Proteomic Profiling Identifies Candidate Diagnostic Biomarkers of Hydrosalpinx in Endometrial Fluid: A Pilot Study.

Hydrosalpinx is a fluid occlusion and distension of the fallopian tubes, often resulting from pelvic inflammatory disease, which reduces the success of artificial reproductive technologies (ARTs) by 50%. Tubal factors account for approximately 25% of infertility cases, but their underlying molecular mechanisms and functional impact on other reproductive tissues remain poorly understood. This proteomic profiling study applied sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to study hydrosalpinx cyst fluid and pre- and post-salpingectomy endometrial fluid. Among the 967 proteins identified, we found 19 and 17 candidate biomarkers for hydrosalpinx in pre- and post-salpingectomy endometrial fluid, respectively. Salpingectomy significantly affected 76 endometrial proteins, providing insights into the enhanced immune response and inflammation present prior to intervention, and enhanced coagulation cascades and wound healing processes occurring one month after intervention. These findings confirmed that salpingectomy reverses the hydrosalpinx-related functional impairments in the endometrium and set a foundation for further biomarker validation and the development of less-invasive diagnostic strategies for hydrosalpinx.

Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells.

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.

Cervical pregnancy in assisted reproduction: an analysis of risk factors in 91,067 ongoing pregnancies.

RESEARCH QUESTION: What is the frequency of cervical pregnancy in women undergoing assisted reproductive technologies (ART) and what are the risk factors? DESIGN: Case-control study of women undergoing assisted reproductive technology (ART) at 25 private assisted reproduction clinics run by the same group in Spain. Two control groups (tubal ectopic pregnancies and intrauterine pregnancies) were established. The main outcome measure was frequency of cervical pregnancy. Demographic, clinical factors and IVF parameters were assessed for their influence on cervical pregnancy risk. RESULTS: Thirty-two clinical pregnancies were achieved out of 91,067 ongoing pregnancies, yielding a rate of 3.5/10,000. Cervical pregnancies represented 2.02% of all ectopic pregnancies (32/1582). The main risk factors two or more previous pregnancies (OR 2.68; CI 1.18 to 6.07); two or more previous miscarriages (OR 4.21; CI1.7 to 10.43), one or more previous curettages (OR 3.99, CI 1.67 to 9.56), two or more previous curettages (OR 4.71; CI 1.19 to 18.66) and smoking (OR 2.82 CI 1.14 to 6.94). History of caesarean sections and tubal pregnancy was not associated with an elevated cervical pregnancy risk. Infertility conditions and endometrial thickness were similar across the three groups. The proportion of women from whom fewer than 10 oocytes were retrieved was higher in the clinical pregnancy group than in the IUP group. CONCLUSIONS: In ART, the main risk factors for cervical ectopic pregnancy are a history of at least two pregnancies, miscarriages, at least one curettage and smoking. IVF parameters do not seem to influence the development of clinical pregnancies. Cervical pregnancies are less common in ART than previously reported, attributable to improvements in ART; a publication bias in early IVF reports cannot be ruled out.

Proteomic pattern of implantative human endometrial fluid in in vitro fertilization cycles.

PURPOSE: To assess whether there are proteins in endometrial fluid aspirate (EFA) that predict implantation. METHODS: The population under study consisted of 285 women undergoing embryo transfer (ET). Endometrial fluid aspiration was performed immediately before ET. Results of proteomic analysis of EFA were compared between 33 cases who achieved pregnancy and 33 who did not. Samples were analysed by 2D electrophoresis and mass spectrometry. Blood samples were studied by ELISA Pregnancy rates and maternal complications were compared to those in women refusing aspiration. RESULTS: We found 23 proteins differentially expressed in the EFA in conception cycles: 4 up-regulated proteins and 19 down-regulated (FC = 0.31 0.78) (among others, arginase-1, actin B, PARK-7, cofilin-1, stathmin, annexin-2 and CAPZB). Among the five studied proteins that were differentially expressed in EFA, none was differentially expressed in serum. The aspiration procedure had no impact on pregnancy rate. No maternal complications were reported. CONCLUSIONS: We found a very different protein profile in implantative cycles, the majority of proteins being down-regulated. This probably reflects a different endometrial functional status, more favourable to implantation. EFA proteomic analysis could be a useful tool in the planning ET strategies.

In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.

