Effects of dynamic oxygen concentrations on the development of mouse pre- and peri-implantation embryos using a double-channel gas supply incubator system / 대한생식의학회지

OBJECTIVE: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture.METHODS: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets.RESULTS: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group.CONCLUSION: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos / Suplementação com soro fetal bovino altera a modificação de histona H3R26me2 durante o período pré-implantacional em embriões bovinos produzidos in vitro

In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation...

A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação...

Effect of different activators on development of activated in vitro matured caprine oocytes

This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR[2]aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes [COC's] were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes [n=226] were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR[2]aa medium. Group 2 in vitro matured oocytes [n=294] were exposed to 7% ethanol for 5 min followed by treatment with 10 micro g/ml CHX for 4 h in mCR[2]aa medium. Group 3 in vitro matured oocytes [n=325] were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 micro g/ml CHX for 4 h in mCR[2]aa medium. Group 4 in vitro matured oocytes [n=108] were cultured for 4 h without any chemical treatment in mCR[2]aa medium [control]. The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR[2]aa is most favorable for parthenogenetic caprine embryos production

Effect of laser-assisted multi-point zona thinning on development and hatching of cleavage embryos in mice / 대한생식의학회지

OBJECTIVE: This study aimed to examine the effect of laser-assisted zona thinning (LAZT) at one or four-points on the blastocyst formation and hatching process in mice with respect to female age. METHODS: Eight-cell or morula embryos collected from superovulated C57BL female mice with different ages (6-11 and 28-31 weeks) were treated with LAZT at one-point (LAZT1) or four-points (LAZT4). The zona pellucida was thinned to more than 70% of its initial thickness by making two holes of 15-20 microm. RESULTS: In the young mice, LAZT resulted in a significant increase in early hatching and hatching rates compared to the control group (p<0.05). However, in the old mice, LAZT significantly increased blastocyst formation as well as early hatching and hatching compared to the controls (p<0.05). These effects were more remarkable in LAZT4 than in LAZT1 and in aged mice than in young ones. CONCLUSION: These results show that multi-point LAZT leads to a significant improvement of blastocyst formation and hatching in mice compared to controls.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn / Efeitos da sazonalidade e da condição ovariana sobre a produção in vitro de embriões de gatos domésticos em uma região entre o Equador e o Trópico de Capricórnio

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. [...] During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Do Trópico de Capricórnio ao Equador, sabe-se que a sazonalidade no gato domestico é ausente, i.e., estes animais são considerados reprodutores não sazonais nestas regiões. [...] O objetivo deste experimento foi determinar a porcentagem de clivagem e formação de mórulas e blastocistos produzidos a partir de oócitos recuperados de ovários de gatas em três condições - folicular, lútea ou inativa - durante duas estações reprodutivas pelas quais gatas passam na região sudeste do Brasil (22°53'09" S e 48°26'42" O) - estação não reprodutiva (ENR), que compreende os meses de janeiro a março; e estação reprodutiva (ER), agosto à outubro. Trinta gatas foram castradas. [...] Durante a ENR, de um total de 272 (inativo), 162 (lútea) e 134 (folicular) oócitos fertilizados, a porcentagem de clivagem de zigotos, formação de mórulas e de blastocistos derivados de ovários inativos foi 24,63, 16,54 e 8,09 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 21,6, 12,96 e 8,64, e para aqueles provenientes de ovários na fase folicular, foi de 24,62, 16,41 e 8,21. Considerando a ER, de um total de 102 (inativo), 198 (lútea) e 86 (folicular) oócitos fertilizados, a frequência relativa (%) de zigotos clivados, mórulas e blastocistos derivados de ovários na condição inativa foi de 64,7, 41,17 e 23,53 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 64,14, 40,41 e 23,73, e para aqueles provenientes de ovários na fase folicular, foi de 63,95, 39,54 e 24,41. Os resultados deste experimento demonstraram que não houve diferença estatística significante (P < 0.05) na frequência de embriões clivados e na formação de mórulas e blastocistos quando comparadas as três condições ovarianas dentro da mesma estação. Entretanto, a ER apresentou resultados melhores considerando as taxas de clivagem e formação de mórula e de blastocisto se comparada à ENR.

