RESUMO
BACKGROUND: Herlitz junctional epidermolysis bullosa (H-JEB) is an extremely rare genodermatosis characterized by lethality owing to severe blister formation. We report two unrelated Japanese patients with H-JEB. Genetic analyses detected a single nonsense mutation on the LAMC2 gene in these two patients. AIM: To identify the mutation involved and describe the first reported Japanese recurrent mutation in the LAMC2 gene. METHODS: Direct sequencing was performed of DNA from either peripheral blood or fetal cells in amniotic fluid. Reverse transcriptase PCR was used to confirm that an aberrant transcript resulted from the splice site mutation. A haplotype analysis was performed to define the origin of the recurrent mutation. RESULTS: Both patients had blisters and erosions on the trunk and limbs at birth, with nail dystrophy. Patient 1 died as a result of sepsis at 30 weeks of age, and patient 2 died as a result of disseminated intravascular coagulation at 20 weeks of age. Mutation analysis of the LAMC2 gene revealed that patient 1 was compound heterozygous for a nonsense mutation (p.Cys553X) and a novel splice site mutation (c.2868+1delG), and patient 2 was a homozygous for p.Cys553X. Prenatal diagnosis performed during a subsequent pregnancy in family 2 revealed that this second child was heterozygous for p.Cys553X, and was thus not affected. Haplotype analysis suggested that a p.Cys553X allele derived from the same origin had been independently inherited by these two unrelated families. CONCLUSIONS: p.Cys553X in the LAMC2 gene may be a Japanese-specific recurrent mutation as a result of a founder effect, and it may therefore be useful for initial screening in the mutation analysis of H-JEB.
Assuntos
Códon sem Sentido/genética , Análise Mutacional de DNA/métodos , Epidermólise Bolhosa Juncional/genética , Laminina/genética , Povo Asiático/genética , Epidermólise Bolhosa Juncional/fisiopatologia , Evolução Fatal , Feminino , Haplótipos , Humanos , Lactente , Masculino , Linhagem , Gravidez , Diagnóstico Pré-NatalRESUMO
The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.
Assuntos
Angiotensina II/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Sistema Renina-Angiotensina , Angiotensina II/farmacologia , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Cinética , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Perfusão , Coelhos , Análise de Regressão , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Maturidade Sexual , Estreptoquinase/farmacologia , Fatores de TempoRESUMO
The relationship between progesterone and prostaglandin (PG) secretion in the pseudopregnant rabbit corpus luteum was investigated using isolated in vitro perfused ovaries. Progesterone and PG secretion were measured on days 1, 11, and 18 of hCG-induced pseudopregnancy. The mean progesterone secretion increased significantly from days 1 to 11, and then decreased significantly by day 18. PG secretion was inversely correlated with progesterone secretion, suggesting that PG might inhibit progesterone secretion. To test this hypothesis, indomethacin, an inhibitor of PG secretion, was administered to intact rabbits from days 11-18 of pseudopregnancy and/or on day 18 ovaries were perfused in vitro with indomethacin. Indomethacin administered in vivo, in vitro, or both in vivo and in vitro significantly reduced PG secretion compared to that in controls, but did not affect progesterone secretion. In addition, perfusion of ovaries in vitro with PGF2 alpha did not alter progesterone secretion on either day 11 or day 18. Thus, although there is an inverse relationship between progesterone and PG secretion during pseudopregnancy, PGF2 alpha alone had no effect on progesterone secretion. These results question the hypothesis that PGF2 alpha alone is the luteolytic factor.
