Sperm proteomic changes associated with early embryo quality after ICSI.

RESEARCH QUESTION: Do alterations of human sperm protein profile affect embryo quality? DESIGN: Sperm proteins from 27 infertile couples undergoing intracytoplasmic sperm injection (ICSI) were extracted and digested. The resulting peptides were labelled using tandem mass tags, separated by two-dimensional liquid chromatography, and identified and quantified using tandem mass spectrometry. Subsequently, sperm protein and peptide abundance were statistically analysed for correlation with ICSI-derived embryo quality in the subset of idiopathic infertile couples. Detected correlations were further assessed in the subset of infertile patients with a known factor. RESULTS: The abundance of 18 individual sperm proteins was found to correlate with embryo quality after ICSI. Of note, a high percentage of poor-quality ICSI-derived embryos was associated with alterations in several components of the eight-membered chaperonin-containing T-complex, which plays an important role in the folding of many essential proteins. Additionally, the abundance of sperm proteins with known functions in embryogenesis, such as RUBVL1, also correlated with early embryo quality (r = -0.547; P = 0.028). Some of the correlations found in this study were validated using either proteomic data from infertile patients with a known factor or data from similar published studies. Analysis at the peptide level revealed the association of some correlations with specific post-translational modifications or isoforms. CONCLUSIONS: Our results support the hypothesis that the sperm proteome plays a role in early embryogenesis. Moreover, several sperm proteins have emerged as potential biomarkers that could predict the outcome of in-vitro assisted reproductive technologies, leading to the possibility of improved diagnosis of couples with idiopathic infertility.

Increased circulating cell-derived microparticle count is associated with recurrent implantation failure after IVF and embryo transfer.

Cell-derived microparticles (cMPs) are small membrane vesicles that are released from many different cell types in response to cellular activation or apoptosis. Elevated cMP counts have been found in almost all thrombotic diseases and pregnancy wastage, such as recurrent spontaneous abortion and in a number of conditions associated with inflammation, cellular activation and angiogenesis. cMP count was investigated in patients experiencing unexplained recurrent implantation failure (RIF). The study group was composed of 30 women diagnosed with RIF (RIF group). The first control group (IVF group) (n = 30) comprised patients undergoing a first successful IVF cycle. The second control group (FER group) included 30 healthy women who had at least one child born at term and no history of infertility or obstetric complications. cMP count was significantly higher in the RIF group compared with the IVF and FER groups (P < 0.05 and P < 0.01, respectively) (RIF group: 15.8 ± 6.2 nM phosphatidylserine equivalent [PS eq]; IVF group: 10.9 ± 5.3 nM PS eq; FER group: 9.6 ± 4.0 nM PS eq). No statistical difference was found in cMP count between the IVF and FER groups. Increased cMP count is, therefore, associated with RIF after IVF and embryo transfer.

Assisted reproductive technologies are associated with cardiovascular remodeling in utero that persists postnatally.

BACKGROUND: Assisted reproductive technologies (ARTs) have been shown to be associated with general vascular dysfunction in late childhood. However, it is unknown whether cardiac remodeling is also present and if these changes already manifest in prenatal life. Our aim was to assess fetal and infant (6 months of age) cardiovascular function in ART pregnancies. METHODS AND RESULTS: This prospective cohort study included 100 fetuses conceived by ART and 100 control pregnancies. ART fetuses showed signs of cardiovascular remodeling, including a more globular heart with thicker myocardial walls, decreased longitudinal function (tricuspid ring displacement in controls: median, 6.5 mm [interquartile range, 6.1-7.1 mm]; tricuspid ring displacement in ART: 5.5 mm [interquartile range, 5.1-6.1]; P<0.001), impaired relaxation, and dilated atria (atrial area in controls, 1.46 cm(2) [interquartile range, 1.2-1.5 cm(2)]; atrial area in ART, 1.6 cm(2) [interquartile range, 1.3-1.8 cm(2)]; P<0.001). Additionally, ART infants showed persistence of most cardiac changes and a significant increase in blood pressure and aortic intima-media thickness (systolic blood pressure in controls, 74 mm Hg [interquartile range, 67-83 mm Hg]; systolic blood pressure in ART, 83 mm Hg [interquartile range, 75-94 mm Hg]; P<0.001; aortic intima-media thickness in controls, 0.52 mm [interquartile range, 0.45-0.56 mm]; aortic intima-media thickness in ART, 0.64 mm [interquartile range, 0.62-0.67]; P<0.001). We could not demonstrate that our findings were directly caused by ART because of their association with various confounding factors, including intrauterine growth restriction or factors related to the cause of infertility. CONCLUSIONS: Children conceived by ART manifest cardiac and vascular remodeling that is present in fetal life and persists in postnatal life, suggesting opportunities for early detection and potential intervention. The underlying mechanisms and the effect of potential confounders such as growth restriction or prematurity remain to be elucidated.

Reduced plasma fibrinolytic potential in patients with recurrent implantation failure after IVF and embryo transfer.

