Effects of vitrification and cryostorage duration on single-cell RNA-Seq profiling of vitrified-thawed human metaphase II oocytes / 医学前沿

Oocyte cryopreservation is widely used for clinical and social reasons. Previous studies have demonstrated that conventional slow-freezing cryopreservation procedures, but not storage time, can alter the gene expression profiles of frozen oocytes. Whether vitrification procedures and the related frozen storage durations have any effects on the transcriptomes of human metaphase II oocytes remain unknown. Four women (30-32 years old) who had undergone IVF treatment were recruited for this study. RNA-Seq profiles of 3 fresh oocytes and 13 surviving vitrified-thawed oocytes (3, 3, 4, and 3 oocytes were cryostored for 1,2, 3, and 12 months) were analyzed at a single-cell resolution. A total of 1987 genes were differentially expressed in the 13 vitrified-thawed oocytes. However, no differentially expressed genes were found between any two groups among the 1-, 2-, 3-, and 12-month storage groups. Further analysis revealed that the aberrant genes in the vitrified oocytes were closely related to oogenesis and development. Our findings indicated that the effects of vitrification on the transcriptomes of mature human oocytes are induced by the procedure itself, suggesting that long-term cryostorage of human oocytes is safe.

Evaluación de la tasa de blastocistos euploides en fase folicular y lútea (DuoStim) en mujeres con pobre respuesta ovárica: estudio preliminar en México / Euploid blastocyst rate in follicular vs luteal phase (DuoStim) in women with poor ovarian response: Preliminary study in Mexico

Resumen: OBJETIVO: Comparar la tasa de blastocistos euploides obtenida después de la estimulación ovárica en fase folicular con la fase lútea en un mismo ciclo menstrual en pacientes con deficiente respuesta ovárica. MATERIALES Y MÉTODOS: Estudio clínico, prospectivo y comparativo llevado a cabo en el Centro de Reproducción Arcos, Nascere, entre los meses de enero a julio de 2019. Se incluyeron pacientes con pobre respuesta ovárica según los criterios de Bologna y con indicación de PGT-A. Las estimulaciones en fase folicular y lútea se efectuaron con antagonista de la GnRH y FSHr/LHr (2:1) a partir del día 3 del ciclo y 5 días después de la primera recuperación de los ovocitos. Para completar el proceso de maduración ovocitaria se utilizaron análogos de GnRH, se tomó una biopsia de trofoectodermo en día 5-7. RESULTADOS: Se estudiaron 20 pacientes. Al comparar la fase folicular con la lútea la tasa de fertilización fue de 79% (IC95%: 29-46) vs 55% (IC95%: 34-53), la tasa de blastocistos 42% (IC95%: 19-44) vs 45% (IC95%: 24-55) y la tasa de blastocistos euploides 100% (IC95%: 44-53) vs 70% (IC95%: 38-46), respectivamente. Solo la tasa de recuperación de ovocitos en metafase II mostró diferencias significativas entre ambas fases 40% (IC95%: 18-37) vs 59% (IC95%: 31-59), p = 0.0333 en la fase folicular y lútea, respectivamente. CONCLUSIONES: La estimulación ovárica bifásica (folicular-lútea), en el mismo ciclo menstrual (DuoStim), resultó en mayor tasa de recuperación de ovocitos en metafase II durante la fase lútea. Sin embargo, las tasas de desarrollo embrionario a día 5-6 (blastocistos) y de embriones euploides fueron similares entre ambas fases.

Abstract: OBJECTIVE: Euploid blastocyst rate comparison between ovarian stimulation in follicular vs luteal phase performed in the same menstrual cycle in patients with poor ovarian response. MATERIALS AND METHODS: Clinical, prospective and comparative study conducted at Centro de Reproducción Arcos S.C., "Nascere", during january-july, 2019. Patients with PGT-A indication and poor ovarian response according to Bologna criteria were included. Under a short GnRH-antagonist protocol, stimulations, both in follicular and luteal phase were performed using rFSH/rLH (2:1) from day 3 of the cycle and 5 days after the first oocyte retrieval. In addition, ovulation trigger with an GnRH agonist was used, finally, on day 5-6 of embryo development, trophoctoctoderm biopsy was performed. RESULTS: In this study, 20 patients were included; when comparing follicular phase vs luteal phase, we found that fertilization rate was 79% (95%CI 29-46) vs 55% (95%CI 34-53), blastocysts rate was 42% (95%CI 19-44) vs 45% (95%CI 24-55) and euploid embryo rate was 100% (95%CI 44-53) vs 70% (95%CI 38-46). Only the oocyte recovery rate in metaphase II showed significant differences between both phases 40% (IC 95% 18-37) vs 59% (IC 95% 31-59), p=0.0333. CONCLUSION: Biphasic ovarian stimulation (follicular/ luteal) in the same menstrual cycle (DuoStim) resulted in a higher metaphase II ooctye recovery rate during the luteal phase in comparison with the follicular phase. However, the rates of blastocysts and euploid blastocysts were similar between both phases.

