Maternal body mass index affects embryo morphokinetics: a time-lapse study.

PURPOSE: To assess the effect of body mass index (BMI) on morphokinetic parameters of human embryos evaluated with time-lapse technology during in vitro culture. METHODS: A retrospective analysis of ART cycles utilizing time-lapse technology was undertaken to assess the potential impact of maternal BMI on morphokinetic and static morphological parameters of embryo development. The cohort of patients was divided into four groups: 593 embryos from 128 underweight women in group A; 5248 embryos from 1107 normal weight women in group B; 1053 embryos from 226 overweight women in group C; and 286 embryos from 67 obese women in group D. RESULTS: After adjusting for maternal age, paternal age, and cause of infertility, time to reach five blastomeres (t5) and time to reach eight blastomeres (t8) were longer in obese women compared with normoweight women [50.84 h (46.31-55.29) vs. 49.24 h (45.69-53.22) and 57.89 h (51.60-65.94) vs. 55.66 h (50.89-62.89), adjusted p < 0.05 and adjusted p < 0.01, respectively]. In addition, t8 was also delayed in overweight compared with normoweight women [56.72 h (51.83-63.92) vs. 55.66 h (50.89-62.89), adjusted p < 0.01]. No significant differences were observed among groups with regard to embryo morphology and pregnancy rate. Miscarriage rate was higher in underweight compared with normoweight women (OR = 2.1; 95% CI 1.12-3.95, adjusted p < 0.05). CONCLUSION: Assessment with time-lapse technology but not by classical static morphology evidences that maternal BMI affects embryo development. Maternal obesity and overweight are associated with slower embryo development.

Prognostic significance of the echocardiographic estimate of pulmonary hypertension and of right ventricular dysfunction in acute decompensated heart failure. A pilot study in HFrEF patients.

BACKGROUND: Mortality following an admission for acute decompensated heart failure (ADHF) is high and risk stratification in this context remains a challenge. The objective of the present study was to assess whether a simple echocardiographic assessment of pulmonary hypertension (PH) and/or of right ventricular (RV) dysfunction is associated with cardiovascular events in a 1-year follow-up after hospital discharge. METHODS AND RESULTS: The present prospective longitudinal study included 214 patients admitted to hospital with a cardiologist-adjudicated diagnosis of ADHF and a left ventricular ejection fraction (LVEF) at echocardiography < 40%. Echocardiography was performed at admission and at discharge and included pulmonary artery systolic pressure (PASP) and RV function as defined by the tricuspid annular plane systolic displacement (TAPSE). The primary end-point was the combination of all-cause mortality and re-hospitalization for worsening heart failure at 1 year after hospital discharge. During an average follow-up period of 230 ±â€¯130 days, 40 patients died and 41 patients underwent re-hospitalization due to ADHF. At multivariate analysis the independent predictors were LVEF, PASP at discharge and creatinine plasma levels (all p < 0.001). At ROC analysis the best threshold of PASP to discriminate low-risk from high-risk patients was 40 mm Hg. CONCLUSIONS: In ADHF patients with reduced LVEF, PH at discharge is a pivotal prognostic feature to predict morbidity/mortality within the first year after the acute episode.

Morphokinetics of embryos developed from oocytes matured in vitro.

PURPOSE: In in vitro maturation (IVM) cycles primed with human chorionic gonadotropin (hCG), both immature and mature oocytes are retrieved from antral follicles sized 8-12 mm. Using time-lapse microscopy, we compared the morphokinetic behavior of embryos developed from oocytes matured in vivo and in vitro, testing the hypothesis that IVM affects preimplantation development. Furthermore, we extended the morphokinetic analysis of these embryos by a comparison with embryos obtained in stimulated assisted reproduction technology (ART) cycles. METHODS: In IVM cycles primed with follicle-stimulating hormone (FSH)/hCG, prior to sperm microinjection, oocytes surrounded by an expanded cumulus at retrieval and presumably mature (EC-MII) were incubated for 6 h, while immature oocytes enclosed in a compact cumulus (CC) were matured in vitro for 30 h. The morphokinetics of embryos selected for transfer or cryopreservation, derived from EC-MII and CC oocytes, were comparatively and retrospectively analyzed in terms of cleavage times (t2, t3, t4, t5, and t8) and intervals (cc2, cc3, s2, s3). For further comparison, the morphokinetics of embryos selected for transfer or cryopreservation (ICSI) or giving rise to ongoing pregnancies (model) in stimulated ART cycles was also assessed. RESULTS: The morphokinetic behavior of EC-MII and CC embryos was entirely comparable, as suggested by the absence of statistical differences in the averages of all cleavage times and intervals. Almost all cleavage and interval times were also similar between EC-MII, CC, ICSI, and model groups, with the exception of t4 and s2, which were delayed and longer, respectively, in embryos generated in IVM cycles (EC-MII and CC). CONCLUSIONS: These findings do not support the hypothesis that maturation in vitro affects embryo morphokinetics, while they suggest only marginal differences in the morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles and embryos developed from mature oocytes recovered in stimulated cycles.

Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization.

BACKGROUND: In a growth phase occurring during most of folliculogenesis, the oocyte produces and accumulates molecules and organelles that are fundamental for the development of the preimplantation embryo. At ovulation, growth is followed by a phase of maturation that, although confined within a short temporal window, encompasses modifications of the oocyte chromosome complement and rearrangements of cytoplasmic components that are crucial for the achievement of developmental competence. Cumulus cells (CCs) are central to the process of maturation, providing the oocyte with metabolic support and regulatory cues. METHODS: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning oocyte maturation in mammals. Searches were performed adopting 'oocyte' and 'maturation' as main terms, in association with other keywords expressing concepts relevant to the subject. The most relevant publications, i.e. those concerning major phenomena occurring during oocyte maturation in established experimental models and the human species, were assessed and discussed critically to offer a comprehensive description of the process of oocyte maturation. RESULTS: By applying the above described search criteria, 6165 publications were identified, of which 543 were review articles. The number of publications increased steadily from 1974 (n = 7) to 2013 (n = 293). In 2014, from January to the time of submission of this manuscript, 140 original manuscripts and reviews were published. The studies selected for this review extend previous knowledge and shed new and astounding knowledge on oocyte maturation. It has long been known that resumption of meiosis and progression to the metaphase II stage is intrinsic to oocyte maturation, but novel findings have revealed that specific chromatin configurations are indicative of a propensity of the oocyte to resume the meiotic process and acquire developmental competence. Recently, genetic integrity has also been characterized as a factor with important implications for oocyte maturation and quality. Changes occurring in the cytoplasmic compartment are equally fundamental. Microtubules, actin filaments and chromatin not only interact to finalize chromosome segregation, but also crucially co-operate to establish cell asymmetry. This allows polar body extrusion to be accomplished with minimal loss of cytoplasm. The cytoskeleton also orchestrates the rearrangement of organelles in preparation for fertilization. For example, during maturation the distribution of the endoplasmic reticulum undergoes major modifications guided by microtubules and microfilaments to make the oocyte more competent in the generation of intracellular Ca(2+) oscillations that are pivotal for triggering egg activation. Cumulus cells are inherent to the process of oocyte maturation, emitting regulatory signals via direct cell-to-cell contacts and paracrine factors. In addition to nurturing the oocyte with key metabolites, CCs regulate meiotic resumption and modulate the function of the oocyte cytoskeleton. CONCLUSIONS: Although the importance of oocyte maturation for the achievement of female meiosis has long been recognized, until recently much less was known of the significance of this process in relation to other fundamental developmental events. Studies on chromatin dynamics and integrity have extended our understanding of female meiosis. Concomitantly, cytoskeletal and organelle changes and the ancillary role of CCs have been better appreciated. This is expected to inspire novel concepts and advances in assisted reproduction technologies, such as the development of novel in vitro maturation systems and the identification of biomarkers of oocyte quality.

Clinical outcomes from mature oocytes derived from preovulatory and antral follicles: reflections on follicle physiology and oocyte competence.

PURPOSE: The aim of this retrospective study was to compare the competence of oocytes obtained from preovulatory and antral follicles. METHODS: Mature oocytes from preovulatory follicles were retrieved from women selected for standard IVF treatment (Group A). Mature oocytes from antral follicles were recovered from women undergoing hCG-primed in vitro maturation (IVM) treatment (Group B). Patients groups were matched for age, BMI, FSH, AMH and antral follicle count (AFC) values. In vivo matured oocytes from both groups were microinjected and resulting embryos were culture and selected on day 3 for embryo transfer. RESULTS: Oocyte pick-ups (OPU) were 315 and 204 in Groups A and B, respectively. Fertilization rates were comparable (72.8 and 75.9 %, respectively; P = 0.137). In Group A, in which the average number of embryos transferred was higher, clinical pregnancy rates per OPU (37.5 %) and embryo transfer (38.4 %) were superior in comparison to Group B (27.0 %, P = 0.013; 29.4 %, P = 0.041; respectively). On the other hand, implantation rates (Group A, 23.7 %; Group B, 20.8 %) and proportions of babies born per transferred embryo (Group A, 19.5 %; Group B, 16.9 %) were similar (P = 0.528 and 0.332, respectively). CONCLUSIONS: Overall, this suggests that oocyte competence is already achieved at the antral stage of follicle development.

A comprehensive electrocardiographic, molecular, and echocardiographic study of Brugada syndrome: validation of the 2013 diagnostic criteria.