BACKGROUND: Recurrent implantation failure (RIF) following embryo transfer (ET) is a major continuing problem in IVF. Women with haemostatic defects may be at increased risk of miscarriage and preclinical pregnancy loss. The fibrinolytic system is considered, at present, the key to new thrombotic pathogenic mechanisms. Patients with unexplained recurrent miscarriage have an impairment of fibrinolysis, as demonstrated by prolonged clot lysis time (CLT) in association with increased plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI). In this study, we investigated fibrinolytic potential in patients with RIF. METHODS: Three groups of patients were studied: 30 women with RIF (RIF group), 60 patients undergoing a first successful IVF-ET cycle (IVF group) and 60 healthy fertile women (FER group). Plasma CLT was measured using a global fibrinolysis assay. TAFI antigen plasma levels and polymorphisms in the TAFI gene (+505A/G and +1542C/G) were analysed using enzyme-linked immunosorbent assay and allele-specific PCR, respectively. RESULTS: CLT was significantly longer (P < 0.0001 and P < 0.0009, respectively) and TAFI antigen levels were significantly higher (both P < 0.0001) in the RIF versus the IVF and FER groups. A direct relationship between CLT and TAFI antigen levels (r = 0.40; P = 0.001) was detected in the whole study population. There were no differences in distribution of TAFI polymorphisms between groups. CONCLUSIONS: Patients with RIF have reduced plasma fibrinolytic potential, as shown by a prolonged CLT, and this may be explained, at least in part, by increased TAFI antigen levels.

Increased messenger ribonucleic acid expression of the cyclin-dependent kinase inhibitor p27Kip1 in cleavage-stage human embryos exhibiting developmental arrest.

OBJECTIVE: To quantify p27 messenger RNA (mRNA) levels in human arrested and normally developing embryos and nonfertilized oocytes to determine whether the p27 protein abundance, reported in cleavage-stage embryos exhibiting developmental arrest, is regulated at the mRNA expression level. DESIGN: Real-time reverse transcription quantitative polymerase chain reaction was used to quantify the expression of p27 in three samples: arrested embryos (group A, n = 29), normally developing embryos (group D, n = 34), and nonfertilized oocytes (group O, n = 20). SETTING: Research laboratory working closely with a clinical IVF practice. PATIENT(S): Oocytes and embryos were obtained from patients undergoing assisted fertilization. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantification of mRNA transcripts. RESULT(S): The amount of p27 mRNA was statistically significantly higher in group A (mean +/- SEM, 86,143 +/- 42,496 relative units [RU]) compared with groups D (10,680 +/- 3,850 RU) and O (3,555 +/- 1,458 RU). Furthermore, in a group of 13 two- to four-cell arrested embryos, high levels of p27 mRNA (51,481 +/- 31,120 RU) were found in comparison with the nonfertilized oocyte group (3,555 +/- 1,458 RU). CONCLUSION(S): Cleavage-stage human embryos exhibiting developmental arrest show increased p27 mRNA expression. This probably is due to increased transcriptional activity.

Air in the transfer catheter does not affect the success of embryo transfer.

OBJECTIVE: To investigate the effect on embryo transfer (ET) success of air loaded into the transfer catheter to bracket the embryo-containing medium. DESIGN: Prospective, randomized study. SETTING: University teaching hospital. PATIENT(S): One hundred two consecutive patients undergoing ET after IVF. INTERVENTION(S): In group 1 (n = 52), embryos were loaded as follows: 200 microL of air in the syringe, 100-125 microL of air in the proximal part of the catheter, 20-25 microL of medium containing the embryos to be transferred, and 10 microL of air at the tip of the catheter. In group 2 (n = 50), the syringe and the entire catheter were filled with medium and the embryo-containing medium (20-25 microL) was aspirated without being bracketed by air spaces. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): No differences were found between groups 1 and 2 with respect to implantation and pregnancy rates. CONCLUSION(S): The air loaded into the transfer catheter to bracket the embryo-containing medium has no negative effect on ET success.

Ultrasonography as a predictor of embryo implantation after in vitro fertilization: a controlled study.

OBJECTIVE: To investigate the predictive value of ultrasonographic parameters as prognostic indicators of implantation after IVF when measured on the day of embryo transfer. DESIGN: Comparative, observational study. SETTING: University teaching hospital. PATIENT(S): Two hundred eighty patients undergoing IVF. INTERVENTION(S): Ovarian stimulation, IVF. MAIN OUTCOME MEASURE(S): Variables related to patients' clinical characteristics, treatment characteristics, ovarian response, ovum retrieval, outcome of IVF and ICSI, embryo transfer, ultrasonographic and Doppler endometrial measurements, and uterine blood flow that have been proposed as potential predictive factors of implantation. All transvaginal ultrasonographic assessments were performed on the day of embryo transfer. RESULT(S): Among 240 patients finally evaluable, 67 (group 1) became pregnant after IVF, and 173 (group 2) failed to conceive. The 111 nonpregnant patients who had the same embryo score per replacement (group 3) as did patients in group 1 were selected for comparison purposes. The only significant differences between groups 1 and 3 were the type A endometrium and the absence of a protodiastolic notch in the uterine arteries, both of which were more frequently found in group 1. However, a considerable overlap existed between conception and nonconception cycles regarding both variables. CONCLUSION(S): Ultrasonographic parameters as predictors of implantation in assisted reproduction have a limited value in the clinical setting.