The spindle of oocytes observed by polarized light microscope can predict embryo quality

Background: The aim is to evaluate spindle position of metaphase II oocyte and the development of embryos originated from oocytes with spindle and without spindle.Methods: Cross-sectional analysis Research: 250 MII oocytes were analyzed with polarized microscope in Military Institute of Clinical Embryology and Histology, Vietnam Military Medical University.Results: Spindles were detected in 170 (77.98%) of 218 metaphase II oocytes, 115 spindles (67.65%) of MII oocytes is beneath or adjacent to the first polar body, 55 oocytes had the spindle located between 300 and 1800 away from the first polar body. Fertilization rate and the rate of good quality embryos in oocytes with a visible spindle (77.98% and 61.02%) were higher than those in oocytes without a visible spindle (22.02% and 36.84%), the difference was statistically significant with p <0.001 and p <0.05.Conclusions: The spindle position of metaphase II oocytes is not always beneath or adjacent to the first polar body. Fertilization rate and the rate of good quality embryos in oocytes with a visible spindle were higher than those in oocytes without a visible spindle.

Cytogenetic test and clinical study on cryptic acute promyelocytic leukemia with ins (15; 17) / 中华血液学杂志

Objective@#To investigate the genetic screening methods for cryptic acute promyelocytic leukemia (APL) to further explore its clinical prognosis.@*Methods@#From June 2016 to November 2018, we collected 373 newly diagnosed APL cases. The patients were retrospected by the results of PML-RARα detections both by RT-PCR and i-FISH, those who harbored positive PML-RARα detection by RT-PCR and negative by i-FISH were chosen. Metaphase FISH and Sanger sequencing were further performed to verify these results.@*Results@#A total of 7 cryptic APL cases were discovered. These cases had tiny fragment of RARα inserted into PML in chromosome 15, formed ins (15;17) . The 7 cryptic APL cases had no PML-RARα gene subtype specificity, involving 5 cases in L subtype, 1 case in S subtype and 1 case in V subtype respectively. After the treatment of retinoic acid and arsenic or anthracyclines, 6 cases achieved complete remission, 1 case died of intracranial hemorrhage on the 6th day of therapy.@*Conclusion@#The size and covering position of PML-RARα probe should be taken into account when PML-RARα was performed by FISH on APL patients. Furthermore, combination with Metaphase FISH could improve the recognition of cryptic APL. There were no differences between the cryptic and common APL patients in terms of clinical features and treatment choices. Cryptic APL patients also had a good response to the therapy of retinoic acid and arsenic or anthracyclines.

Anaplastic Large Cell Lymphoma with Massive Eosinophilia and Complex Karyotype Initially Misdiagnosed as Chronic Eosinophilic Leukemia

We report a patient with massive eosinophilia and a complex karyotype that was initially misdiagnosed as chronic eosinophilic leukemia (CEL), but later diagnosed as anaplastic large cell lymphoma (ALCL) masked by massive eosinophilia. The complex karyotype observed at initial diagnosis remained unchanged later, after the evidence of bone marrow involvement of ALCL was obtained. At diagnosis, genetic aberrations corresponding to metaphase cytogenetics were not identified by interphase fluorescence in situ hybridization, although abnormal results were noted at follow-up. Together, these observations indicate that the complex karyotype at initial work-up has been derived from a low proportion of lymphoma cells with high mitotic ability that were not identified by microscopy, rather than from massive eosinophils. These findings suggest that our patient had ALCL with secondary eosinophilia rather than CEL since initial diagnosis.

