Comparison of different methods for exogenous DNA uptake by bovine spermatozoa / Comparação de diferentes métodos de incorporação de DNA exógeno pelo espermatozoide bovino

Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal propósito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência gênica, na qual a célula espermática tem habilidade espontânea de se ligar à molécula de DNA e internalizá-la. Dado o potencial da transferência gênica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência gênica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência gênica mediada por espermatozoides.

Association between infertile women's anxiety with art success rates

Anxiety increases blood cortisol and prolactin concentrations which may lead to infertility, but in contrast lower anxiety levels may help natural fertility. However, there is not much evidence that lower anxiety levels may lead to higher success rates in Assisted Reproductive Techniques [ART] among infertile women. This study aimed to determine the association between infertile women's anxiety and ART success rates. This cohort study was done on 180 infertile women who enrolled as candidates for ART. The cases were recruited in their last visit before starting the treatment cycle by quota sampling in two select infertility treatment centers in Tehran, Iran. The individuals' anxiety was assessed by using State and Trait Anxiety Inventories. They were allocated to high and low anxiety groups according to the achieved scores of 20-49 and 50-80, respectively. A positive pregnancy test was considered the criterion for treatment success. The individuals' state and trait anxiety scores were 47.33 +/- 10.6 and 43.89 +/- 9.8, respectively. Nineteen [10.6%] out of 180 women became pregnant. Pregnancy rates in the group with high and low levels of state anxiety were 11.1% and 10.1% and in groups with high and low levels of trait anxiety they were 14.5% and 9%, respectively. Pregnancy rates between high and low state or trait anxieties were not significantly different [p>0.05]. There were no relationship between infertile women's state and trait anxiety status with assisted reproductive technology outcomes. The results of this study can ensure infertile women that their anxiety would not affect their treatment success rates, although counseling and reducing infertile women's anxiety are necessary to improve their quality of life

Crianças nascidas após o emprego de técnicas de reprodução assistida: aspectos maternos e resultados perinatais / Children born after assisted reproduction techniques: maternal aspects and perinatal results

Saúde Reprodutiva é um estado de completo bem-estar físico, mental e social em todas as matérias concernentes ao sistema reprodutivo, suas funções e processos, e não a simples ausência de doença ou enfermidade. Desde tempos remotos o homem pensou na possibilidade de fecundação fora do ato sexual. Atualmente é altamente divulgada e realizada. Encontram-se os procedimentos tanto de baixa complexidade (relação programada e inseminação artificial), como de alta complexidade (fertilização "in vitro", tranferência intratubária de gametas, injeção intracitoplasmática de espermatozóides). Contudo são poucos os trabalhos brasileiros demonstrando a repercussão materno-infantil desses procedimentos. Objetivo: Comparar as características maternas e resultados perinatais de crianças concebidas por diferentes modalidades de técnicas de reprodução assistida da Unidade de Reprodução Humana do Hospital Israelita Albert Einstein. Método: Estudo foi descritivo, retrospectivo para coleta dos dados e prospectivos para análise dos mesmos. Utilizando dados de mulheres que foram submetidas às diferentes técnicas de reprodução assistida na Unidade de Reprodução Humana do Hospital Israelita Albert Einstein no período de janeiro de 1995 a dezembro de 2003.Foram selecionados para o estudo todos os casos de sucesso gestacional cujos partos ocorreram na própria instituição. Resultados: Neste período o Serviço de Reprodução Humana base deste estudo realizou 2448 procedimentos, sendo FIVc 439 e ICSI 2009. Em média a taxa de gravidez variou de 25-30 por cento. Não houve diferenças significativas entre os dois métodos estudados, em relação aos resultados perinatais. Encontrou-se maior morbi-mortalidade entre os múltiplos em relação aos únicos. Conclusão: As técnicas de reprodução assistida não interferiram nos resultados perinatais. A prematuridade dos múltiplos foi o fator determinante de maior morbi-mortalidade.

A Case of False Positive Amniotic Fluid Acetylcholinestserase in One Fetus of Twin Pregnancy conceived by Intracytoplasmic Sperm Injection and Zygote Intrafallopian Tube Transfer / 대한주산의학회잡지

Maternal serum alpha-fetoprotein(MSAFP) has been a world wide screening test for open neural the tube defect. But elevation of MSAFP is related to not only neural tube defect, but also incorrect gestational age, congenital anomalies such as congenital nephrosis, esophageal and intestinal obstruction, low birth weight, oligohydroamnios, fetal death and chromosomal anomalies. If MSAFP is elevated, gestational age, congenital anomalies such as neural tube defect, multiple pregnancy and fetal death must be evaluated by ultrasound. When the ultrasound is nondiagnostic, amniotic fluid AFP(AFAFP) levels are measured and if AFAFP is elevated, presence or absence of aetylchoineststarase(AChE) is determined to rule out the false positive of amniotic AFP. Amniotic AChE test yielded detection rate of open spina bifida of 99%, 98% for anecephaly and a false-positive rate of 0.34%. We report a case with elevated AFAFP and positive amniotic AChE result in one fetus of the twin pregnancy conceived by ICSI and ZIFT, but in which targeted ultrasound findings were normal, maintained the pregnancy to term and normal twin was delivered by elective cesarean section.