Assuntos
Ovário/metabolismo , Progesterona/metabolismo , Prostaglandinas/metabolismo , Pseudogravidez , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Gonadotropina Coriônica , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Dinoprostona/metabolismo , Feminino , Técnicas In Vitro , Indometacina/farmacologia , Ovário/efeitos dos fármacos , Perfusão , Pseudogravidez/induzido quimicamente , Coelhos , Fatores de TempoRESUMO
Rihachi Iizuka has contributed strong leadership for the remarkable development of reproductive medicine which has undergone a complete transformation in the previous half century. The Keio University Hospital introduced artificial insemination as the first assisted reproductive technology in Japan. As it follows, lizuka and his colleagues first reported the live birth of a female infant in August 1949 after heterologous insemination: AID. Iizuka and his colleagues were also among the first to successfully inseminate a woman with sperm that had been frozen. He developed the new cryopreservation medium for human semen called "KS Cryo-medium". He also developed semen preparation methods of washing and concentrating sperm counts by centrifugation with Percoll (colloidal silica derivative) solution for oligozoospermic patients. These methods are broadly used in the clinical field. Furthermore, he developed the X-, Y-bearing sperm preseparation method using Percoll which is the so-called "gender selection" procedure for preventing X-linked genetic disorders. The most striking assisted reproductive technology was in vitro fertilization first carried out in Britain. Prior to the clinical application in Japan, the Japan Society of Fertilization and Implantation was established as the main organ for the exchange of official scientific information by lizuka in 1982. As rapid development and spreading of in vitro fertilization and its implicated technologies, lizuka and his colleague of the department had the first success of offspring following embryo freezing and thawing in Japan which was performed at the Tokyo Dental College Ichikawa General Hospital. Already the numbers of offspring following in vitro fertilization treatment has risen to approximately 1% of births in Japan. Rihachi lizuka still undertakes the responsibility for reproductive medicine as he has done so far.
Assuntos
Medicina Reprodutiva , Feminino , História do Século XX , Humanos , Recém-Nascido , Infertilidade/história , Infertilidade/terapia , Japão , Masculino , Gravidez , Medicina Reprodutiva/história , Técnicas de Reprodução Assistida/história , Preservação do Sêmen/históriaRESUMO
OBJECTIVE: To review the available information regarding the role of integrins in reproductive physiology and to discuss their potential clinical implications. DESIGN: Studies that specifically relate to the expression and modulation of integrins in fertilization, embryogenesis, and implantation were identified through the literature and Medline searches. RESULT(S): Integrins are a class of adhesion molecules that participate in cell-to-cell and cell-to-substratum interactions and are present on essentially all human cells. All mammalian eggs express integrins at their surface, and the integrin alpha 6 beta 1 serves as a sperm receptor that mediates sperm-egg binding. In addition, certain integrin moieties appear to be regulated within the cycling endometrium. Specifically, the expression of beta 1 integrins in the early proliferative phase is restricted to the glandular epithelium, whereas stromal cells also express beta 1 integrins in the midsecretory phase. The expression of beta 1 integrins increases at the time of implantation and remains elevated in the decidua during early pregnancy. A disruption of integrin expression is associated with certain types of infertility in women. The apical surface of the mural trophectoderm does indeed possess functional integrins, and trophoblast interactions with extracellular matrix proteins largely depend on the integrin family of adhesion receptors. CONCLUSION(S): Integrins play particularly important roles in both fertilization and embryogenesis, including the process of implantation.
Assuntos
Implantação do Embrião/fisiologia , Desenvolvimento Embrionário e Fetal , Fertilização/fisiologia , Integrinas/fisiologia , Reprodução/fisiologia , Animais , Decídua/química , Embrião de Mamíferos/metabolismo , Endométrio/química , Feminino , Humanos , Integrinas/análise , Masculino , GravidezRESUMO
The success ratio of live birth after human in vitro fertilization and embryo transfer program is still around 12% Per oocyte retrieval cycle. A large number of mature oocytes became degenerate and unable to develop after insemination or implantation. In order to determine this discrepancy, assessment of maturation process as well as cytoplasmic maturity of the oocyte was performed. Initiation and completion of maturation were dependent on steroid metabolism and cyclic AMP in the oocyte cytoplasm. Extracellular calcium is another determinant factor of oocyte maturation. Phosphorylation of 23.5kD and 29.5kD protein was observed in the cytoplasm during maturation. In addition to cAMP, nuclear maturation is regulated by protein tyrosine phosphorylation in the cytoplasm. An inhibitor of Na+/H+ antiport appears to be effective in the procedure of cryopreservation but has no relationship with oocyte maturation. Quality of the oocyte consists of nuclear and cytoplasmic maturation.
