Zona thinning with a noncontact diode laser in ICSI embryos from women of advanced age

Purpose: the objective of this study was to determine if the zona thinning (ZT) technique improved the rates of implantation and clinical pregnancy for patients aged, greater than or equal to38 years submitted to an ICSI program.Methods: A total of 100 patients submitted to ICSI and aged, greater than or equal to38 years were divided in a prospective and randomized manner into two groups: Group I - patients submitted to ZT (n = 50); a laser diode with 1.48 mum wavelength (Fertilaser) was used for the ZT procedure with 1-2 irradiations of 10 ms applied to four different positions on the zona pellucida (ZP) of each embryo to thin 60-90% of the ZP (each point with a 15-20 mum length of ZT). Group II - patients with no ZT (n = 50). In both groups, embryo transfer was performed on the second or third day.Results: the age of Group I patients (39.8 +/- 1.3) did not differ (p = 0.67) from that of Group II patients (40 +/- 1.9). The number of oocytes retrieved at metaphase II from Group I (6.4 +/- 4.2) and Group II (6.8 +/- 5) was similar (p = 0.94). Normal fertilization rates and cleavage rates were similar (p = 0.78 and p = 0.63, respectively) for Group I (71.5 +/- 22% and 96.7 +/- 11%) and Group II (73.5 +/- 19.7% and 96 +/- 11%, respectively). The number of embryos transferred was similar (p = 0.53) for the two groups (Group I = 3.1 +/- 1.3; Group II = 2.9 +/- 1.1). The thickness of the ZP of Group I embryos (16.9 +/- 2.4 mum) did not differ (p = 0.97) from that of Group II embryos (16.9 +/- 2.3 mum). The rates of embryo implantation and clinical pregnancy per embryo transfer were similar (p = 0.67, p = 0.61) for Group I (7 and 16%, respectively) and for Group II (8.2 and 22%, respectively).Conclusions: These results suggest that ZT in the population aged, 38 years may have no impact on ICSI success rates. However, this conclusion is limited to a situation in which length of the laser ZT was less than or equal to 20 mum and the laser was applied to four different positions.

Development of a real-time PCR method for rapid sexing of human preimplantation embryos

Genes on the X chromosome are known to be responsible for more than 200 hereditary diseases. After IVF, the simple selection of embryo sex before uterine transfer can prevent the occurrence of affected offspring among couples at risk for these genetic disorders. The aim of this investigation was to develop a rapid method of preimplantation genetic diagnosis (PGD) using real-time polymerase chain reaction (PCR) for the sexing of human embryos, and to compare it to the fluorescence in-situ hybridization technique, considered to be the gold standard. After biopsies were obtained from 40 surplus non-viable embryos for transfer, a total of 98 blastomeres were analysed. It was possible to analyse 24 embryos (60%) by both techniques, generating a total of 70 blastomeres (35 per technique), white 28 blastomeres from 16 embryos (40%) were analysed only by real-time PCR. A rapid and safe method was developed in the present study for the sexual diagnosis of a single human cell (blastomere and buccal cell) using the emerging technology of real-time PCR. (C) 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Are poor responders patients at higher risk for producing aneuploid embryos in vitro?

Purpose To test the hypothesis that aged women with poor ovarian response express an increase on embryo chromosomal alterations when compared to aged women who presented normal response.Methods Couples undergoing intracytoplasmic sperm injection cycles with preimplantation genetic screening, were subdivided into two groups: Poor Responder group (n = 34), patients who produced a parts per thousand currency sign4 oocytes; and Normoresponder group (n = 50), patients who produced a parts per thousand yen5 oocytes. Groups were compared regarding cycles' outcomes and aneuploidy frequency.Results There were no significant differences between and groups regarding the fertilization rate (p = 0.6861), clinical pregnancy (p = 0.9208), implantation (p = 0.6863), miscarriage (p = 0.6788) and the percentage of aneuploid embryos (p = 0.270). Embryo transfer rate was significantly lower on poor responder group (p = 0.0128) and logistic regression confirmed the influence of poor response on the chance of embryo transfer (p = 0.016).Conclusions Aged females responding poorly to gonadotrophins are not at a higher risk for producing aneuploid embryos in vitro.