BACKGROUND: The debate on the diagnostic value of high intercostal spaces (ICSs) and of the number of diagnostic leads in Brugada syndrome (BrS) has been settled by a recent expert consensus statement. OBJECTIVE: To test the validity, and the underlying anatomy, of the new electrocardiographic (ECG) diagnostic criteria using echocardiographic, molecular, and clinical evidence in 1 clinical study population with BrS. METHODS: We analyzed 114 patients with BrS and with a spontaneous or drug-induced type 1 ECG pattern recorded in 1 or more right precordial leads in fourth, third, and second ICSs. The right ventricular outflow tract (RVOT) was localized by using echocardiography. All probands were screened on the SCN5A gene. RESULTS: The percentage of mutation carriers (MCs) and the event rate were similar regardless of the diagnostic ICS (fourth vs high ICSs: MCs 23% vs 19%; event rate 22% vs 28%) and the number of diagnostic leads (1 vs ≥2: MCs 20% vs 22%; event rate 22% vs 27%). The concordance between RVOT anatomical location and the diagnostic ICSs was 86%. The percentage of the diagnostic ECG pattern recorded was significantly increased by the exploration of the ICSs showing RVOT by echocardiography (echocardiography-guided approach vs conventional approach 100% vs 43%; P < .001). CONCLUSION: The high ICSs are not inferior to the standard fourth ICS for the ECG diagnosis of BrS, and the interindividual variability depends on the anatomical location of the RVOT as assessed by using echocardiography. This approach significantly increases diagnostic sensitivity without decreasing specificity and fully supports the recently published new diagnostic criteria.

Cleavage kinetics analysis of human embryos predicts development to blastocyst and implantation.

Cleavage kinetics of human embryos is indicative of ability to develop to blastocyst and implant. Recent advances in time-lapse microscopy have opened new and important research opportunities. In this study involving infertile couples requiring standard IVF/intracytoplasmic sperm injection treatment, zygotes were cultured by integrated embryo-culture time-lapse microscopy to analyse cleavage times from the 2- to the 8-cell stages in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after 8-cell stage, times of cleavage to 7- and 8-cell stages of embryos developing to blastocyst were shorter (56.5 ± 8.1 versus 58.8 ± 10.4h, P=0.03 and 61.0 ± 9.4 versus 65.2 ± 13.0 h, P=0.0008, respectively). In embryos developing to blastocyst, absence of blastocoele expansion on day 5 was associated with progressive cleavage delay. Implanting embryos developed to 8-cell stage in a shorter period compared with those unable to implant (54.9 ± 5.2 and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from 2- to 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on days 2 and 3 are inappropriate for accurate embryo evaluation. The speed at which human embryos cleave is known to be suggestive of their ability to develop in vitro to the blastocyst stage and implant after transfer into the uterus. Recent advances in time-lapse microscopy, which allows acquisition of images every 15-20 min, have opened new and important research opportunities. In a retrospective study involving infertile couples requiring standard IVF or intracytoplasmic sperm injection treatment, fertilized oocytes were cultured by an integrated embryo-culture time-lapse microscopy system in order to perform an analysis of cleavage times from the 2- to the 8-cell stage in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after the 8-cell stage, times of cleavage to the 7- and 8-cell stage of embryos that developed to blastocyst were significantly shorter (56.5 ± 8.1h versus 58.8 ± 10.4h and 61.0 ± 9.4h versus 65.2 ± 13.0 h, respectively). In embryos developing to the blastocyst stage, absence of blastocoele expansion on day 5 was associated with a progressive cleavage delay. Implanting embryos developed to the 8-cell stage in a shorter period compared to those unable to implant (54.9 ± 5.2h and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from the 2- to the 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on day 2 and 3 are appropriate for accurate embryo evaluation.

Human oocyte ultravitrification with a low concentration of cryoprotectants by ultrafast cooling: a new protocol.

OBJECTIVE: To evaluate the efficacy of a new ultravitrification technique with a low concentration of cryoprotectants. DESIGN: Ultravitrification research. SETTING: Private assisted reproduction center. PATIENT(S): Oocytes donated voluntarily with the aim of research. INTERVENTION(S): Ultravitrification with different protocols of 100 mature oocytes and 100 immature oocytes divided in four groups to determine which is the adequate cryoprotectant concentration and the appropriate cooling solution. MAIN OUTCOME MEASURE(S): Human oocytes survival rate with low concentration of cryoprotectants by ultravitrification technique. RESULT(S): We obtained higher survival rates with slush nitrogen than with liquid nitrogen (92% vs. 56%) and better results with 2 M of cryoprotectants than with 1.5 M (92% vs. 60%). The best protocol was 2 M PrOH + 0.5 M sucrose + slush nitrogen with a mature oocytes survival rate of 92% (23 of 25) and immature of 88% (22 of 25). CONCLUSION(S): This ultravitrification technique is a new option to preserve human oocytes that avoids the use of a high cryoprotectant concentration while obtaining a high survival rate with a concentration of cryoprotectants typical of slow freezing.