Assuntos
Oócitos/fisiologia , Animais , Cálcio/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Feminino , Fertilização in vitro , Humanos , Mesocricetus , Oócitos/metabolismo , Fosforilação , Proteínas/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Esteroides/metabolismoRESUMO
OBJECTIVE: To determine the effect of danazol on in vitro fertilization-embryo transfer (IVF-ET) patients who failed to conceive in previous attempts despite having embryos with optimal morphology, whether endometriosis is present or not. STUDY DESIGN: In this prospective, randomized, controlled study, of 81 patients who experienced unexplained failures of IVF-ET despite having good-morphology embryos, 40 received danazol (400 mg/d orally for 12 weeks) following the unsuccessful IVF-ET cycle. The next IVF-ET was performed within three months of the first spontaneous ovulation after danazol administration. The remaining 41 patients constituted the control group, and in them the next IVF-ET was performed within six months after the previous failed cycle. RESULTS: Conception occurred in 16 of 40 (40%) danazol-treated patients at the subsequent cycle and showed a significant increase when compared with 8 of 41 (19.5%) control subjects (P < .05), though the number of embryos with optimal morphology decreased after danazol treatment. CONCLUSION: Danazol may be used for patients who have had repeated failures of IVF-ET despite having morphologically optimal embryos and may be useful for increasing receptivity of the endometrium in these patients.
Assuntos
Danazol/uso terapêutico , Transferência Embrionária , Antagonistas de Estrogênios/uso terapêutico , Fertilização in vitro , Infertilidade/terapia , Falha de Tratamento , Autoanticorpos/sangue , Feminino , Humanos , Gravidez , Estudos ProspectivosRESUMO
The human early pregnancy factor (EPF) has been solidly isolated under the conditions without loosing its rosette inhibition activity. The activity fraction has been purified as a glycoprotein with 24-30KD molecular weight. The peptide structure was determined in 16 amino acid sequences from N-terminal which was demonstrated to have a strong homology with a part of human epidermal growth factor precursor. This molecule has been identified from the thioredoxin homologous peptide originated from human placenta which has been recently reported as a EPF molecule by Clarke et al. The rabbit in vitro-perfusion experiments have been performed to elucidate the expression mechanisms of EPF activity. EPF has been first detected within 3 hours after fertilization in the local circulation of ovary and oviduct contained embryos. Although the embryo-derived platelet activating factor (PAF) has been known as another preimplantation factor, the exposure of synthetic PAF induced EPF activity. Many other factors should implicate to express the activity and biofunction of EPF. The datas of EPF activity on the human in vitro fertilization and artificial insemination cases suggested that it demonstrated the conceptive circumstances and that EPF might be implicated in some regurations for the pregnancy establishment.
Assuntos
Desenvolvimento Embrionário e Fetal , Peptídeos/isolamento & purificação , Proteínas da Gravidez , Fatores Supressores Imunológicos , Sequência de Aminoácidos , Animais , Chaperonina 10 , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Ativação Linfocitária , Linfócitos/imunologia , Peso Molecular , Ovário/metabolismo , Peptídeos/química , Peptídeos/fisiologia , Gravidez , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Formação de RosetaRESUMO
Early pregnancy factor, a glycoprotein, and platelet activating factor, a phospholipid, are early signals of pregnancy appearing during the preimplantation period. The aim of this study was to determine if the administration of platelet activating factor to rabbits would stimulate the production of early pregnancy factor and, if so, to ascertain the organs responsible. Early pregnancy factor was assayed after the administration of synthetic platelet activating factor to intact rabbits, rabbits that had undergone bilateral salpingo-oophorectomy, and isolated in vitro perfused rabbit ovary and/or oviduct. Although serum early pregnancy factor activity was detectable 30 minutes after platelet activating factor injection in intact rabbits, no activity was found in rabbits whose ovaries and oviducts had been removed. Perfusate early pregnancy factor activity was detectable only in the presence of both ovary and oviduct, and was first evident 120 minutes after platelet activating factor exposure. However, combined samples of perfusates from the ovary alone and oviduct alone expressed positive early pregnancy factor activity. Furthermore, the addition of platelet activating factor to either single or combined perfusates did not increase early pregnancy factor activity. Our data imply that platelet activating factor induces the production of ovary and oviduct early pregnancy factor components independently.
