Presumptive germ cells derived from mouse pluripotent somatic cell hybrids.

The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.

Cabergoline reduces the early onset of ovarian hyperstimulation syndrome: a prospective randomized study.

Prophylactic use of cabergoline has been associated with a decrease in the severity of ovarian hyperstimulation syndrome (OHSS). A prospective randomized study was designed to evaluate the potential of cabergoline to decrease the incidence of OHSS in high-risk patients undergoing assisted reproductive technology treatment; 166 patients with oestradiol concentrations over 4000 pg/ml on the day of human chorionic gonadotrophin (HCG) administration were evaluated. They all received 20 g routine preventive intravenous human albumin on the day of oocyte retrieval. They were then randomized into two groups: group A (n = 83) received 0.5 mg oral cabergoline per day for 3 weeks beginning on the day after oocyte retrieval, and group B (n = 83) received no medication. 'Early' OHSS was defined as being when the onset of the syndrome was initiated during the first 9 days after HCG administration, and 'late' OHSS was defined as being when the onset of the syndrome was initiated from 10 days after HCG administration. In group A, no patients progressed to 'early' OHSS and nine patients (10.8%) developed 'late' OHSS; in group B, 12 patients (15.0%) progressed to 'early' OHSS and three (3.8%) to 'late' OHSS. Although the risk of 'early' OHSS decreased significantly (P < 0.001), the risk of 'late' onset OHSS did not. The two groups presented no changes in pregnancy, implantation or miscarriages rates.

In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes.

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.

ICSI and day 5 embryo transfers: higher implantation rates and lower rate of multiple pregnancy with prolonged culture.

This retrospective review study, carried out in a private IVF clinic, compared pregnancy and implantation rates with day 3 versus day 5 embryo transfers in a selected group of patients. Participants were patients who failed to achieve pregnancy in at least one previous attempt with embryo transfer on days 2 or 3, and had more than five oocytes fertilized. A total of 296 patients who had undergone day 3 (group A) transfers were compared with 154 who had undergone day 5 transfers (group B). Interventions were intracytoplasmic sperm injection (ICSI), day 3 and day 5 embryo transfer. Outcome measures were pregnancy, implantation, multiple gestation and blastocyst formation rates. Overall, 86.4% of embryos were at the six- to eight-cell stage at 72 h and 30% developed to blastocyst by day 5. The mean number of embryos transferred was 4.0 on day 3 and 3.0 on day 5. Pregnancy and implantation rates were 34.8 and 11.5% in group A, versus 45.3 and 18.5% in group B. Multiple gestation rate was 47.1% in group A and 28.5% in group B. Prolonging embryo culture in vitro to day 5 improved embryo selection and implantation rates. A significant decrease in high order gestations was achieved by reducing the number of embryos transferred, without compromising the pregnancy rates.

The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin.

Fertilization rates increased continuously with the time elapsed after administration of hCG, reaching a peak of 84% when intracytoplasmic sperm injection (ICSI) was performed >41 hours after hCG administration. However, the highest implantation rate, 24%, was achieved when ICSI was performed 37-41 hours after hCG administration.

Location of the embryo-transfer catheter guide before the internal uterine os improves the outcome of in vitro fertilization.

The objective of this study was to assess whether positioning the embryo transfer (ET) catheter guide at the time of embryo expulsion, before or beyond the internal os, has an impact on IVF cycle outcome. We performed a retrospective study comparing IVF outcomes in relation to the ET guide position relative to the internal uterine os. We analyzed ultrasound-guided ETs in IVF-intracytoplasmic sperm injection (ICSI) cycles, performed with the tip of the ET catheter guide just before the internal os (group 1) and beyond the internal os (group 2). Implantation and pregnancy rates were significantly better in group 1 than in group 2, at 14.8% versus 11.8% and 57.3% versus 43.1, respectively. We conclude that passing the ET catheter guide beyond the internal os reduces implantation and pregnancy rates.

Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia.

OBJECTIVE: To determine whether the injection of testicular spermatozoa results in more viable embryos (higher implantation rate) than injection of epididymal spermatozoa in cases of obstructive azoospermia. DESIGN: Retrospective analysis of 265 cases of testicular sperm aspiration (TESA) and percutaneous sperm aspiration (PESA), including 185 cases of obstructive azoospermia. SETTING: Private Infertility clinic. PATIENT(S): None, charts review. INTERVENTION(S): None, charts review. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR), implantation rate. RESULT(S): Although fertilization rates were higher in the PESA group, implantation rates were significantly better in the TESA group. There was also a trend to higher ongoing PR and lower miscarriage rates in TESA cases. CONCLUSION(S): In cases of obstructive azoospermia, embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa.