OBJECTIVE: To determine the clinical value of preimplantation genetic diagnosis for aneuploidy screening (PGD-A) in women of advanced maternal age (AMA; between 38 and 41 years). DESIGN: This was a multicenter, randomized trial with two arms: a PGD-A group with blastocyst transfer, and a control group with blastocyst transfer without PGD-A. SETTING: Private reproductive centers. PATIENT(S): A total of 326 recruited patients fit the inclusion criteria, and 205 completed the study (100 in the PGD-A group and 105 in the control group). INTERVENTION(S): Day-3 embryo biopsy, array comparative genomic hybridization, blastocyst transfer, and vitrification. MAIN OUTCOME MEASURE(S): Primary outcomes were delivery and live birth rates in the first transfer and cumulative outcome rates. RESULT(S): The PGD-A group exhibited significantly fewer ETs (68.0% vs. 90.5% for control) and lower miscarriage rates (2.7% vs. 39.0% for control). Delivery rate after the first transfer attempt was significantly higher in the PGD-A group per transfer (52.9% vs 24.2%) and per patient (36.0% vs. 21.9%). No significant differences were observed in the cumulative delivery rates per patient 6 months after closing the study. However, the mean number of ETs needed per live birth was lower in the PGD-A group compared with the control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). CONCLUSION(S): Preimplantation genetic diagnosis for aneuploidy screening is superior compared with controls not only in clinical outcome at the first ET but also in dramatically decreasing miscarriage rates and shortening the time to pregnancy.

Lipidomic profiling of endometrial fluid in women with ovarian endometriosis†.

The proteomic content of the endometrial fluid (EF) from patients with endometriosis has been investigated, but the lipidomic profile has not been analyzed yet in detail.This study is a comparative untargeted lipidomic analysis of human EF obtained from 35 patients (12 endometriosis and 23 controls). Global differential lipidomic profile was analyzed in both groups by ultrahigh performance liquid chromatography coupled to mass spectrometry. A total of 123 out of the 457 metabolites identified in the EF were found to be significantly differentially expressed between ovarian endometriosis (OE) versus controls. Univariate statistical analysis showed reduced levels of saturated diacylglycerols and saturated triacylglycerols in endometriosis patients. A predictive model was generated using the 123 differentially expressed metabolites. Using this model, we were able to correctly classify 86% of the samples. This study identified the lipidomic profile in the EF associated with OE, suggesting that EF analysis could be considered as a minimally invasive approach for the diagnosis of endometriosis. In conclusion, the lipidomic profile of the EF is different between samples from patients with OE and controls.

Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

OBJECTIVES: To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. STUDY DESIGN: PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). RESULTS: We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. CONCLUSIONS: The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation.

Female obesity impairs in vitro fertilization outcome without affecting embryo quality.

OBJECTIVE: To compare embryo quality and reproductive outcome in our IVF program according to the women's body mass index (BMI). DESIGN: Retrospective study. SETTING: University-affiliated infertility clinic, between January 2001 and April 2007. PATIENT(S): Women undergoing a total of 6,500 IVF-intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): Six thousand five hundred IVF-ICSI cycles were included and divided into four groups: lean (<20 kg/m(2); n = 1,070; 16.5%); normal (20-24.9 kg/m(2); n = 3,930; 60.5%); overweight (25-29.9 kg/m(2); n = 1,081; 16.6%); and obese (> or =30 kg/m(2); n = 419; 6.4%). MAIN OUTCOME MEASURE(S): Comparison of embryo quality and reproductive outcome (implantation, pregnancy, miscarriage, and live birth rates) among BMI groups. RESULT(S): No difference in insemination procedure, fertilization rate, day of ET, mean number of transferred and cryopreserved embryos, percentage of blastocyst transfers, or embryo quality on day 2 and 3 was found among groups. However, implantation, pregnancy, and live birth rates were poorer in obese women. In fact, pregnancy and live birth rates were reduced progressively with each unit of BMI (kilograms per square meter) with a significant odds ratio of 0.984 (95% confidence interval 0.972-0.997) and 0.981 (95% confidence interval 0.967-0.995), respectively. In addition, the cumulative pregnancy rate after four IVF cycles was reduced as BMI increased. CONCLUSION(S): Female obesity impairs IVF outcome, but embryo quality is not affected, pointing to an alteration in the uterine environment.