Assuntos
Ovário/metabolismo , Oviductos/metabolismo , Peptídeos/sangue , Fator de Ativação de Plaquetas/farmacologia , Proteínas da Gravidez , Fatores Supressores Imunológicos , Animais , Chaperonina 10 , Feminino , Perfusão , CoelhosRESUMO
Two peaks of rabbit serum EPF activity were seen over the course of pregnancy. Rabbit ovaries with or without attached oviducts were perfused in vitro for 5 h beginning 12, 16, 24, 48, 72 and 120 h after mating. Perfused isolated ovaries did not produce EPF in vitro, but significant EPF activity was detected in the perfusate of the ovary together with oviduct. Pseudopregnant animals and those rabbits that did not ovulate exhibited no perfusate EPF activity. Perfusate EPF activity was highest at the time embryos were at the pronuclear stage and continued through the morula stage. Although the location of embryos at 72 h after mating varied between oviduct and uterus, EPF activity was maintained over the perfusion period. The results suggest that EPF release occurs within 3 h of fertilization and that the presence of the preblastocyst embryo is crucial for EPF release.
Assuntos
Tubas Uterinas/metabolismo , Ovário/metabolismo , Biossíntese Peptídica , Proteínas da Gravidez , Fatores Supressores Imunológicos , Animais , Chaperonina 10 , Embrião de Mamíferos/fisiologia , Feminino , Peptídeos/sangue , Perfusão , Gravidez , CoelhosRESUMO
Two types of rabbit early pregnancy factor (EPF) components, prepared from the in-vitro perfused ovary and oviduct and from serum ammonium sulphate fractions, were investigated to elucidate the source of EPF production. The state of the animals, i.e. pregnant, pseudopregnant, unstimulated or platelet activating factor (PAF)-treated, and order of addition of the components to the assay lymphocytes were varied to characterize conditions of production and expression of EPF activity. Although each component alone had no EPF activity, combination of two components, i.e. ovary and oviduct components, or serum precipitate and supernatant components, expressed EPF activity. The oviduct and serum supernatant components were produced in pregnancy and pseudopregnancy but the ovary and serum precipitate components were produced only in pregnancy. The similarity of the production pattern and ability of the perfusate and serum components to yield EPF activity when combined suggests that they are similar or the same. The sources of stimulation of EPF production did not appear to affect component production because the activity produced by the perfused ovary and oviduct in pregnancy or in response to PAF stimulation appeared similar. Oviduct and supernatant components apparently bound directly to lymphocytes. These results suggest that EPF components are produced in the ovary and oviduct individually and that the combination of the two components expresses EPF activity in the rabbit.
Assuntos
Tubas Uterinas/metabolismo , Imunossupressores/análise , Ovário/metabolismo , Peptídeos/análise , Proteínas da Gravidez , Fatores Supressores Imunológicos , Animais , Chaperonina 10 , Tubas Uterinas/efeitos dos fármacos , Feminino , Imunossupressores/metabolismo , Linfócitos/metabolismo , Ovário/efeitos dos fármacos , Peptídeos/metabolismo , Perfusão , Fator de Ativação de Plaquetas/farmacologia , Gravidez , Pseudogravidez , CoelhosRESUMO
Ultrapremature babies accompanied by intrauterine growth retardation may require supplemental calcium and phosphorus far above the recommended doses.