Correlation of Pregnant Metaphase Serum CRP and Premature Rupture of Membranes and Preterm Birth / 医学研究杂志

Objective To explore the correlation of pregnant metaphase serum CRP and premature rupture of membranes and preterm birth.Methods Totally 300 cases maternals in pregnant metaphase were sampled to collected their fasting venous blood in the middle of elbow to detected the levels of serum CRP by immunoturbidimetry,and allof the maternals were received follow-up until end of the delivery.Of the 300 cases,there were 16 cases with premature rupture of membranes,28 cases with preterm birth,and 256 cases with full -term birth.The levels of serum CRP in pregnant metaphase of the three groups were compared,and the value of threshold to predict premature rupture of membranes and preterm birth was analyzed.Results The levels of serum CRP in pregnant metaphase of the groups of premature rupture of membranes and preterm birth were hoth higher than the group of full-term birth(P ＜ 0.05),but there were not significant difference between the two groups(P ＞ 0.05),and the threshold of the serum CRP were 13.9,10.8mg/L respectively.The sensitivity of serum CRP in pregnant metaphase to predict premature rupture of membranes was 68.75％,and the specificity was 98.94％.The sensitivity of serum CRP in pregnant metaphaseto predict preterm birth was 75.00％,and the specificity was 98.90％.Conclusion The level of pregnant metaphase serum CRP beyond a certain range may increase the possibility of premature rupture of membranes and preterm birth,which is valuable to predict premature rupture of membranes and preterm birth.

Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation / 대한생식의학회지

OBJECTIVE: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca²⁺ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). METHODS: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca²⁺ chelator to investigate the effect of Ca²⁺ oscillations on their maturation. RESULTS: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca²⁺ chelator-treated group. CONCLUSION: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca2+ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca²⁺ oscillations driven by fertilization.

Dysregulation of Telomere Lengths and Telomerase Activity in Myelodysplastic Syndrome

BACKGROUND: Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA. METHODS: The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated. RESULTS: MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively). CONCLUSIONS: The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.

Chromosomal differences in Sabaletas (Brycon henni) from the upper basins of the Cauca and Patia rivers / Diferencias cromosómicas en sabaletas (Brycon henni) procedentes de las cuencas altas de los ríos Cauca y Patía / Diferenças cromossômicas de sabaletas (Brycon henni) procedentes das bacias altas dos rios Cauca e Patía

This research chromosomically compared Sabaletas (Brycon henni) from the upper basins of the Cauca and Patia rivers in the Cauca department. For the study 6 specimens were captured in each site and transported to the Genetic toxicology laboratory of the Universidad del Cauca. Once there, kidney cells were extracted and cultivated, spreads of cells in metaphase were done, pictures were taken with an optical microscope, and the modal chromosome number and types of chromosomes were determined according to the position of the centromere, manually and using an assisted segmentation system for image processing. The results showed that Brycon henni has a chromosomal number of 2n=52, and two karyological formulas specific to each of the basins studied: Brycon henni from the Cauca river had the kariotype formula 22m+16sm+14st, while from the Patia river basin the formula was 8m+10sm+26st+8t. These differences suggest that the specimens studied from both basins are probably in a speciation process due to geographical isolation. Moreover, no differences in the chromosome number were found between males and females on both basins. Additionally, the physico-chemical parameters of the water from the upper basins of the Cauca and Patia rivers have not shown significant changes that could affect the physiological conditions of these fishes.

Esta investigación realizó la comparación cromosómica de la sabaleta (Brycon henni) pertenecientes a las cuencas altas de los ríos Cauca y Patía en el departamento del Cauca. Para el estudio fueron capturados seis ejemplares de cada sitio y se trasladaron al Laboratorio de Toxicología Genética de la Universidad del Cauca, una vez allí se procesaron cultivos celulares in vitro extraídos del riñón 5, 9, 16, realizados los extendidos de las metafases, se tomaron las fotografías con microscopio óptico y se determinó el número cromosómico modal y el tipo de cromosomas según la posición del centrómero, en forma manual y con ayuda de un sistema asistido de segmentación para el procesamiento de imágenes 23 (López y Pinto, 2007), mediante este método se obtuvo: que la especie Brycon henni presenta un número cromosómico de 2n=52 y dos fórmulas cariológicas específicas para cada una de las cuencas estudiadas así: Brycon henni del rio Cauca su fórmula cariológica fue: (22m+16sm+14st) y para Brycon henni de la cuenca del rio Patía (8m+10sm+26st+8t). Estas diferencias sugieren que los ejemplares estudiados en las dos cuencas probablemente se encuentren en proceso de especiación debido al aislamiento geográfico. Por otra parte no se presentaron diferencias en el número de cromosomas entre machos y hembras de Brycon henni en las dos cuencas estudiadas. Asimismo los parámetros físico-químicos del agua de las cuencas altas de los ríos Cauca y Patía no han mostrado cambios significativos que afecten las condiciones fisiológicas de estos peces.