Comparison of pronuclear stage embryo transfer and in vitro fertilization-embryo transfer following intracytoplasmic sperm injection.

To determine whether the mode of embryo transfer (PROST vs IVF) affected the outcome in intracytoplasmic sperm injection (ICSI) cycles, 237 ICSI cycles (106 PROST and 131 IVF) were analyzed. Several parameters, including patient age, duration of infertility, amounts of hMG used, number of mature eggs retrieved and injected, fertilization rate, number of embryos transferred, and clinical pregnancy rate, were compared. Most of the variable factors were not significantly different, except the mean numbers of transferred embryos which were significantly higher in the PROST group. The clinical pregnancy rate showed no statistical difference between PROST and IVF cycles (25.5 and 16.8%; p = 0.139). This study suggests that even the pregnancy rate in PROST cycles was slightly higher than IVF cycles, but there was no statistically significant difference between the two groups.

Effect of Coculture System with Autologous Cumulus Cells on Embryo Quality and Pregnancy Rates / 대한불임학회지

Despite the rapid development of assisted reproductive technologies (ART) in recent years, implantation rates after replacement of embryos into the uterine cavity remains low. Several techniques such as culture conditions based on formulations of human tubal fluid and various ART techniques as GIFT, ZIFT, TET have been adopted in recent years to improve embryo viability in vitro and implantation rates. Also, coculture of human IVF-derived embryos have been used in an effort to increase the number of viable embryos following IVF and to improve synchrony between the developing embryo and the uterine environment. The aim of this study was to evaluate whether the use of coculture with autologous cumulus cells has a significant beneficial effect on the development of embryos in vitro and its relation to the pregnancy rates in 120 patients with previous failed IVF-ET from September, 1995 to January 1998. We obtained the results from which significant improvement in the quality of viable embryos were observed using a coculture system with autologous cumulus cells, but pregnancy rates in this group of patients did not differ from the rate in the standard IVF group during the same period. Our study shows that a simplified short-term coculture system with autologous cumulus cells may help rescue moderate quality embryos to cleave regularly.

Inicio de la vida humana: la necesidad de una reflexión filosófica / Benginning of human life: the need for a philosophical reflection

The question about the identification of the exact moment when human life begins has a special ethic transcendence. Elements afforded by science are insufficient this objective, because of the nature of the object in study and of the cognoscent subject. This article makes a proposal with arguments afforded by the philosophical reflection

A prospective randomized crossover comparison of zygote intrafallopian transfer and in vitro fertilization-embryo transfer in unexplained infertility.

60 couples aged under 38 with unexplained infertility were recruited in this prospective randomized crossover study to compare the efficacy of treatment between ZIFT and IVF-ET. A total of 150 stimulation cycles were commenced and 132 proceeded to successful egg collection (88%). Fertilization successfully occurred in 114 cycles (86.36%). A total of 110 embryo replacements were carried out, (52 uterine transfer and 58 tubal transfer). This prospective randomized cross over trial demonstrated the trend for higher implantation rate, pregnancy rate, and ongoing pregnancy rate with ZIFT over IVF-ET for patients with unexplained infertility. The actual implantation rate, pregnancy rate, and ongoing pregnancy rate with ZIFT were 13.7, 34.5, 31.0 per cent respectively compared to the results from IVF-ET which were 12.1, 26.9, 25.0 per cent respectively. However, the difference was not statistically significant. Although this study showed a 5 per cent ectopic pregnancy rate with ZIFT and none with IVF-ET, the difference was not statistically significant. Similarly, there was no difference in the overall miscarriage rate and multiple pregnancy rate between the two protocols. In conclusion, this prospective randomized crossover trial demonstrated the trend for higher reproductive outcomes in term of implantation rate, pregnancy rate, and ongoing pregnancy rate with ZIFT over IVF-ET in unexplained infertility but the difference was not statistically significant.

Transcervical GIFT & ZIFT by tactile sensation / 대한산부인과학회잡지

No abstract available.

Studies on the Phosphorylase Activity in Ciliary Muscle

The author studied the effects of adrenergic and its blocking agents, ACTH, methylprednisolone, 5-hydroxytryptamine, and prostaglandin E1 and E2 on the phosphorylase activity in bovine ciliary muscle in vitro and obtained the following results. 1. The phosphorylase activity in bovine ciliary muscle was enhanced by the adrenergic agents. such as epinephrine. norepinephrine and isoproterenol, but was not affected by adrenergic alpha receptor blocking agents, such as phenoxybenzamine and phentolamine, and adrenergic beta receptor blockades. such as propranolol and pronethalol. Pretreatment with propranolol prevented partly the epinephrine-induced increase of the phosphorylase activity in bovine ciliary muscle. 2. The phosphorylase activity in bovine ciliary muscle was shown to be stimulated by ACTH, methylprednisolone and 5-hydroxytryptamine but not by prostaglandin E1 and E2. 3. The phosphorylase activity in bovine ciliary muscle which was stimulated by epinephrine, ACTH, methylprednisolone or 5-hydroxytryptamine was markedly blocked by pretreatment with prost aglandin E1 but less by prostaglandin E2. 4. Prostaglandin E1 exerted no effects on the phosphorylase activity in bovine ciliary muscle following the addition of cyclic AMP or MgH++-ATP, but markedly depressed that stimulated by epinephrine.