Assuntos
Compostos de Cálcio/administração & dosagem , Desenvolvimento Infantil/fisiologia , Suplementos Nutricionais , Recém-Nascido Prematuro , Recém-Nascido de muito Baixo Peso , Fósforo/administração & dosagem , Adulto , Osso e Ossos/efeitos dos fármacos , Cesárea , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Seguimentos , Humanos , Recém-Nascido , Gravidez , Resultado do Tratamento , Ultrassonografia Pré-Natal/métodos , Aumento de PesoRESUMO
In the present study we investigated the role of angiotensin II (Ang II) receptor subtypes in gonadotropin-induced ovulation, oocyte maturation, and ovarian steroidogenesis and prostaglandin (PG) production in in vitro-perfused rabbit ovaries. The addition to the perfusate of PD123319, a nonpeptide Ang II antagonist with a high affinity for AT2 receptors, inhibited hCG-induced ovulation in a dose-dependent manner, whereas CV-11974, a nonpeptide AT1 receptor antagonist, had no effect. The majority of ovulated ova and follicular oocytes resumed meiotic maturation in response to hCG; and PD123319, but not CV-11974, significantly inhibited hCG-induced oocyte maturation. The addition of both Ang II receptor antagonists to the perfusate had no significant effect on the concentration of progesterone in the perfusate of hCG-treated ovaries, whereas PD123319 inhibited the hCG-stimulated production of estradiol. The production of PGE2 and PGF2 alpha was significantly increased at 6 h in hCG-treated ovaries compared with ovaries before hCG administration. PD123319 inhibited the hCG-stimulated production of PGs by perfused rabbit ovaries in a dose-dependent manner, indicating that hCG-induced PG synthesis is mediated, at least in part, via the activation of AT2 receptors. Ovulatory efficiency in ovaries perfused with or without PD123319 in the presence of hCG was significantly correlated with PG production by perfused rabbit ovaries 12 h after exposure to hCG (r = 0.6553 for PGE2, p < 0.001; r = 0.4758 for PGF2 alpha, p < 0.05). In conclusion, Ang II exerts complex and coordinated control on at least two distinct aspects in the normal ovulatory process, ovulation and oocyte maturation. Ang II produced locally by gonadotropin exposure may be a part of a novel intraovarian paracrine or autocrine control mechanism that operates via the AT2 receptor in the ovary.
Assuntos
Angiotensina II/fisiologia , Gonadotropina Coriônica/farmacologia , Indução da Ovulação , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Membrana Celular/metabolismo , Dinoprosta/biossíntese , Dinoprostona/biossíntese , Feminino , Células da Granulosa/metabolismo , Imidazóis/farmacologia , Radioisótopos do Iodo , Piridinas/farmacologia , Coelhos , Saralasina/farmacologiaRESUMO
For gender determination of preimplantation embryos or circulating fetal cells in maternal blood, we developed a multiplex polymerase chain reaction assay from a single cell. This assay which co-amplifies X (DXZ1)- and Y (DYZ1)-specific repeat sequences, yields a 308-bp band in females and two bands of 154 and 308 bp in males. In a randomized, blinded assay of 100 isolated single amniocytes, 99 (99%) were amplified successfully. All 50 of the XY cells were correctly diagnosed as male (100%), whereas 49 of the 50 XX cells were diagnosed as female (98%). This accurate and efficient assay may be applicable in these clinical settings.