In vitro development of murine embryos in presence of campylobacter fetus / Desarrollo de embriones murinos in vitro en presencia de campylobacter fetus

Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CFF (cell-free filtrate) and Brucella broth (BB). Blastocysts were cultured in MEM (CG); challenge group were constituted by the addition of Cfv 27. Differentiation, hatching, hatched, adhesion and expansion were evaluated. Results were analyzed by Chi2 test. In two-cell embryo, the differentiation rate was not modified when the study strains were added (p > 0.05). The differentiation rate at 24 h for embryos at the morula stage was lower for BB, Cfv, and CFF, compared with CG (p < 0.05). After 48 h culture, no differences were observed in blastocyst formation for Cfv and BB, compared to CG (p > 0.05). However, the differentiation rate for the CFF group was lower than for CG (p < 0.05). At 48 and 72 h, the hatching rate was higher in CFF and Cfv groups than in CG (p < 0.05). Differences were not detected in blastocyst cultures. In conclusion, under these experimental conditions, Cf was not detrimental to the development of murine embryos. Efforts will be intensified to establish in vitro infection models that reproduce their pathogenicity.

La campilobacteriosis bovina caudada por Campylobacter fetus produce pérdidas reproductivas existiendo poca información de los mecanismos de patogenicidad de dicha bacteria, por lo cual se propone un modelo utilizando ratones BALC/c. Embriones de dos células fueron cultivados en Ham-F10: grupo control (GC), los grupos experimentales fueron adicionados con las cepas Cfv 1, Cfv 3, Cff 2 y Cff 5. Mórulas fueron cultivadas en Ham-F10 (GC); los grupos tratados recibieron Cfv27, CFF (filtrado libre de células) y caldo Brucella (BB). Blastocistos fueron cultivados en MEM (GC) y MEM más Cfv 27 (grupo desafiado). Se evaluó: diferenciación, "hatching", "hatched", adhesión y expansión. Los resultados fueron analizados por Chi2. En embriones de dos células, la diferenciación no fue modificada por acción de las cepas evaluadas (p > 0,05). Para embriones en estadío de mórula, la diferenciación a las 24 h de cultivo fue menor para BB, Cfv, y CFF, comparado con el GC (p < 0,05). Luego de 48 h de cultivo, no hubo diferencias entre Cfv, BB, y CG (p > 0,05), no obstante para el grupo CFF la diferenciación fue menor al CG (p < 0,05). El porcentaje de "hatching" (48 y 72 h de cultivo), fue mayor en los grupos CFF y Cfv comparado con el GC (p < 0,05). La adición de Cfv 27 no modificó el desarrollo de blastocistos. En el modelo propuesto, Cf no afectó negativamente el desarrollo embrionario. Futuros trabajos serán necesarios para establecer un modelo de infección in vitro en pos de reproducir su patogenicidad.

Carcinoma de endometrio: revisión clínico-patológica de 6 años en relación con los factores pronósticos / Endometrial carcinoma: Clinico-pathological revision after 6 years, related to prognostic factors

Los carcinomas de endometrio se dividen en dos categorías mayores (I y II), según los datos clínico-patológicos y las alteraciones genéticas. Los de tipo I se asocian con hiperestimulación estrogénica, obesidad, tratamiento hormonal exógeno y reconocen como lesión precursora a la hiperplasia endometrial con desarrollo de carcinomas endometrioides (CE). Los de tipo II predominan en mujeres postmenopáusicas, de subtipos histológicos más frecuentes serosos y de células claras. El objetivo es realizar una revisión anátomo-clínica de los carcinomas de endometrio, estudiados en nuestra institución entre el periodo comprendido entre 1/01/2005 y 31/12/2010. Se realizó estudio retrospectivo y descriptivo de 234 casos de pacientes con diagnóstico de carcinoma endometrial. La edad promedio de las pacientes fue de 68,6 años. El motivo de la consulta en la mayoría de los casos fue metrorragia de la postmenopausia (91,2 por ciento). El factor de riesgo más frecuente fue hipertensión arterial (22 pacientes). El 67,5 por ciento de las pacientes tenían antecedentes de biopsia previa por videohisteroscopía realizada en nuestra institución, observándose concordancia diagnóstica en un 91 por ciento. Se clasificaron según la OMS en CE (79 por ciento), adenocarcinoma seroso (9 por ciento) y mixtos (12 por ciento). El grado histológico (GH) FIGO fue en el 86 por ciento tipo I (exclusión de carcinomas serosos) y el estadío más frecuente fue el I ( 65 por ciento). La metaplasia (Me) más frecuente observada en estas neoplasias fue la mucinosa (35 por ciento). Las mórulas se presentaron en 2 casos comprobados por IHQ con positividad para CD 10 y CDX-2 y negatividad para p63. Se evidenciaron cambios reactivos en el 26 por ciento. El patrón MELF (glándulas microquísticas, elongadas y fragmentadas), de infiltración en la pared miometrial, se identificó en 3 casos. El CE fue el tipo histológico más frecuente y su presentación (mayoritariamente en estadios tempranos de enfermedad), se asoció en un alto porcentaje con las metaplasias mucinosa y tubaria...