Esta pesquisa realizou a comparação cromossômica da sabaleta (Brycon henni) pertencentes as bacias altas dos rios Cauca e Patía no departamento de Cauca. Para realizar o estudo foram capturados 6 exemplares de cada lugar e se trasladaram ao Laboratório de Toxicologia e Genética da Universidad del Cauca (Popayán, Cauca), uma vez ali, se processaram os cultivos celulares in vitro extraídos do rim, se realizaram os estendidos da metáfase, se tiraram as fotografias com microscópio ótico e se determinou o número cromossômico modal e o tipo de cromossomos segundo a posição do centrômero. Isto se fez em forma manual e com a ajuda de um sistema assistido de segmentação para o processamento de imagens. Com a utilização desta técnica encontrou-se que: a espécie Brycon henni apresenta um número cromossômico de 2n=52 e dois formulas cariológicas especificas para cada uma das bacias estudadas, assim: a formula cariológica do Brycon henni do rio Cauca foi: (22m+16sm+14st) e do Brycon henni do rio Patía: (8m+10sm+26st+8t). Estas diferenças sugerem que os exemplares estudados nas duas bacias provavelmente se encontram em processo de especiação devido ao isolamento geográfico. De outro lado, não se apresentaram diferenças no número de cromossomos entre machos e fêmeas de Brycon henni nas duas bacias estudadas. Do mesmo jeito, os parâmetros físico-químicos da agua das bacias dos rios Cauca e Patía não mostraram mudanças significativas que afetem as condições fisiológicas desta espécie de peixe.

Application of interim 18 F-FDG PET-CT in lymphoma / 国际肿瘤学杂志

Interim fluorine 1 8 fluorodeoxyglucose (1 8 F-FDG)positron emission tomography (PET) integrated with computed tomography (CT) provides both metabolic and morphologic information,which becomes one of the most sensitive tools to evaluate the efficacy of the therapy and predict the prognosis of patients with lymphoma.Meanwhile,it is the basis for guiding trial design and changing clinical practice.How-ever,it is still a controversial issue in the ideal utilization of interim 1 8 F-FDG PET-CT imaging for the patients with lymphoma.

Mitotic activity of the brain ganglia in different subphases of the last larval instar of Melipona quadrifasciata lepeletier (Hymenoptera, apidae, meliponini) / Atividade mitótica do gânglio cerebral em diferentes fases do último instar larval de melipona quadrifasciata (hymenoptera, apidae, meliponini) / MITOTIC ACTIVITY OF THE BRAIN GANGLIA IN DIFFERENT SUBPHASES OF THE LAST LARVAL INSTAR OF MELIPONA QUADRIFASCIATA LEPELETIER (HYMENOPTERA, APIDAE, MELIPONINI)

In recent years, the number of cytogenetic studies on Melipona species has increased considerably. However, most cytogenetic techniques used for these studies require preparations with a great number of metaphase cells for reliable analysis of the karyotypes. The present study seeks to evaluate which subphase of the last larval instar of Melipona quadrifasciata Lepeletier provides the greatest number of metaphases, which is here considered a direct measure of mitotic activity. A total of 25 defecating larvae were selected based on the quantity of feces in their intestines, so as to maintain five larvae in each of the five different developmental subphases. The brain ganglia of each larva were extracted and used for cytogenetic preparation. The number of metaphase mitotic cells per preparation was counted. An analysis of variance (ANOVA) model, with Tukey's post hoc tests, was conducted. It was observed that larvae in the second subphase, defined here as the subphase in which feces were visible below the segment VII, provided the greatest number of metaphases. Therefore, it is the most appropriate developmental subphase for cytogenetic preparations of brain glanglia in M. quadrifasciata and possibly in other Melipona species.

Estudos citogenéticos envolvendo o gênero Melipona vêm aumentando nos últimos anos. Entretanto, a utilização de várias técnicas para o estudo do cariótipo exigem preparações com um grande número de células em metáfase para uma análise confiável das características citogenéticas das espécies. O presente estudo teve como principal objetivo avaliar, para Melipona quadrifasciata, o instar do desenvolvimento larval mais adequado para estudos citogenéticos, no que se refere à atividade mitótica. Foram selecionadas 25 larvas defecantes divididas em cinco subfases de acordo com a quantidade restante de fezes no intestino. Os gânglios cerebrais das larvas foram extraídos e utilizados para a obtenção dos cromossomos mitóticos metafásicos. O número de metáfases por lâmina foi contabilizado para cada indivíduo e os dados submetidos à análise de variância (ANOVA) e ao teste de TUKEY. Foi observado que larvas da segunda subfase, definidas aqui como a subfase na qual as fezes se encontram na altura do VII segmento apresentaram o maior número de metáfases. Logo, esta é a subfase mais indicada para obtenção de grande número de metáfases em células do gânglio cerebral de Melipona quadrifasciata e, possivelmente, para outras espécies do gênero Melipona.