Assuntos
Feto/citologia , Reação em Cadeia da Polimerase/métodos , Processos de Determinação Sexual , Líquido Amniótico/citologia , Feminino , Humanos , Cariotipagem , Masculino , Gravidez , Diagnóstico Pré-Implantação , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
PURPOSE: The effect of paternal age on the nondisjunction of sex chromosomes is controversial. Also, the prevalence of chromosomal anomalies in infertile patients is controversial, it has been reported that the sex chromosomal aneuploidy rate following treatment with intracytoplasmic sperm injection (ICSI) is higher than in naturally conceived pregnancies. We investigated the influence of paternal age and oligozoospermia on the nondisjunction of spermatozoa. METHODS: We determined the rate of aneuploidy for gonosomes and autosomes, using two-color fluorescence in situ hybridization (FISH) of the X and Y chromosomes and chromosomes 12 and 18 in 10 donors under 25 years of age who had a normal sperm count (> or = 20 x 10(6)/ml), 10 donors over the age of 39 years with idiopathic infertility and normozoospermia (> or = 20 x 10(6)/ml), and 5 oligozoospermic donors (< 20 x 10(6)/ml). RESULTS: There was no obvious relationship between increasing age and autosomal disomy (disomy 12 and disomy 18). Neither autosomal disomy nor diploidy was increased in any group. The frequency of X-, Y-, XX-, and YY-bearing sperm did not differ significantly among groups, but the frequency of XY-bearing sperm was significantly higher in the older infertile group than in the control donors. CONCLUSIONS: The incidence of nondisjunction of paternal sex chromosome in meiosis I was higher in older men with idiopathic infertility. The present results suggest that the risk of producing XXY fetuses is higher among men > 39 years of age with idiopathic infertility.
Assuntos
Aberrações Cromossômicas , Infertilidade Masculina/genética , Contagem de Espermatozoides , Espermatozoides/fisiologia , Adulto , Fatores Etários , Aneuploidia , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 18 , Humanos , Hibridização in Situ Fluorescente , Masculino , Meiose , Oligospermia/genética , Oligospermia/patologia , Cromossomo X , Cromossomo YRESUMO
PURPOSE: Our purpose was to investigate the relation between the dominant sperm anomaly and sperm morphology and the outcome of intracytoplasmic sperm injection (ICSI). METHODS: Two hundred ninety-five patients who underwent a total of 181 cycles of in vitro fertilization (n = 168) and/or 217 cycles of ICSI (n = 177) between July 1995 and May 1997 at Keio University Hospital were investigated. RESULTS: The rates of fertilization and pregnancy were 63.3 and 27.8%, respectively, in ICSI cycles with < or = 4% normal forms. When the percentage of strictly normal morphology was < or = 4, the fertilization rate was lower in the case of severely tapered head (13.0%; n = 4) than in the cases of other deformities in ICSI. The acrosomal defect made no difference in the fertilization rate with ICSI. CONCLUSIONS: The predominant abnormal form affects the ICSI outcome in the case of < or = 4% normal forms.
Assuntos
Fertilização in vitro/métodos , Oligospermia/patologia , Espermatozoides/patologia , Adulto , Interpretação Estatística de Dados , Transferência Embrionária , Feminino , Humanos , Masculino , Oócitos , Gravidez , Resultado da Gravidez , Taxa de GravidezRESUMO
PURPOSE: In preimplantation genetic diagnosis (PGD), a rapid and accurate assay has been required. We have therefore developed a capillary polymerase chain reaction (PCR) method using rapid thermal cycling programs to determine the gender of single amniocytes. METHODS: Single amniocytes from each amniotic fluid sample were isolated by micromanipulation and their gender was determined by a multiplex PCR assay in a capillary tube, using primers that amplify a 308-bp DXZ1 and a 154-bp DYZ1 repeat sequence on the X and Y chromosomes, respectively. RESULTS: All four thermal cycling programs, which took 180, 150, 120, and 90 min, were 100% accurate in diagnosing the gender of single amniocytes. No DNA contamination was observed in any samples. CONCLUSIONS: The multiplex PCR assay was rapid and accurate in diagnosing gender in single cells and may be clinically applicable in PGD.