Endometrial carcinomas are divided into two major categories (I and II), according to clinicopathologic and genetic alterations. The type I is associated with estrogen hyperstimulation, obesity, exogenous hormonal treatment, and endometrial hyperplasia is recognized as a precursor lesion, with the consecuent endometroid carcinoma. The type II predominate in post-menopausal women, most common histologic subtypes are serous and clear cell. The aim is to review the anatomical and clinical characteristic of endometrial carcinomas, studied at our institution between 01/01/2005 and 31/12/2010. We performed a retrospective and descriptive study of 34 cases of patients with endometrial carcinoma. The average age was 68.6 years. The reason for consultation in most cases was post-menopausal's metrorrhagia (91.2 percent) Hypertension (22 patients) was the more prevalent risk fctor. 67.5 percent of the patients had a history of prior biopsy (histeroscopy) performed at our institution; diagnostic concordance was observed in 91 percent. Were classified according to the WHO in endometrioid carcinomas (79 percent), serous adenocarcinoma (9 percent) and mixed (12 percent). FIGO histologic grade was 86 percent in type I (excluding serous carcinomas) and stage I was the most frequent (65 percent). Mucinous metaplasia was the most frequently observed (35 percent). The morulae were presented in 2 cases (IHC positive for CDX-2 and CD10, and negative for p63). Reactive changes were in 26 percent. The MELF pattern of myometrial infiltration was identified in 3 cases. Endometrioid carcinoma was the most common histological type and presentation (mostly in early stages of disease). Was associated with a high percentaje of tubal and mucinous metaplasia (this would indicate malignancy attenuated). Not yet known meaning of morulae in carcinomas. While it must be record horns infiltration, the presence or not of the same does not change the treatment.

effect of biopsy during precompacted morula stage on post vitrification development blastocyst derived bovine embryos

Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different precompacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2,3, and 4 post-isemination with different cell numbers [4 to 16-cells]. Embryo cell biopsy was carried out in a 100 micro l drop of H-SOF following pronase drilling by aspiration of one blastomrere. The biopsied embryos were then cutured in SOFFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room temperature after exposure of equilibration [glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min] and vitrification solutions [3.4 M glycerol and 4.6 M ethylene glycol]. The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived from biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrification survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts

Effects of timing on cell biopsy from pre-compacted morula stage bovine embryos on subsequent embryonic development

Embryo biopsy has potential applications in molecular research processes in domestic animals, besides its application in sex determination in embryo transfer programs. The objective of the present study was to assess the in vitro development of bovine embryos biopsied on different days of precompacted morula stage. Slaughterhouse-derived oocytes were matured in vitro, fertilized [Day-0] by frozen-thawed, Percol-separated spermatozoa and cultured on oviductal cell monolayer. The embryos were subjected to cell biopsy on Days 2, 3, and 4 postinsemination at 4-16-cell stages. The data were analyzed using ANOVA and Chi-squared tests [SigmaStat, version 2]. A p-value < 0.05 was considered significant. Biopsies carried out at 16-cell stage [Day-4] resulted in 94% of embryos developing to the blastocyst stage, which was significantly higher [p < 0.05] than the ones biopsied at 8-cell stage on Day-4 [64%], and those undergoing the procedure on Day-3 [49% and 46% at 4-cell and 8-cell stages, respectively] and Day-2 [39% and 33% at 4-cell and 8-cell stages, respectively]. No significant differences were observed between biopsied and non-biopsied embryos on a given day. The total cell number in biopsy-derived blastocysts ranged between 103 and 135. The difference in the number of total cells, dead cells and cell allocation to trophectoderm and inner cell mass between non-biopsied and biopsy-derived blastocysts was insignificant. Biopsy of bovine embryos at 4-16-cell stages had no adverse effects on in vitro developmental potentials and the 16-cell stage embryos, biopsied on Day-4 was the best stage for blastomere removal