Caracterización citogenética de Caesalpinia spinosa de los distritos de Tarma y Palca (Junín)

Se analizan los cromosomas somáticos de Caesalpinia spinosa (Feuillée ex Molina) Kuntze “Tara”, de poblaciones silvestres de las localidades de Huinco y Palca (Junín). La especie es diploide (2n=24). Los cromosomas son de pequeño tamaño. Aunque los cariotipos de ambas localidades muestran el mismo número cromosómico, se encontraron diferencias en los parámetros morfológicos de los mismos, siendo la fórmula cariotípica para la localidad de Huinco: 6m + 6 sm y para la localidad de Palca: 5m + 7 sm.

Somatic chromosomes of Caesalpinia spinosa (Feuillée ex Molina) Kuntze, “Tara”, wild popu- “Tara”, wild populations of Huinco and Palca (Junín) regions were studied. The specie were diploid (2n=24). Chromosomes were small. The karyotypes showed the same chromosome number, they found differences in morphological parameters of the same, with the karyotype formula for the town of Huinco: 6m + 6 sm and the town of Palca: 5m + 7 sm.

Anormalidades cromossômicas entre pacientes com cardiopatia congênita / Chromosomal abnormalities in patients with congenital heart disease

FUNDAMENTO: As anormalidades cromossômicas (ACs) representam importante causa de cardiopatia congênita (CC). OBJETIVO: Determinar a frequência, os tipos e as características clínicas de ACs identificadas em uma amostra prospectiva e consecutiva de pacientes com CC. MÉTODO: Nossa amostra foi composta por pacientes com CC avaliados em sua primeira hospitalização em uma unidade cardíaca de tratamento intensivo de um hospital pediátrico de referência do sul do Brasil. Todos os pacientes foram submetidos à avaliação clínica e citogenética, através do cariótipo de alta resolução. Os defeitos cardíacos foram classificados segundo Botto e cols. Na análise estatística utilizou-se o qui-quadrado, o teste exato de Fisher e odds ratio (p < 0,05). RESULTADOS: Nossa amostra foi composta de 298 pacientes, 53,4% do sexo masculino, com idades variando de um dia a 14 anos. Anormalidades cromossômicas foram observadas em 50 pacientes (16,8%), sendo que 49 deles eram sindrômicos. Quanto às ACs, 44 delas (88%) eram numéricas (40 pacientes com +21, dois com +18, um com triplo X e um com 45,X) e seis (12%) estruturais [dois pacientes com der(14;21), +21, um com i(21q), um com dup(17p), um com del(6p) e um com add(18p)]. O grupo de CCs mais associado a ACs foi o do defeito de septo atrioventricular. CONCLUSÕES: ACs detectadas pelo cariótipo são frequentes entre pacientes com CC. Assim, os profissionais - especialmente aqueles que trabalham em serviços de cardiologia pediátrica - devem estar cientes das implicações que a realização do cariótipo pode trazer, tanto para o diagnóstico, tratamento e prognóstico desses pacientes como para o seu aconselhamento genético.

BACKGROUND: Chromosomal abnormalities (CAs) are an important cause of congenital heart disease (CHD). OBJECTIVE: Determine the frequency, types and clinical characteristics of CAs identified in a sample of prospective and consecutive patients with CHD. METHOD: Our sample consisted of patients with CHD evaluated during their first hospitalization in a cardiac intensive care unit of a pediatric referral hospital in Southern Brazil. All patients underwent clinical and cytogenetic assessment through high-resolution karyotype. CHDs were classified according to Botto et al. Chi-square, Fisher exact test and odds ratio were used in the statistical analysis (p < 0.05). RESULTS: Our sample consisted of 298 patients, 53.4% males, with age ranging from 1 day to 14 years. CAs were observed in 50 patients (16.8%), and 49 of them were syndromic. As for the CAs, 44 (88%) were numeric (40 patients with +21, 2 with +18, 1 with triple X and one with 45,X) and 6 (12%) structural [2 patients with der(14,21), +21, 1 with i(21q), 1 with dup(17p), 1 with del(6p) and 1 with add(18p)]. The group of CHDs more often associated with CAs was atrioventricular septal defect. CONCLUSIONS: CAs detected through karyotyping are frequent in patients with CHD. Thus, professionals, especially those working in Pediatric Cardiology Services, must be aware of the implications that performing the karyotype can bring to the diagnosis, treatment and prognosis and for genetic counseling of patients and families.

