RESUMEN
BACKGROUND: Use of intracytoplasmic sperm injection (ICSI) is generally considered redundant in cases of normozoospermia, or mild male factor cases of infertility and conventional method of insemination is advocated. However, there is a risk of low fertilization or total fertilization failure (TFF) and to avoid this, split in vitro fertilization (IVF)-ICSI method of insemination is advised. In our study, we have shown that not only TFF is avoided with split method of insemination, but also cancellation of embryo transfer (ET) can be avoided in a significant number of IVF cycles. AIMS: This study aimed to assess whether the IVF-ICSI split insemination method was able to reduce the risk of ET cancellation in couples with normal or mild sperm characteristics. SETTINGS AND DESIGN: It is a retrospective study including a total of 107 split insemination cycles done at our center. MATERIALS AND METHODS: The female partner's age was under 37 years, and at least ten oocytes were retrieved in all cycles. Sibling oocytes were randomly allocated to IVF or ICSI. Statistical analysis was carried out using Graphpad Prism, Instat. RESULTS AND CONCLUSION: The fertilization rate in oocytes kept in conventional IVF was significantly higher (79.8%) compared to that of oocytes injected through ICSI (69.1%). Only one couple had TFF. In majority of the cycles, i.e., 97 out of 107 cycles, the mode of insemination did not affect the fertilization rate or embryo quality. Nearly 28% of the cycles were saved from ET cancellation by adopting the split insemination method. "Split IVF-ICSI" approach can save a significant number of ART cycles and is found to be cost-effective as it avoids incurring the cost of two ART cycles.
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AIM: This study was carried out to assess the outcome of the intracytoplasmic morphologically selected sperm injection (IMSI) technique compared with the previous failed intracytoplasmic sperm injection (ICSI) attempts in oligoasthenoteratozoospermia (OAT)/severe OAT (SOAT)/teratozoospermia patients. SETTING: Institution-based, in vitro fertilization center. STUDY DESIGN: It was a nonrandomized prospective study including 57 couples who had previous one or two ICSI failures (failure due to no implantation as embryos were transferred in these cycles and had no pregnancy) due to male factor. There was no case of total fertilization failure. IMSI was carried out in these couples and the results were compared with their previously failed ICSI attempts. MATERIALS AND METHODS: Real-time selection of sperms was done using IMSI as it allows the assessment of fine nuclear morphology and vacuoles in the sperm head at a high magnification (>6000×) with differential interference contrast optics. Therefore, IMSI was applied in couples having OAT, SOAT or teratozoospermia as male factor and the results were compared with their previous failed ICSI attempts. Statistical analysis was carried out using GraphPad Prism. RESULTS AND CONCLUSION: Data analysis demonstrated significant difference in the fertilization rate between IMSI and previous ICSI attempts of these patients (30.0% vs. 52.0%; P < 0.05). The embryo quality, implantation and pregnancy rates with IMSI were also significantly higher than those of their previous ICSI cycles (32% vs. 56.4%; 30.2% vs. 68.5%; 0.0% vs. 62.4%; P < 0.05). Our conclusion is that the IMSI procedure improved embryo development and the clinical outcomes in the same infertile couples with male infertility and poor embryo development over their previous ICSI attempts and can be taken up as the treatment of choice in cases of severe male factor infertility.
RESUMEN
Spermatids are the earliest male germ cells with haploid set of chromosomes. Spermatid injection was introduced in human assisted reproduction for the treatment of men with non-obstructive azoospermia. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of spermatids for intracytoplasmic injection (ICSI) has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following ICSI using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells has been proposed as a means to improve this poor outcome. Oocyte activation rarely occurs when injected with a spermatid. Therefore, spermatid injection requires use of calcium ionophores for oocyte activation which is otherwise carried out by PLC zeta from mature sperms. This is the only option available for the nonobstructive azoospermic patients to have their own biological child.
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Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.
Asunto(s)
Alelos , Culicidae/clasificación , Insectos Vectores , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Culicidae/genética , ADN Ribosómico , Femenino , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Anopheles culicifacies Giles is a complex of five sibling species, provisionally designated as species A, B, C, D and E. Species A, C, D and E are vectors of malaria in India. Species A, B, C and D can be identified by polytene chromosome examination except in areas where species B and E are sympatric. Species B and E share the same configuration of the polytene chromosomes but can be differentiated by examining the mitotic chromosomes of F(1) progeny from field collection. Further, polytene chromosome examination method requires the mosquitoes to be at the semigravid stage, which limits on use of this method to a very small proportion of the population. The present study investigated whether the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method can be used to differentiate the members of this complex. Complete ITS2 region along with part of the 5.8S and 28S rDNA sequences (512 bp) and the mitochondrial cytochrome oxidase II (530 bp) were amplified and digested with different restriction endonucleases. The Alu I digest of the COII amplicon and Rsa I digest of the ITS2 amplicon could distinguish two categories: species A and D forming one category and species B, C and E forming another. Further, Dde I digestion of the COII amplicon could distinguish species E from species B and C within the latter category. The PCR-RFLP techniques developed in this study can be applied to areas where species A and B and species B and E are sympatric.
Asunto(s)
Anopheles/genética , Complejo IV de Transporte de Electrones/genética , Animales , Anopheles/clasificación , ADN Ribosómico/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminates An. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally identified specimens of An. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.