Results of in vitro fertilization in Italy after the introduction of a new law.

OBJECTIVE: To investigate the consequences of a law introduced in Italy in 2004 that forbids the fertilization or injection of more than three oocytes for assisted reproduction and does not allow any embryo selection or cryopreservation. DESIGN: Retrospective observational analysis. SETTING: Subfertile patients enrolled in an assisted reproduction program. PATIENT(S): Before the introduction of the law there were 1,179 cycles and after its enactment there were 1,860 cycles in 1,619 subfertile couples. INTERVENTION(S): Ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI) attempts. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rate. RESULT(S): Pregnancy rates (PR) per cycle (24.34% vs. 23.11%), per retrieval (28.64% vs. 25.65%), per transfer (31.37% vs. 27.74%), and the take-home babies per started cycle (19.1% vs. 18%) was not significantly different between the two periods. After introduction of the law, the PR significantly decreased in patients whose total motile sperm count was <1 x 10(6) (40.85% vs. 23.62%) and in patients receiving two embryos (35.71% vs. 23.53%). This difference was mostly the result of a reduced PR in patients <36 years old receiving two unselected embryos (41.16% vs. 30.90%). This result was, however, obscured by the higher proportion of patients <36 years (3.9% vs. 45.12%) receiving three embryos after the enactment of the law, which lead to a significantly higher PR (28.73% vs. 37.56%) and a consequent significantly higher triplet rate (0.58% vs. 4.71%). CONCLUSION(S): Women in whom elective transfer of two embryos was allowed before passing the law and couples with a severe male infertility factor had significantly reduced success rates. Although the overall PR did not change after the new law, if the transfer of frozen embryos is not considered, this was mainly the result of a higher number of embryos transferred into women <36 years old. This study shows how the negative effects of the new law are obscured by the transfer of a higher number of embryos in younger patients, resulting in a higher PR at the cost of a significantly higher triplet rate.

Normal birth after transfer of cryopreserved human embryos generated by microinjection of cryopreserved testicular spermatozoa into cryopreserved human oocytes.

OBJECTIVE: To report the first birth after transfer of cryopreserved embryos generated by intracytoplasmic sperm injection of cryopreserved testicular spermatozoa into cryopreserved human oocytes. DESIGN: Case report. SETTING: Tertiary center for reproductive technology. PATIENT(S): A 36-year-old woman with primary infertility of 3 years' duration and a 37-year-old man with congenital bilateral absence of the vas deferens. INTERVENTION(S): Cryopreservation of human embryos after oocytes and sperm thawing. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): A healthy, normal female infant with a birth weight of 2,950 g was born by cesarean section at 38 weeks' gestation, with normal 6-month follow-up. CONCLUSION(S): Embryo cryopreservation can lead to successful results, even with the use of cryopreserved gametes.

Cryopreservation of a small number of spermatozoa in yolk-filled human zonae pellucidae.

OBJECTIVES: Conventional sperm freezing procedures need the addition of a relatively large volume of cryoprotectant. The dilution of extremely poor sperm suspensions from ejaculate or testicular tissue may make the recovery of viable spermatozoa difficult at the moment of the intracytoplasmic sperm injection (ICSI) procedure. The cryopreservation of a few spermatozoa in empty zonae pellucidae is an interesting solution for crypto-azoospermic infertile men. We have modified this procedure by filling empty human zonae with TEST Yolk Buffer, an optimal cryoprotective medium, in order to analyse the number of zonae lost after thawing, the number of recovered spermatozoa per zona after thawing, and the sperm motility rate before freezing and after thawing. MATERIALS AND METHODS: Fifty empty human zonae pellucidae previously filled with TEST Yolk Buffer were injected with 750 motile spermatozoa from ten infertile men (15 spermatozoa per zona). Sterile straws containing two zonae each were frozen following a two-phase protocol. RESULTS: All of the zonae and 445/750 spermatozoa (59%) were recovered. The mean number (+ SD) of spermatozoa per zona was 8.9 +/- 1.9 (range: 5-12). The recovery rate of motile spermatozoa was 73% (327/445), with a mean number of motile spermatozoa per zona of 6.5 +/- 1.7 (range: 3-10). CONCLUSIONS: The cryopreservation of a small number of motile spermatozoa within empty zonae pellucidae using TEST Yolk Buffer as a freezing medium is possible without any major loss of spermatozoa and with an appreciable maintenance of sperm motility. This procedure seems to avoid: i) uncertain sperm retrieval after a laborious and time-consuming search on the day of oocyte aspiration; ii) the need for a repeated testicular biopsy; and iii) the need for heterologous insemination or oocyte cryopreservation (11).