Toward pre-conceptual genetic analysis of human spermatozoa.

Nuclei of mature mammalian spermatozoa are extraordinarily resistant to chemical and thermal injury. Additionally, decondensation of spermatozoa DNA can be accompanied by little or no visual changes of the sperm head. This study tested whether human spermatozoa could be recovered following several cycles of primer extension preamplification (PEP) and used to achieve fertilization and subsequent development of human oocytes. An attempt was also made to amplify PEP buffer after spermatozoon removal. The results demonstrate that the sperm head can be successfully recovered following treatment with KOH or proteinase K followed by one to four cycles of PEP. It is also shown that following this treatment, the spermatozoa can be injected into the oocytes and will transform into a pronucleus if the oocyte is activated by sperm cytosolic fraction. In some cases, it was also possible to obtain polymerase chain reaction signals using a buffer after sperm cells were removed following several cycles of PEP. Although sperm participation in development was confirmed by fluorescence in-situ hybridization, light microscopy revealed some degree of damage to spermatozoal chromosomes. It is concluded that pre-conceptual analysis of sperm cells may be possible, but more research is necessary to determine the optimal conditions that would preserve sperm DNA integrity while allowing accurate diagnoses.

Evidence of parthenogenetic origin of ovarian teratoma: case report.

This case report represents one of the few documented cases of parthenote embryo retrieval from an IVF patient with a history of ovarian teratomas. A 29-year-old woman presented at our centre with a history of primary infertility for 6 years due to male factor. She had undergone left oophorectomy 4 years before due to an ovarian teratoma. An ultrasound scan performed during basal evaluation revealed two complex images in the right ovary suggesting teratomas, measuring 2.5 x 2.4 and 1.7 x 1.3 cm. A significant extent of sonographically normal ovarian parenchyma was present, and the patient underwent the long leuprolide acetate protocol of ovarian stimulation with recombinant FSH for an IVF-ICSI cycle. She had 13 metaphase II (MII), four metaphase I (MI), two germinal vesicle (GV) oocytes and one 4-cell embryo retrieved. Eight out of nine injected oocytes were fertilized normally while one was unfertilized. Embryo transfer was carried out 72 h after retrieval. The 4-cell (parthenote) embryo recovered at oocyte retrieval continued to cleave in culture, developing into a 7-cell embryo by the next day. The embryo was morphologically normal, presenting an evident nucleus in each blastomere. Fluorescent in situ hybridization (FISH) returned two signals for the X chromosome in each blastomere that was analysed. Of the eight normally fertilized embryos, three were transferred, resulting in a normal singleton pregnancy and the birth of a healthy baby.

O papel da histeroscopia em infertilidade / The role of hysteroscopy in infertility

Os autores apresentam uma revisäo de vários trabalhos publicados na literatura no período de janeiro de 1978 a julho de 1997 sobre histeroscopia, em particular o seu papel na infertilidade. Discutem-se as principais indicaçöes da histeroscopia, (HSC) nos pacientes que consultam por infertilidade e além disso apresentam-se a relaçäo da HSC com a histerosalpingografia (HSG), Laparoscopia e Ultra-sonografia Transvaginal (USGTV) que säo exames utilizados na rotina da mulher infértil. Aspectos relativos ao diagnóstico e tratamento histeroscópico das doenças da cavidade uterina presentes nesse grupo de pacientes, tais como septo uterino, sinéquias uterinas, miomas submucosos, pólipos endometriais e endometrite säo comentados de maneira sumária. Os autores descrevem ainda as indicaçöes da HSC nas pacientes submetidas à Fertilizaçäo In vitro (FIV) e a outras Técnicas de Reproduçäo Assistida.