Materno-fetal immunogenetic disparity: the biological basis for in vitro fertilization treatment in patients with unexplained recurrent abortion?

PROBLEM: The majority of women with recurrent miscarriage have no discernible cause but it has been postulated that immunologic aberrations may be the cause in most of such cases. Also, it has been stressed that deliberate modification of the maternal host defense system can improve the chances of success. We tested the hypothesis that it is possible to potentiate maternal immune functions so as to improve reproductive performance by replacing several embryos into the uterus, thus favoring the recognition of fetal antigens. METHOD OF STUDY: A total of 57 couples with three or more (mean 5.52; range 3-12) consecutive first-trimester spontaneous clinical abortions of unknown etiology were treated with in vitro fertilization (IVF) and embryo transfer for a total of 84 cycles. Patients underwent IVF after combined gonadotropin-releasing hormone agonist/gonadotropin treatment for ovarian stimulation, and up to four embryos were replaced into the uterus. RESULTS: There were 32 pregnancies (three of them after frozen-thawed embryo transfers) and 26 (81%) of them were viable gestations. Overall, patients had a previous obstetric history of 315 pregnancy losses and 15 live-born babies. Thus, the probability of having a live baby before treatment was 4.54% (95% CI, 2.78-7.36) a figure significantly lower (P < 0.0001) than that observed under IVF treatment (81%; 95% CI, 64.53-91.01). None of selected variables potentially related with the outcome of pregnancy after IVF and embryo transfer in recurrent aborters (including pre-implantation genetic diagnosis) was found to be associated with miscarriage. CONCLUSIONS: This study shows that replacement of several embryos after IVF is a useful treatment in the prevention of unexplained recurrent spontaneous abortion thus providing further evidence for immunologically modifiable pregnancy loss.

Early hormonal markers of pregnancy outcome after in vitro fertilization and embryo transfer.

PURPOSE: To investigate the relative power of HCG, estradiol, and progesterone determinations in the prediction of pregnancy outcome after IVF. These prognostic hormonal factors were studied as single and combined predictors. METHODS: Serum concentrations of beta-HCG, progesterone, and estradiol were measured 12-13 days after embryo transfer (study point 1) and 7 days later (study point 2) in a series of 20 consecutive infertile patients having a first-trimester spontaneous clinical abortion after an IVF-embryo transfer cycle. As a control group (n = 60), the next three IVF-embryo transfer cycles resulting in an ongoing pregnancy after each miscarried IVF cycle in our assisted reproduction program was used. The discrimination attained between the two study groups (ongoing pregnancies and miscarriages) was evaluated by logistic regression and receiver operating characteristic (ROC) curve analysis. RESULTS: Mean hormone concentrations at study points 1 and 2 were higher in the ongoing pregnancy than in the abortion group. Regarding pregnancy outcome the percentage increment of HCG serum levels (> or = 1321%), with an accuracy (predictive value of pregnancy outcome) of 81.2% (sensitivity 98%, specificity 50%), had the best prognostic reliability but no significant differences were found when this parameter was compared with the predictive value of HCG concentration (> or = 72 IU/l) at study point 1 (diagnostic accuracy 80.5%; sensitivity 70%; specificity 80%). When ROC analysis was used, the best predictor of ongoing pregnancy according to the AUC(ROC) was HCG concentration at study point 2 but again no significant differences were found when this parameter was compared with the predictive value of HCG serum levels at study point 1. A multiple marker strategy did not help distinguish viable from nonviable pregnancies. CONCLUSION: A single, early (days 12-13 after embryo transfer) HCG quantitative serum measurement in IVF cycles not only is diagnostic but also has good predictive value for pregnancy outcome.

Increased expression of the cyclin-dependent kinase inhibitor p27 in cleavage-stage human embryos exhibiting developmental arrest.

It is accepted that approximately 50% of embryos obtained after IVF arrest during the first week. Traditionally, chromosome abnormality and suboptimal culture conditions have been proposed as factors commonly associated with embryo arrest. However, even when considering 'ideal' conditions and embryos of only excellent morphology in vitro, there is still a significant incidence of embryonic arrest. There is considerable evidence that the nuclear protein p27, a member of the Cip/Kip family of CDK inhibitors, plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis. The present investigation, using immunocytochemical techniques coupled with confocal microscopy, was undertaken to determine whether p27 could play a role in the arrest of 4-8-cell human embryos. A total of 28 preimplantation embryos at the 4-8-cell stage were investigated. Of these, 16 were diploid embryos showing cleavage arrest with no further progression, and 12 were normally developing embryos. There was a 2-fold increased expression of the cell-cycle inhibitor p27 in arrested embryos compared with control normally developing embryos. This study represents the first demonstration of an increased expression of p27 in cleavage-stage human arrested embryos.