In vitro maturation, fertilization, embryo development & clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles.

Background & objectives: The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII) arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM) metaphase I (MI) oocytes subjected to intracytoplasmic sperm injection (ICSI) at different time intervals after extrusion of the first polar body (1PB) in in vitro fertilization (IVF) cycles. Methods: A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII) (27.1%) matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10), 8-11 h (n=4) and 23-26 h (n=10). Fertilization and development potential were evaluated in both studies. Results: Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I) and 8-11 h (group II) after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born. Interpretation & conclusions: Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes. Further, human IVM oocytes need between 2-6h after the 1PB extrusion to complete its maturation.

Comparación de la calidad embrionaria entre fertilización in vitro (FIV) y cultivo intravaginal de ovocitos (INVO) en el Centro Colombiano de Fertilidad y Esterilidad - CECOLFES, Bogotá, Colombia / Comparing embryo quality between in vitro fertilization (IVF) and intravaginal ovocyte culture (INVO) in the Colombian Centre for Fertility and sterility - CECOLFES, Bogotá, Colombia

Objetivo: comparar la calidad embrionaria y describir las tasas de implantación, embarazo y aborto en las técnicas de fertilización in vitro (FIV) y el cultivo intravaginal de ovocitos. Materiales y métodos: cohortes históricas de pacientes con tratamiento de fertilización in vitro y el cultivo intravaginal de ovocitos en el Centro Colombiano de Fertilidad y Esterilidad (Cecolfes) durante el año 2010. Se incluyeron 137 pacientes aspiradas dentro de los cuatro grupos de estudio: Grupo 1, FIV/Incubadora; Grupo 2, FIV/INVO; Grupo 3, ICSI/INVO, y Grupo 4, ICSI/Incubadora. Se midió el peso de la paciente, el número de ovocitos recuperados y óvulos maduros (MII), la tasa de implantación y la tasa de embarazo y aborto en cada uno de los grupos. Se realizó análisis mediante la prueba de Kruskal-Wallis; la calidad embrionaria fue evaluada con un análisis de covarianza multivariado (MANOVA). Resultados: se observó diferencia significativa en la calidad embrionaria entre las dos técnicas FIV e INVO (p = 0,0388). En la técnica INVO se presentaron mayores tasas de división embrionaria (μ = 7,35/INVO frente a 6,64/Incubadora) y menor fragmentación (μ = 4,67/INVO frente a 4,59/ Incubadora). En cuanto a la tasa de implantación, embarazo y aborto se obtuvieron más altos porcentajes en los grupos INVO. Conclusión: la técnica INVO se asoció a mejor calidad embrionaria. Las tasas de implantación, embarazo y bajas tasas de aborto son semejantes a las descritas en la técnica FIV.

Objective: Comparing embryo quality and describing implantation, pregnancy and abortion rates regarding in vitro fertilization (IVF) and intravaginal oocyte culture (INVO) techniques. Materials and methods: The study involved historical cohorts of patients undergoing IVF and INVO treatment in the Colombian Fertility and Sterility Centre (Centro Colombiano de Fertilidad y Esterilidad – Cecolfes) during 2010. It involved 137 aspirated patients, covering four study groups: Group 1 IVF/incubator, Group 2 IVF/INVO, Group 3 ICSI/INVO and Group 4 ICSI/incubator. The patients’ weight, the number of ovocytes retrieved, mature ovules (M2), implantation rate, pregnancy and abortion rates were measured in each group. The Kruskal-Wallis test was used for statistical analysis; embryo quality was evaluated by multivariate covariance analysis (MANOVA). Results: A significant difference was observed regarding embryo quality between IVF and INVO (p = 0.0388), the INVO technique having higher embryo cleavage rates (μ = 7.35/INVO cf 6.64/ incubator) and lower embryo fragmentation (μ = 4.67/INVO cf 4.59/incubator). INVO groups also had higher percentages concerning their implantation, pregnancy and abortion rates. Conclusion: The INVO technique led to obtaining better quality embryos; implantation, pregnancy and abortion rates were similar to those described for the IVF technique.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida / Apparently matured oocytes injected in telophase I have worse outcomes from assisted reproduction