Gravidez com FSH-recombinante: relato de caso / Recombinant FSH and pregnancy: case report

OBJETIVO: relatar um caso de gravidez obtida após tratamento de fertilizaçäo "in vitro" usando FSH-recombinante. DESENHO: estudo restrospectivo de caso clínico. MATERIAL E MÉTODO: foi realizada técnica de fertilizaçäo in vitro (FIV), associada a técnica de injeçäo intracitoplasmática de espermatozóide (ICSI) em paciente de 42 anos de idade. Foram obtidos dois óvulos após induçäo da ovulaçäo com protocolo longo usando acetato de leuprolide e FSH-recombinante. Após a transferência dos embriöes o suporte da fase lútea foi feito com estradiol transdérmico e progesterona oleosa. RESULTADO: foram aspirados 2 oócitos M2 com 100 por cento de fertilizaçäo após o uso da ICSI. No 12§ dia pós transferência de 02 embriöes com 0 por cento de fragmentaçäo, foi constatada gravidez através de dosagem sérica de b-HCG e confirmada através de ultra-sonografia transvaginal realizada no 26§ dia pós transferência quando visualizou-se saco gestacional único e tópico com embriäo com comprimento crânio nádega de 2,8 mm, atividade cardíaca presente e vesícula vitelínica normal, compatível com gestaçäo única de 5 semanas de 3 dias. CONCLUSÃO: os autores relatam um caso de gravidez obtida através da técnica de FIV-ICSI em paciente de 42 anos onde foi utilizado FSH recombinante na induçäo da ovulaçäo.

Gravidez quádrupla e nascimento após injeçäo intracitoplasmática de espermatozóides (ICSI) totalmente imóveis / Quadruplets pregnancy after intracytoplasmic sperm injection (ICSI) of immobile sperm

OBJETIVO: relatar um caso de gravidez quadrigemelar após Injeçäo Intracitoplasmática de Espermatozóide (ICSI) com espermatozóides imóveis, obtidos através de Aspiraçäo direta do testículo (TESA), que resultou no nascimento de quatro crianças saudáveis. DELINEAMENTO: estudo retrospectivo de caso clínico. Local: Clínica e Centro de Pesquisa em Reproduçäo Humana Roger Abdelmassih. MÉTODO: Foi realizada técnica de Fertilizaçäo "in vitro" (FIV), associada a técnica de Injeçäo Intracitoplasmática de Espermatozóide (ICSI) em paciente de 37 anos de idade, utilizando espermatozóides totalmente imóveis recuperados por aspiraçäo direta do testículo. Os óvulos foram obtidos após induçäo da ovulaçäo com protocolo longo usando acetato de leuprolide e FSH altamente purificado associado com hMG (Pergonal, Serono laboratórios). Após a transferência dos embriöes, o suporte da fase lútea foi feito com estradiol transdérmico e progesterona oleosa. RESULTADOS: com a injeçäo de espermatozóides imóveis, 6 dos 7 oócitos injetados apresentaram fertilizaçäo e clivagem normais. No 17§ dia após transferência de 4 embriöes, foi constatada gravidez através de dosagem sérica de b-HCG. Através de ultra-sonografia foi constatada gestaçäo quadrigemelar com todos embriöes viáveis que resultou no nascimento de quatro crianças saudáveis através de TESA, resultando no nascimento de 4 crianças normais, demonstrando a viabilidade do tratamento proposto.

Influência da qualidade seminal no resultado da injeçäo intracitoplasmática de espermatozóides (ICSI) / Influence of semen quality in the result of the intracytoplasmatic sperm injection technique (ICSI)

OBJETIVOS: Analisar os resultados obtidos com a técnica de ICSI utilizando amostras seminais alteradas qualitativa e quantitativamente, classificadas em graus severo, moderado e normal. DELINEAMENTO: Estudo comparativo. MATERIAL E MÉTODOS: Foram realizados 256 ciclos de ICSI em 232 pacientes. Os parâmetros de classificaçäo para sêmen severo foram concentraçäo <5.0 X 106 sptz/ml - <20 por cento de motilidade e/ou < 10 por cento de morfologia normal (Kruger), moderado: entre 5 a 20.0 X 106 sptz/ml e entre 20 a 50 por cento de motilidade; normal: acima de 20.0 X 106sptz/ml, acima de 50 por cento de motilidade e acima de 14 por cento de morfologia normal. PRINCIPAIS MEDIDAS: Foram analisados: o número de ciclos, total de oócitos aspirados, número de oócitos maduros e injetados, taxa de fertilizaçäo normal, número de embriöes clivados e transferidos, taxa de gravidez clínica, a termo ou evolutiva e a taxa de implantaçäo nos diferentes parâmetros seminais. RESULTADOS: No grupo onde o sêmen apresentava parâmetros severos foram realizados 78 ciclos com a presença de 22 gestaçöes a termo, no grupo em que o sêmen apresentava parâmetro moderado foram realizados 50 ciclos com 17 gestaçöes a termo e no grupo com parâmetro severo foram realizados 125 ciclos com 36 gestaçöes a termo. Foram aspirados 1884 oócitos e destes, 1663 foram injetados. A fertilizaçäo com presença de 2 prónucleos ocorreu em 1341 oócitos (80.6 por cento). Foram transferidos 971 embriöes. A gravidez evolutiva ou nascidos foi confirmada em 75 ciclos (por ciclo-29.2 por cento e por transferência - 31.1 por cento). Foram detectados 138 sacos gestacionais apresentando uma taxa de implantaçäo de 14.2 por cento. CONCLUSÖES: Observamos que os diferentes parâmetros seminais näo influenciaram as taxas de fertilizaçäo, clivagem gravidez evolutiva e a termo e nas taxas de implantaçäo.

