The use of morphokinetics as a predictor of  implantation: a multicentric study to define and validate an algorithm for embryo selection.

STUDY QUESTION: Can we use morphokinetic markers to select the embryos most likely to implant and are the results likely to be consistent across different clinics? SUMMARY ANSWER: Yes, morphokinetic markers can be used to select the embryos most likely to implant and the results were similar in different IVF clinics that share methods and organization to some extent. WHAT IS KNOWN ALREADY: With the introduction of time-lapse technology several authors have proposed the use of kinetic markers to improve embryo selection. The majority of these markers can be detected as early as Day 2 of development. Morphology remains the gold standard but kinetic markers have been proven as excellent tools to complement our decisions. Nevertheless, the majority of time-lapse studies are based on small data sets deriving from one single clinic. STUDY DESIGN, SIZE, DURATION: Retrospective multicentric study of 1664 cycles of which 799 were used to develop an algorithm (Phase 1 of the study) and 865 to test its predictive power (Phase 2 of the study). PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility centres patients undergoing first or second ICSI cycle using their own or donated oocytes. Embryo development was analysed with a time-lapse imaging system. Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation (IR) and clinical pregnancy (CPR) rates were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: During Phase 1 of the study we identified three variables most closely related to implantation: t3 (34-40 h), followed by cc2 (9-12 h) and t5 (45-55 h). Based on these results we elaborated an algorithm that classified embryos from A to D according to implantation potential. During Phase 2 of the study the algorithm was validated in a different group of patients that included 865 cycles and 1620 embryos transferred. In this phase of the study, embryos were categorized based on the algorithm and significant differences in IR were observed between the different categories ('A' 32%, 'B' 28%, 'C' 26%, 'D' 20% and 'E' 17%, P < 0.001). In addition we identified three quality criteria: direct cleavage from one to three cells, uneven blastomere size in second cell cycle and multinucleation in third cell cycle. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study limits its potential value, although the use of one database to generate the algorithm (embryos from this database were not selected by any morphokinetic criteria) and one database to validate it reinforces our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The elaboration of an algorithm based on a larger database derived from different (albeit related) clinics raises the possibility that such algorithms could be applied in different clinical settings.

[On the classification of the cleavage patterns in amphibian embryos].

This paper presents a brief survey and preliminary classification of embryonic cleavage patterns in the class Amphibia. We use published data on 41 anuran and 22 urodele species concerning the character of the third cleavage furrow (latitudinal or longitudinal) and the stage of transition from synchronous to asynchronous blastomere divisions in the animal hemisphere (4-8-celled stage, 8-16-celled stage or later). Based on this, four patterns of amphibian embryonic cleavage are recognized, and an attempt to elucidate the evolutionary relationships among these patterns is undertaken. The so-called "standard" cleavage pattern (the extensive series of synchronous blastomere divisions including latitudinal furrows of the third cleavage) with the typical model species Ambystoma mexicanum and Xenopus laevis seems to be derived and probably originated independently in the orders Anura and Caudata. The ancestral amphibian cleavage pattern seems to be represented by species with longitudinal furrows of the third cleavage and the loss ofsynchrony as early as the 8-celled stage (such as in primitive urodele species from the family Cryptobranchidae).

Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos.

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.

[Pregnancy rate in cryopreserved embryos based on the classical morphologic scoring and the integrity of blastomeres at thawing]. / Tasa de embarazo en embriones criopreservados según la calificación morfológica clásica y la integridad de las blastómeras a la descongelación.

Two different thaw embryo classifications were evaluated, the morphologic classic evaluation (Veeck, 1990) and all blastomeres intact at the time of embryo transfer. We also discuss the clinic parameters and the physiopathological causes implicated in the successful of this treatment. 176 ovarian stimulation cycles and 513 cryopreserved embryos were reviewed. We found better pregnancy rate and delivery rate in embryo transfers were at least one embryo had all blastomeres intact compare with the transfers with good quality embryos (1+, 2+) of the morphologic classic evaluation without statistic significance (p > 0.05). Pregnancy and delivery rate were higher in cases with embryo in pronuclear stage compare with cleavage stage (p < 0.05). The blastomeric harm disturbs the embryo implantation phase and the future is oriented to the microsurgical remove of the damaged blastomeres and the use of assisted hatching.

Human embryos derived from in vitro and in vivo matured oocytes: analysis for chromosomal abnormalities and nuclear morphology.

PURPOSE: To determine whether embryos resulting from oocytes matured in vitro have a higher incidence of nuclear and/or genetic abnormalities compared to embryos resulting from oocytes matured in vivo. METHODS: Fluorescence in situ hybridization analysis for chromosomes X, Y, and 18 was used to compare the rates of aneuploidy, mosaicism, and nuclear abnormalities in embryos derived from oocytes that were prophase I at aspiration (immature group) to that observed in embryos resulting from oocytes that were metaphase I or II at aspiration (mature group). RESULTS: Based on nuclear morphology, significantly more embryos in the mature group (23%) were classified as normal compared to embryos in the immature group (3%). No difference was found in the rate of aneuploidy or in the incidence of mosaicism involving these chromosomes. CONCLUSIONS: These findings suggest that few embryos derived from prophase I oocytes collected following ovarian stimulation are morphologically normal.

Blastocystis hominis: prevalencia en alumnos de una escuela básica. Maracaibo, Estado Zulia, Venezuela / Blastocystics hominis: prevalence in scholars from basic school Maracaibo, Zulia State, Venezuela

Para determinar la prevalencia del Blastocystic hominis se estudiaron 150 escolares de ambos sexos, aparentemente sanos, cuyas edades oscilaban entre 6 y 14 años que cursan en la Escuela Básica "Dr. Nestor Luis Pérez". Municipio Maracaibo, Estado Zulia, Venezuela. Se recolectó una muestra fecal por alumno a la cual se le practicó un examen al fresco con solución salina, coloración temporal con lugol y la técnica de concentración formol-éter. La prevalencia de B.hominis fue de 24,0 por ciento; al relacionar las variables parasitosis, edad, grado de instrucción y sexo y aplicarle la prueba de significancia estadística del Chi cuadrado, se observó dependencia significativa entre parasitosis-edad parasitosis grado de instrucción y una relación de independencia entre parasitosis-sexo. B.hominis se encontró asociación a otras especies de enteroparásitos en un 55.6 por ciento, al aplicar el índice de Fager y la prueba de "t" se demostró asociación significativa entre B.hominis con Endolimax nana y B.hominis con Entamoeba coli. Los resultados obtenidos revelan que la frecuencia de B.hominis es alta, por lo que se considera importante su informe en los exámenes de heces, continuar con los estudios sobre patogenicidad y realizar campañas de educación sanitaria en la población

[Quality of embryos in unexplained sterility]. / Qualité des embryons dans les stérilités inexpliquées.

In our study, the rate of pregnancy by transfer and puncture was not significantly different in unexplained and in tubal infertility, but the mean number of transferred embryos was significantly higher in the first group. To explain these data, we compared the quality of embryos in 32 punctures realized among 29 women with unexplained infertility and in 171 punctures planned among 156 women with tubal infertility. The percentage of embryos with 4 or more blastomeres was significantly lower in the unexplained infertility group than in the pure tubal infertility group.

Mass isolation and culture of sea urchin micromeres.

A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of greater than 99% pure, viable micromeres (2.3 to 4.6 X 10(8) cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development.