OBJETIVO: Avaliar o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozoide (ICSI) e comparar os resultados da injeção intracitoplasmática de espermatozoide entre os oócitos em telófase I (TI) e metáfase II (MII), e entre aqueles em metáfase II com e sem fuso celular visível. MÉTODOS: Estudo prospectivo que incluiu 106 pacientes inférteis submetidas à injeção intracitoplasmática de espermatozoide. Foram incluídas pacientes com idade menor ou igual a 38 anos, hormônio folículo estimulante (FSH) basal menor que 10 mIU/mL e índice de massa corpórea (IMC) menor que 30 kg/m². Foram excluídas pacientes com doenças sistêmicas, com qualquer infecção ativa, tabagistas ou que fizeram uso de medicações hormonais e anti-inflamatórias hormonais e não hormonais nos últimos dois meses, previamente à programação para o procedimento de reprodução assistida. Os oócitos com extrusão do primeiro corpúsculo polar foram avaliados pela microscopia de polarização, imediatamente antes da realização da injeção intracitoplasmática de espermatozoide, e caracterizados quanto ao estágio de maturação nuclear (telófase I ou metáfase II). Os oócitos em metáfase II foram avaliados de acordo com a presença ou não do fuso meiótico. Foram analisadas as taxas de fertilização, clivagem e o número de embriões de boa qualidade no segundo dia (D2) de desenvolvimento. Os dados foram analisados comparativamente através do teste exato de Fisher. Em todas as análises foi considerado o nível de significância de 5% (p<0,05). RESULTADOS: O fuso meiótico de 516 oócitos foi visualizado através da microscopia de polarização. Dezessete dos 516 oócitos avaliados estavam em telófase I (3,3%) e 499 (96,7%) em metáfase II. Os oócitos injetados em telófase I apresentaram taxas de fertilização significativamente menores do que os injetados em metáfase II (53 e 78%, respectivamente) e não produziram nenhum embrião de boa qualidade no segundo dia. Comparando-se os oócitos com e sem fuso celular visível, não foi observada diferença significativa nos resultados de injeção intracitoplasmática de espermatozoide. CONCLUSÕES: Oócitos injetados em telófase I apresentaram menores taxas de fertilização quando comparados aos em metáfase II. É possível que a análise do estágio de maturação nuclear oocitária, por meio da microscopia de polarização, possa ser utilizada como fator de predição das taxas de fertilização pós-injeção intracitoplasmática de espermatozoide.

PURPOSE: To evaluate the nuclear maturation stage and the presence of meiotic spindles of in vivo matured oocytes from infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI) and compare intracytoplasmic sperm injection outcomes between oocytes in telophase I (TI) and metaphase II (MII), and the ones with and without visible meiotic spindle. METHODS: A prospective and controlled study with 106 infertile patients who underwent ovarian stimulation for intracytoplasmic sperm injection purposes. Patients aged 38 years or less, with basal follicle stimulating hormone (FSH) less than 10 mIU/mL and body mass index (BMI) less than 30 kg/m². Were included patients presenting any systemic diseases, any active infection, smokers or patients who had been using hormonal medications and hormonal and nonhormonal anti-inflammatory drugs for the past two months prior to the assisted reproduction procedure were excluded. The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged by polarization microscopy immediately before intracytoplasmic sperm injection and characterized according to nuclear maturation stage (telophase I and metaphase II) and to the presence of a meiotic spindle. We analyzed the fertilization rates, cleavage, number of good quality embryos on the second day (D2) from oocytes on telophase I versus those in metaphase II, and metaphase II visible spindle versus non-visible ones. Data were analyzed comparatively by Fisher's exact test. The level of significance was set at 5% in all analyses (p<0.05). RESULTS: The meiotic spindles of 516 oocytes were imaged using polarization microscopy. From the 516 oocytes analyzed, seventeen were in telophase I (3.3%) and 499 (96.7%) in metaphase II. The oocytes injected in telophase I had significantly lower fertilization rates than those injected in metaphase II (53 and 78%, respectively) and produced no good quality embryos on day 2. When the oocytes with and without a visible meiotic spindle were compared, there was no significant difference in the intracytoplasmic sperm injection results. CONCLUSIONS: Oocytes injected in telophase I showed lower fertilization rates when compared to those in metaphase II. It is possible that the analysis of oocyte nuclear maturation by polarization microscopy can be used as a predictor of fertilization after intracytoplasmic sperm injection.

