = 73, 60, P = 0.021, RR of 0.22 (RR 95% CI 0.05-0.96) (F-HB: 0.0%, 18.5%, n = 27, 32, P = 0.016, RR not applicable due to 0.0% value) over control patients. The study was originally planned to have 200 participants per arm providing 86.1% power to detect an increase in CPR from 35 to 50% at a = 0.05 but was stopped early for financial reasons. As a pilot study had demonstrated that sperm preparation protocols may increase the HB score, the design of the current study incorporated a priori collection and analysis of the data by both the I-HB and the F-HB scores. Analysis by both the I-HB and F-HB score acknowledged the potential impact of sperm preparation protocols.BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTIONSelection bias was controlled by randomization. Geographic and seasonal bias was controlled by recruiting from 10 geographically unique sites and by sampling over a 2-year period. The potential for population effect was controlled by adjusting for higher prevalence rates of \u3e65% I-HB that naturally occur by adding the NP arm and to concurrently recruit \u3e65% and ≤65% I-HB subjects. Monitoring and site audits occurred regularly to ensure standardization of data collection, adherence to the study protocol and subject recruitment. Subgroup analysis based on the F-HB score was envisaged in the Study Design .GENERALIZABILITY TO OTHER POPULATIONSThe study included clinics using different sperm preparation Method s, located in different regions of the USA and proceeded in every month of the year. Therefore, the results are widely applicable. Study Funding/Competing Interest (S)This study was funded by Biocoat, Inc., Horsham, PA, USA. The statistical analysis plan and subsequent analyses were performed by Sherrine Eid, a biostatistician. The manuscript was prepared by Kathryn C. Worrilow, Ph.D. and the study team members. Biocoat, Inc. was permitted to review the manuscript and suggest changes, but the final decision on content was exclusively retained by the authors. K.C.W is a scientific advisor to Biocoat, Inc. S.E. is a consultant to Biocoat, Inc. D.W. has nothing to disclose. M.P., S.S., J.W., K.I., C.K. and T.E. have nothing to disclose. G.D.B. is a consultant to Cooper Surgical and Unisense. J.L. is on the scientific advisory board of Origio. Trial Registration Number NCT00741494. © 2012 The Author

Additional loss of MSH2 and MSH6 expression in sporadic deficient mismatch repair colorectal cancer due to MLH1 promoter hypermethylation

Colorectal cancer (CRC) is common with 3% of cases associated with germline mutations in the mismatch repair pathway characteristic of Lynch syndrome (LS). The UK National Institute for Health and Care Excellence recommends screening for LS in all patients newly diagnosed with CRC, irrespective of age. The Yorkshire Cancer Research Bowel Cancer Improvement Programme includes a regional LS screening service for all new diagnoses of CRC. In the first 829 cases screened, 80 cases showed deficient mismatch repair (dMMR) including four cases showing areas with loss of expression of all four mismatch repair proteins by immunohistochemistry. The cases demonstrated diffuse MLH1 loss associated with BRAF mutations and MLH1 promoter hypermethylation in keeping with sporadic dMMR, with presumed additional double hit mutations in MSH2+/−MSH6 rather than underlying LS. Recognition and accurate interpretation of this unusual phenotype is important to prevent unnecessary referrals to clinical genetics and associated patient anxiety