Aspectos epidemiológicos, clínicos e hematológicos de 251 cães portadores de mórula de Ehrlichia spp. naturalmente infectados / Epidemiological, clinical, and hematological aspects of 251 dogs naturally infected with Ehrlichia spp. morulae

Realizou-se um estudo retrospectivo dos aspectos epidemiológicos, sinais clínicos, dados de exame físico e alterações hematológicas da erliquiose em 251 cães naturalmente infectados por Ehrlichia spp. Dos 4407 casos atendidos em hospital veterinário no período de janeiro de 2002 a dezembro de 2003, verificou-se que 251 cães eram portadores de mórula de Ehrlichia spp. em leucócitos de sangue periférico. Destes, 48 foram eliminados das avaliações por apresentarem patologias concomitantes. Nos 203 cães restantes, verificou-se que houve maior ocorrência em fêmeas (61,1 por cento) e que a doença manteve-se constante durante todo o período avaliado. Observou-se que 38 por cento encontravam-se na faixa etária entre um e 23 meses e 58,6 por cento eram de raça definida. As principais alterações clínicas observadas foram apatia, anorexia/hiporexia, vômito, secreção oculonasal e esplenomegalia. Cento e cinco cães apresentaram temperatura retal entre 38 e 39,5ºC. As alterações observadas com maior frequência no hemograma foram anemia, predominando o tipo normocítica normocrômica (58,2 por cento); desvio nuclear de neutrófilos para a esquerda (67 por cento) e eosinopenia (58,1 por cento).

A study of epidemiological and clinical aspects, alterations of physical exams, and hematological changes of canine ehrlichiosis was performed. A retrospective study was performed in 4,407 dogs referred to a Veterinary Hospital from January 2002 to December 2003. Of all cases, 251 dogs showed Ehrlichia spp. morulae. Among these, 48 were excluded from the study due to other co-infection by other pathologies. In the other 203 evaluated dogs, females (61.1 percent) were more infected than males. The dogs aged from one to 23 months (68.6 percent) and 58.6 percent were definite breed. Emesis, apathy, anorexia/hypoxeria, spleenomegaly, and nasal discharge were the most common signs presented. Rectal temperature was 38 - 39.5ÚC in 105 dogs. The most usual changes seen during the hematological tests were normochromic and normocitic anemia (58.2 percent), a left shift of the neuthrophils (67 percent), and eosinopenia (58.1 percent).

The effectiveness of ethylene glycol as cryoprotectant in mouse embryo freezing with slow freezing method / 대한산부인과학회지

OBJECTIVE: We intended to know how the cryoprotectant ethylene glycol (EG) would affect the outcome of the embryo development when used in slow freezing method. And to know if there is any difference in the outcome of frozen-thawed embryos according to freezing methods and the timing. METHODS: We used 5-6 weeks old ICR female mice and T6 containing 0.4% BSA for basic culture media. The embryos at the developmental stages of 1-cell, 8-cell and blastocyst were cryopreserved respectively by slow freezing method using EG, propylene glycol (PROH), and glycerol as a cryoprotectant. We also compared the results of slow freezing and vitrification methods with the same cryoprotectant, EG. And finally, we evaluated the quality of blastocysts by counting the cell numbers in each group. RESULTS: The post-thaw embryo development were better in EG group when they were frozen at 1-cell and blastocyst stage (P<0.05). Although there were no differences in the recovery rate, the survival rate in vitrification group was significantly higher (P<0.05). Post-thaw embryo development to morula and blastocyst were better in vitrification group when frozen at 1-cell embryo (P<0.05), not at 8-cell and blastocyst group. The cell counts of blastocyst derived from 1-cell stage frozen EG group were significantly increased than that of PROH-glycerol groups (P<0.05), however, there was no difference between the two freezing methods. CONCLUSION: These results suggest that EG may be advantageous comparing with the conventional cryoprotectants, PROH and glycerol in slow freezing method for mouse embryo cryopreservation. In terms of freezing method, vitrification is better than slow freezing.