Efeito do soro de cadela em estro na maturação in vitro de ovócitos caninos / Effect of estrus bitch serum on in vitro maturation of canine oocyte

Avaliou-se o efeito do soro de cadela em estro na maturação in vitro de ovócitos caninos, utilizando-se 92 ovócitos de cadelas, submetidas à cirurgia eletiva de ovarioisterectomia. Os ovócitos foram selecionados e distribuídos em dois tratamentos: T1 (n = 48), ovócitos cultivados in vitro durante 96 horas utilizando meio base - TCM199 + 5µg/mL de LH + 20µg/mL de FSH - mais 10 por cento de soro inativado de vaca em estro e T2 (n = 44), ovócitos cultivados em meio base mais 10 por cento de soro inativado de cadela em estro. O percentual de ovócitos observados em metáfase I não indicou diferenças (P>0,05) entre T1 (2,1 por cento) e T2 (0,0 por cento), porém a taxa de ovócitos maduros (metáfase II) foi diferente (P<0,05), sendo 27,1 por cento em T1 e 47,7 por cento em T2. O mesmo fato ocorreu com a taxa de cromatina condensada (P<0,01), com 14,6 e 0,0 por cento, respectivamente. Nos ovócitos sem configuração cromossômica, não foram observadas diferenças (P>0,05), sendo 56,3 por cento em T1 e 52,3 por cento em T2. Estes resultados indicam que a adição de soro de cadela em estro no meio de cultivo oferece melhores condições para o desenvolvimento in vitro, quando comparado à de soro de vaca em estro.

This study aimed to evaluate the effect of estrus on in vitro canine oocyte. A total of 92 oocyte from bitches under ovary-hysterectomy surgery was used. The oocytes were selected and randomly assigned to two different treatments, being T1 (n = 48) in vitro cultured for 96h using basic medium (TCM199 + 5µg/mL of LH + 20µg/mL of FSH), plus 10 percent of cow inactive serum in estrus and T2 (n = 44) basic medium plus 10 percent of bitch inactive serum in estrus. The percentage of oocyte observed on metaphase I do not indicate a difference (P>0.05) between T1 (2.1 percent) and T2 (0.0 percent). However, the rate of mature oocyte (metaphase II) was different (P<0.05), being 27.1 percent for T1 and 47.7 percent for T2. There was difference (P<0.05) in the condensed chromatin rate for T1 (14.6 percent) and T2 (0.0 percent), respectively. There was no difference (P>0.05) between T1 (56.3 percent) and T2 (52.3 percent) in oocyte with no chromosome configuration. These results indicate that supplementation with estrus bitch serum on culture media offer better conditions to in vitro development, when compared to estrus cow serum.

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.

Variant Philadelphia Chromosome Identified by Interphase Fluorescence In Situ Hybridization (FISH) without Evidence on G-banded Karyotyping and Metaphase FISH / 대한진단검사의학회지

A variant Philadelphia chromosome (Ph) is generated from translocation of one or more partner chromosomes in addition to chromosomes 9 and 22. We have described the cases of 2 patients bearing variant Ph detected by interphase FISH but not detected by G-banded karyotyping and metaphase FISH. FISH was performed using BCR/ABL dual color dual fusion translocation probes (Abbott Molecular, USA). A 52-year-old man was diagnosed with acute leukemia of mixed phenotype. G-banded karyotyping showed 46,XY,t(9;22)(q34;q11.2)[12]/47,idem,+der(22)t(9;22)[5]/46,XY[3]. Interphase FISH revealed nuc ish(ABL1,BCR)x3(ABL1 con BCRx2)[329/450]/(ABL1,BCR)x4(ABL1 con BCRx3)[5/450]/(AL1,BCR)x3(ABL1 con BCRx1)[44/450]. Metaphase FISH showed ish (9;22)(ABL1+,BCR1+;BCR+,ABL+)[22]/der(22)(BCR+,ABL1+)[3]. The other case was that of a 31-yr-old male patient diagnosed with CML in the blastic phase. G-banded karyotyping of all 20 metaphase cells showed 47,XYYc,dup(1)(q21q32),del(7)(p11.2),t(9;22)(q34;q11.2). Interphase FISH revealed nuc ish(ABL1,BCR)x3(ABL1 con BCRx2)[254/600]/(ABL1,BCR)x3(ABL1 con BCRx1)[191/600]. Metaphase FISH showed ish t(9;22)(ABL1+,BCR+;BCR+,ABL1+)[16]. These results suggest that typical t(9;22) and variant Ph may coexist in the same patient, and interphase FISH may facilitate the detection of the variant Ph that cannot be detected by G-banded karyotyping alone.

Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.