Assuntos
Âmnio/citologia , Reação em Cadeia da Polimerase/métodos , Processos de Determinação Sexual , Feminino , Humanos , Masculino , Diagnóstico Pré-Implantação/métodosRESUMO
The linear everting (LE) catheter has been developed to safely guide a Falloposcope into the entire length of Fallopian tube in order to observe the tubal lumen. It may also be useful therapeutically for the recanalization of occluded tubes. Fifty infertility patients who had been diagnosed with proximal, mid and distal tubal occlusion by hysterosalpingogram, Rubin test and hysteroscopic selective hydrotubation, were selected to undergo Falloposcopic tuboplasty (FT). Patients having hydrosalpinges were excluded from the study group. The total number of tubes treated was 102 during 53 FT procedures. On the basis of tubes attempted, the LE catheter successfully accessed 85.3% (87/102). A follow-up hysterosalpingogram was completed 1-3 months following the FT procedure, which revealed an overall patency rate of 79.4% (81/102). During FT, a high incidence of multiple adhesions was observed in the entire length of tubal lumen in patients having bilateral occlusions. To date, the total number of pregnancies following FT treatment is 11 over a follow-up period of 2 months to 3 years. FT has been established as a highly useful, less invasive and novel treatment for tubal infertility. This technique may be useful in selected patients with tubal infertility.
Assuntos
Doenças das Tubas Uterinas/cirurgia , Fertilização in vitro/instrumentação , Infertilidade Feminina/cirurgia , Laparoscopia , Cateterismo , Testes de Obstrução das Tubas Uterinas , Feminino , Seguimentos , Humanos , Taxa de Gravidez , Resultado do TratamentoRESUMO
Indomethacin blocks ovulation in human chorionic gonadotropin-stimulated rabbits. Experiments were done with an in vitro ovarian perfusion system to investigate whether indomethacin affects luteinization and steroidogenesis. Indomethacin (10 mg/kg) was administered in combination with human chorionic gonadotropin (100 IU) via a marginal ear vein, and a second dose of indomethacin was given 8 hours later. Control animals received vehicle in place of indomethacin. Laparotomy was performed 24 hours after the initial treatment. The presence of unruptured follicles and corpora lutea was recorded and the ovaries were perfused in vitro for 3 hours. Progesterone, prostaglandin F2 alpha, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in samples obtained at 0, 30, 60, 120, and 180 minutes from the circulating perfusion medium entering and exiting the ovary. At the end of the perfusion all ovaries (12 treated and 10 controls) were fixed for histologic analysis. Ovulation occurred in all control ovaries but in none of the indomethacin-treated ovaries. The mean number of unruptured follicles per ovary in the treated group was not significantly different from the number of corpora lutea plus unruptured follicles per ovary in the controls. Cells in both groups were qualitatively similar in ultrastructure; abundant lipid droplets, smooth endoplasmic reticulum, and mitochondria were seen. Secretion rates of progesterone and prostaglandin did not differ between the two groups during the 3-hour perfusion period. These results suggest that transformation of granulosa cells into fully functional luteal cells can occur in the absence of follicular rupture.
Assuntos
Corpo Lúteo/fisiologia , Indometacina/farmacologia , Ovário/fisiologia , Ovulação/efeitos dos fármacos , Progesterona/biossíntese , Prostaglandinas/biossíntese , Animais , Corpo Lúteo/citologia , Corpo Lúteo/ultraestrutura , Feminino , Injeções Intravenosas , Microscopia Eletrônica , Folículo Ovariano/efeitos dos fármacos , Ovário/metabolismo , CoelhosRESUMO
The potential role of oxygen free radicals in hCG-induced ovulation was investigated using the free radical scavenging enzymes superoxide dismutase (SOD) and/or catalase with the in-vitro perfused rabbit ovary preparation. SOD (25 micrograms/ml) and SOD + catalase (25 micrograms/ml) significantly reduced the % of large follicles that ovulated during perfusion (P less than 0.005). Neither maturity nor degeneration of ovulated ova and follicular oocytes was affected by SOD and/or catalase. Progesterone concentration in the perfusate was significantly increased in the SOD + catalase treatment group (P less than 0.01). These results indicate a significant role for oxygen free radicals in the process of ovulation.