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro.

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.

Vitrification of Mouse Embryos in Ethylene Glycol-based Solutions / 대한불임학회지

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols / 生物工程学报

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Assessment of Epidermal growth factor [EGF] effects on development of vitrified mouse morulae to the blastocyst stage

Many investigators are interested in finding the new cultural systems that can support the in vitro development of pre-implantation embryos better. Previous studies suggested that growth factors such as epidermal growth factor [EGF] are important in pre-implantation embryo development and implantation process. On the other hand, it is very important to support post thaw development of frozen embryos. The purpose of this study was to determine if the developmental potential of mouse morulae survived after vitrification could be increased using medium containing EGF. Mouse morulae were divided into vitrified and non-vitrified groups. Vitrification procedure was carried out using a combination of 40% ethylene glycol, 30% ficoll and 0.5 M sucrose [EFS40] as cryoprotectant. The embryos were warmed rapidly using 0.5 M sucrose. The survived embryos were cultured either on T6 or T6+EGF media. Accordingly, the embryos of the non-vitrified group were also cultured. The developmental rates in all groups were daily recorded and compared statistically using Chi-square test. The results showed that after 4 days of culture, the developmental potential of non-vitrified embryos cultured on T6+EGF was significantly increased. There was no significant difference between vitrified embryos cultured on T6 and T6+EGF media. In conclusion, the developmental potential of vitrified-warmed embryos does not increase in the medium containing EGF, even though there was significant increased developmental potential of non-vitrified embryos after culture on medium containing EGF. It is needed to do more study about the changes which will probably happen on the embryo EGF receptors following vitrification

Comparative study on the development of frozen-thawed mouse embryo using vitrification at each developmental stage / 대한산부인과학회잡지

OBJECTIVE: To find out the optimal freezing time in terms of developmental stage of mouse embryo and the effectiveness of vitrification method for freezing them. METHODS: Superovulation induction was performed using PMSG and hCG. Total 1,437 mouse embryos (vitrification group: 743, slow-freezing group: 694) were obtained and cultured with the T6 containing 0.4% BSA medium. Each developmental stage of embryos (1-, 2-, 4-, 8-cell, morula and blastocyst) were cryopreserved by vitrification and also by slow freezing method for comparison of the results. After thawing, the recovery rate, the survival rate and the blastocyst developmental rate were analysed and compared in two different settings. Student's t-test was used for statistical analysis. RESULTS: The survival and developmental rates at all subgroups of vitrification method are significantly higher than those of slow-freezing groups, but not in the recovery rates. In vitrification group, the survival rate and the blastocyst developmental rate are highest when frozen at morula stage, 98.4% and 86.4%, respectively. In slow-freezing group, the survival rate is also highest when frozen at morula stage, 87.2%, and the blastocyst developmental rate is highest when frozen at 8-cell stage, 78.1%. CONCLUSION: The vitrification method is more efficient for mouse embryo freezing compared with slow freezing one. Among various developmental stages of mouse embryos, morula stage seems to be the optimal stage for cryopreservation, whatever the freezing method applied. Therefore, we recommend embryo freezing at morula stage by vitrification method.

[Effects of different media on fallopian tube epithelial cells culture and human]

The advantage of fallopian tube epithelial cell [FTEC] co-culture system on development of human embryo has been shown. However, lack of appropriate media for both FTEC and embryo is one of the disadvantages of this system. The aim of the present study was to determine the effects of different culture media on both FTEC and human embryo development. Healthy fallopian tube samples were removed from 19 previously fertile women under 40 ages undergoing total abdominal hysterectomy. Epithelial cell-rich suspension was prepared mechanically and enzymatically and cultured in three different media [DMEM/F12, RPMI-1640 and Ham's F10]. After four passages, the cells were cultured on plastic to obtain unpolarised confluent cells, or seeded into matrigel to produce polarized cells for 7 days. Viability of cells during and after passages was evaluated by neutral red and trypan blue vital staining. In addition, 117, 45 and 48 surplus 4-8 cell human embryos were cultured in Ham's F10, RPMI and DMEM/F12 for 120 hours, respectively. Their morphology were assessed daily. Cellular vitality during culture in DMEM/F12 was 100%, which was significantly different from that in RPMI and Ham's F10 [95% and 93%, respectively] [P<0.05]. However, after cellular proliferation and passages, no significant difference was observed for different media. Moreover, a significantly higher ratio of embryos reached the morula stage in Ham's F10 [79.4%] than RPMI [68.8%] and DMEM/F112 [62.5%] [P<0.05]. It is concluded that DMEM/F12 was superior to other media during cell proliferation and passage of FTEC. However, after cell passage and during co-culture of embryos with FTEC, Ham's F10 was a more appropriate medium than the other two