Injeçäo intracitoplasmática de espermatozóides após tratamento da amostra seminal com soluçäo hipoosmótica de Swelling: relato de caso / Intracytoplasmatic sperm injection after semen washing with hypoosmotic swelling solution: case report

OBJETIVO: Relatar 2 casos onde foi utilizada a soluçäo hipoosmótica de Swelling para identificar espermatozóides imóveis porém vivos, em casos com astenospermia total. Estes espermatozóides foram injetados através da técnica de ICSI após identificaçäo de sua vitalidade. RESULTADOS: Ambos os casos foram injetados com espermatozóides imóveis pós tratamento com a soluçäo hipoosmótica de Swelling.Foram injetados 15 oócitos, e ocorreu a fertilizaçäo de 5 oócitos com clivagem e transferência de todos. Näo houve gravidez. CONCLUSÄO: O teste com soluçäo hipoósmotica de Swelling é indicado para identificar espermatozóides imóveis porém viáveis para injeçäo.

Incidência de lesöes oocitárias após a injeçäo intracitoplasmática de espermatozóides (ICSI) / Oocyte damage incidence after intracytoplasmatic sperm injection (ICSI)

OBJETIVO: Revisar na literatura e comparar os nossos resultados com as taxas de sobrevivência dos oócitos submetidos à técnica de injeçäo intracitoplasmática de espermatozóides (ICSI). DELINEAMENTO: Revisäo bibliográfica e estudo retrospectivo comparativo. MATERIAL E MÉTODOS: Analisar 554 ciclos realizados em nosso centro com a técnica ddo ICSI, comparando os resultados de lesäo oocitária pós injeçäo com os dados da literatura. RESULTADOS: Em 554 ciclos de ICSI realizados em nosso centro, foram injetados 3043 oócitos. Destes, apenas 90 oócitos degeneraram após a injeçäo do espermatozóide. A taxa de degeneraçäo observada foi de 3,0 po cento. CONCLUSÄO: Observamos que é necessário o aperfeiçoamento da técnica de injeçäo do espermatozóide, cuidado na quebra da membrana do oócito e material correto para realizar a técnica da microinjeçäo para evitar o aumento nas taxas de degeneraçäo dos oócitos, aumentando assim o número de embriöes a serem transferidos e futuras gestaçöes.

Gestaçäo por ovodoaçäo em paciente com 57 anos de idade: relato de caso / Ovum donation and pregnancy in a 57 years old patient: case report

OBJETIVO: Relatar um caso de ovodoaçäo, com gestaçäo, em paciente com 57 anos de idade. DELINEAMENTO: Estudo Retrospectivo de Caso Clínico. MATERIAL E MÉTODOS: Foi realizada a técnica de ovodoaçäo em paciente de 57 anos de idade, com conseqüente gravidez. Os óvulos foram obtidos de paciente do banco de doadoras da clínica, após induçäo de ovulaçäo com protocolo longo, usando-se Acetado de Leuprolide (Lupron, Abott, SÒo Paulo-Brasil) e FSH puro (Metrodin HP, Serono, MÚxico). A receptora fez uso de Acetato de Leuprolide e Estradiol transdérmico (Estraderm TTS 100, CIBA, SÄo Paulo-Brasil) até o dia da transferência dos embriöes e, posteriormente, suporte hormonal com Progesterona oleosa (Progesterone in oil, Steries Laboratories, Phoenix-Arizona-USA) e Estradiol transdêrmico. RESULTADO: Os autores relatam um caso de ovodoaçäo em paciente de 57 anos de idade e conseqüente gravidez. A paciente deu à luz de parto cesáreo com 38 1/2 semanas de gestaçäo, a um feto vivo do sexo masculino, medindo 49 cm., pesando 3.400 gr, no dia 19 de setembro de 1996. A paciente, por ocasiäo do parto, já havia completado 58 anos de idade.