IVF/ICSI outcomes after culture of human embryos at low oxygen tension: a meta-analysis

Background: Improved pregnancy, implantation, and birth rates have been reported after the use of reduced O2 concentration during embryo culture, mainly due to a reduction of the cumulative detrimental effects of reactive oxygen species. However, some studies have failed to report any positive effects. The objective of this meta-analysis was to evaluate the effect of a low-O2 environment on IVF/intracytoplasmic sperm injection (ICSI) outcomes.Methods: All available published and ongoing randomised trials that compared the effects of low (similar to 5%; OC similar to 5) and atmospheric (similar to 20%; OC similar to 20) oxygen concentrations on IVF/ICSI outcomes were included. Search strategies included online surveys of databases from 1980 to 2011. The outcomes measured were fertilisation rate, implantation rate and ongoing pregnancy rates. The fixed effects model was used to calculate the odds ratio.Results: Seven studies were included in this analysis. The pooled fertilisation rate did not differ significantly (P = 0.54) between the group of oocytes cultured at low O2 tension and the group at atmospheric O2 tension. Concerning all cycles, the implantation (P = 0.06) and ongoing pregnancy (P = 0.051) rates were not significantly different between the group receiving transferred sets containing only OC similar to 5 embryos and the group receiving transferred sets with only OC similar to 20 embryos. In a meta-analysis performed for only those trials in which embryos were transferred on day 2/3, implantation (P = 0.63) and ongoing pregnancy (P = 0.19) rates were not significantly different between the groups. In contrast, when a meta-analysis was performed using only trials in which embryos were transferred on days 5 and 6 (at the blastocyst stage), the group with transferred sets of only OC similar to 5 embryos showed a statistically significantly higher implantation rate (P = 0.006) than the group receiving transferred sets with only OC similar to 20 embryos, although the ongoing pregnancy (P = 0.19) rates were not significantly different between the groups.Conclusions: Despite some promising results, it seems too early to conclude that low O2 culture has an effect on IVF outcome. Additional randomised controlled trials are necessary before evidence-based recommendations can be provided. It should be emphasised that the present meta-analysis does not provide any evidence that low oxygen concentration is unnecessary.

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cryogenic preservation of embryos of Prochilodus lineatus (Valenciennes, 1836) (Characiforme; Prochilodontidae)

While the freezing techniques of mammal embryos have been providing promising results, the cryopreservation of teleostean eggs and embryos have remained unsuccessful up to now. Therefore, this work aimed to develop a procedure of cryogenic preservation of embryos of Prochilodus lineatus and to observe, at both structural and ultrastructural levels, the morphological alterations that took place after the application of freezing/thawing techniques. The embryos at the morula stage could not tolerate exposure to the cryoprotectants ethylene glycol monomethyl ether, propylene glycol monomethyl ether, methanol, dimethyl sulphoxide and propylene glycol, presenting 100% of mortality. Embryos at the 4- to 6-somites stage tolerated exposure to propylene glycol and dimethyl sulphoxide, and the results revealed no significant differences (alpha = 0.05) regarding survival from both treatments. None of the freezing, thawing and hydration protocols was effective on preserving embryo viability. The ultrastructural analyses of frozen and thawed embryos showed that cells from ectoderm, somites, notochord and endoderm were structurally intact, with well preserved nuclei and mitochondria. The yolk globules were able to tolerate the freezing process, but the yolk syncytial layer was unorganized, displaying an electron-dense and compacted appearance, collapsed reticules, nuclei with modified chromatin and ruptures on the plasmatic membrane at the contact zone with endoderm. It might be concluded that the procedures tested for freezing were unable to avoid the formation of intracellular ice crystals, leading to drastic morphological modifications and making P. lineatus embryos unviable.