Expression of Apoptotic Genes in Mouse Preimplantation Embryo Development / 대한불임학회지

OBJECTIVES: The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. MATERiALS AND METHODS: Two-cell embryos were retrieved from iCR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. RESULTS: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. in contrast, the expression of Mcl-1 and Bok was not significantly different. CONCLUSION: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

Identificación de mórulas de ehrlichia en plaquetas de sangre humana en Venezuela / Identification of morulae of ehrlichia in platelet of blood human ehrlichiasis

Ehrlichiosis humana: enfermedad emergente, transmitida por garrapata, producida por bacteria del género Ehrlichia, Gram-negativa, intracelular obligatoria. En animales, invade leucocitos y plaquetas, donde se multiplica originando una "mórula", visible al microscopio óptico. En humanos, se ha reportado solamente en glóbulos blancos: mononucleares y granulocitos (Walker and Dumler, 2001). Presenta sintomatología diversa, semejante a mononucleares infecciosa, dengue, gripe, entre otras. En Venezuela, el primer reporte de infección en humanos fue publicado en 1994, por Tamí y colaboradores. El objeto del trabajo es la identificación del microorganismo mediante frotis de capa blanca sanguínea, confirmando por técnica de biología molecular. Se estudiaron 37 muestras de sangre de pacientes con sintomatología compatible con ehrlichiosis, mediante revisión minuciosa de frotis, coloreado con Wright. Se practicó técnica de Reacción en Cadena de la Polimerasa (PCR) tipo nested en 15 muestras, empleando oligonucleótidos específicos de la región ribosomal 16S. Se identificó, por frotis, mórulas intraplaquetarias en 8 muestras de las 37 estudiadas. En dos de las muestras procesadas por PCR, se obtuvo amplificación del ADN del microorganismo, con migración electroforética que se corresponde con la de Ehrlichia específica de plaqueta canina (control), siendo ambas positivas en el diagnóstico morfológico. Se identificó y confirmó "Ehrlichia intraplaquetaria"

Comparative study on Development of Mouse Embryos in Conventional Medium versus Vero Cell Coculture / 대한산부인과학회잡지

OBJECTIVE: To evaluate mouse embryos development in conventional medium IVF-20 versus vero cell coculture. METHODS: Female ICR mice aged 6 to 8 weeks, were stimulated with 5IU PMSG and 48 hours later were injected 5IU of hCG, then female and male mice were mated. At 48 hour post-hCG injection, oviducts were dissected out and 2-cell embryos were flushed. The 2-cell embryos were cultured in IVF-20 media or media containing vero cell (African green monkey kidney epithelial cell lines) for 120 hours. Coculture techniques have been applied in mouse 2-cell embryos culture used vero cell lines. RESULTS: 1. After 48 hours culture, 60.7% and 55.7% of 2 cell embryos developed to 4 cell and morulae stage, respectively, in IVF-20 culture medium, but significantly less embryos developed to 4 cell (47.6%, p<0.05) and momlae (42.9%, p<0.05) in vero cell coculture. 2. After 72 hours culture, 51.6% of 2 cell embryos developed to blastocyst and expanded blastocyst in IVF-20 culture medium, but significantly less embryos developed to blastocyst and expanded blastocyst (25.9%, p<0.01) in vero cell coculture. 3. After 96 hours culture, 37.7% and 32.6% of 2 cell embryos similar developed to expanded blastocyst and hatching in IVF-20 culture medium and vero cell coculture, respectively. 4. After 120 hours culture, 36.9% and 37.4% of 2 cell embryos similar developed to expanded blastocyst and hatching in IVF-20 culture medium and vero cell coculture, respectively. CONCLUSION: There was no difference of embryo development rates between the two culture groups. IVF-20 medium alone gives a benefit to the viability of an embryo compared with a vero cell coculture.