EFICÁCIA ADAPTATIVA DE MULHERES E HOMENS INSERIDOS NUM PROGRAMA DE FERTILIZAÇÃO IN VITRO

Este estudo teve como objetivos: caracterizar uma amostra de pacientes inseridos num programa de fertilização in vitro; avaliar a eficácia adaptativa de homens e mulheres inseridos neste programa; analisar dois casos clínicos representativos da amostra, quanto aos micro e macro fatores indicadores de adaptação psicológica. Os dados foram coletados num Centro de Reprodução Humana, tendo sido selecionados 65 homens e 65 mulheres, num período de março a outubro de 2002 e caracterizados segundo dados sócio-econômico-culturais. Foram extraídos 57 pares, sendo 57 homens e 57 mulheres diagnosticados pela Escala Diagnóstica Adaptativa Operacionalizada representando a maior concentração de casos com diagnóstico médico de mulheres inférteis e homens férteis, dos quais foram realizados os diagnósticos adaptativos. Depois, foram selecionados dois casos um homem e uma mulher, representativos deste grupo, que foram estudados em análise qualitativa, clínica, seguindo-se os pressupostos da teoria da adaptação. Os homens encontravam-se em faixa etária de 23 a 56 anos e as mulheres de 23 a 45 anos. Em relação ao diagnóstico, houve as seguintes concentrações: os homens (64,9 %) foram classificados no Grupo 2 Adaptação Ineficaz Leve e as mulheres (52,6 %) no Grupo 4 Adaptação Ineficaz Severa. Durante o diagnóstico, verificou-se que 20,2 % dos sujeitos estavam em crise, sendo 8,8 % dos homens e 31,6 % das mulheres. No estudo de caso representativo sendo H = Grupo 2 em condição adaptativa melhor do que a sua parceira que se encontrava no Grupo 4, verificou-se, que neste homem sua adequação nos setores era: Afetivo-Relacional - resposta pouco adequada; Produtividade - resposta adequada; Sócio-Cultural - resposta adequada, e Orgânico - resposta adequada. Sua parceira, entretanto, com diagnóstico do Grupo 4 Adaptação Ineficaz Severa sem crise, apresentou resposta pouquíssima adequada no setor Afetivo-Relacional, resposta pouco adequada no setor Produtividade, resposta pouco adequada no Sócio-Cultural e resposta pouco adequada no Orgânico. Concluímos que os homens desta amostra possuíam melhor eficácia adaptativa em relação às mulheres durante o processo de fertilização in vitro. Levantamos a necessidade de acompanhamento psicológico preventivo ante possível entrada em crise e de intervenções específicas para o momento de crise.

EFICÁCIA ADAPTATIVA DE MULHERES E HOMENS INSERIDOS NUM PROGRAMA DE FERTILIZAÇÃO IN VITRO

Este estudo teve como objetivos: caracterizar uma amostra de pacientes inseridos num programa de fertilização in vitro; avaliar a eficácia adaptativa de homens e mulheres inseridos neste programa; analisar dois casos clínicos representativos da amostra, quanto aos micro e macro fatores indicadores de adaptação psicológica. Os dados foram coletados num Centro de Reprodução Humana, tendo sido selecionados 65 homens e 65 mulheres, num período de março a outubro de 2002 e caracterizados segundo dados sócio-econômico-culturais. Foram extraídos 57 pares, sendo 57 homens e 57 mulheres diagnosticados pela Escala Diagnóstica Adaptativa Operacionalizada representando a maior concentração de casos com diagnóstico médico de mulheres inférteis e homens férteis, dos quais foram realizados os diagnósticos adaptativos. Depois, foram selecionados dois casos um homem e uma mulher, representativos deste grupo, que foram estudados em análise qualitativa, clínica, seguindo-se os pressupostos da teoria da adaptação. Os homens encontravam-se em faixa etária de 23 a 56 anos e as mulheres de 23 a 45 anos. Em relação ao diagnóstico, houve as seguintes concentrações: os homens (64,9 %) foram classificados no Grupo 2 Adaptação Ineficaz Leve e as mulheres (52,6 %) no Grupo 4 Adaptação Ineficaz Severa. Durante o diagnóstico, verificou-se que 20,2 % dos sujeitos estavam em crise, sendo 8,8 % dos homens e 31,6 % das mulheres. No estudo de caso representativo sendo H = Grupo 2 em condição adaptativa melhor do que a sua parceira que se encontrava no Grupo 4, verificou-se, que neste homem sua adequação nos setores era: Afetivo-Relacional - resposta pouco adequada; Produtividade - resposta adequada; Sócio-Cultural - resposta adequada, e Orgânico - resposta adequada. Sua parceira, entretanto, com diagnóstico do Grupo 4 Adaptação Ineficaz Severa sem crise, apresentou resposta pouquíssima adequada no setor Afetivo-Relacional, resposta pouco adequada no setor Produtividade, resposta pouco adequada no Sócio-Cultural e resposta pouco adequada no Orgânico. Concluímos que os homens desta amostra possuíam melhor eficácia adaptativa em relação às mulheres durante o processo de fertilização in vitro. Levantamos a necessidade de acompanhamento psicológico preventivo ante possível entrada em crise e de intervenções específicas para o momento de crise.

Análise genética da produção in vitro de embriões em bovinos Guzerá

O objetivo dessa pesquisa foi avaliar aspectos genéticos que relacionados à produção in vitro de embriões na raça Guzerá. O primeiro estudo focou na estimação de (co) variâncias genéticas e fenotípicas em características relacionadas a produção de embriões e na detecção de possível associação com a idade ao primeiro parto (AFC). Foi detectada baixa e média herdabilidade para características relacionadas à produção de oócitos e embriões. Houve fraca associação genética entre características ligadas a reprodução artificial e a idade ao primeiro parto. O segundo estudo avaliou tendências genéticas e de endogamia em uma população Guzerá no Brasil. Doadoras e embriões produzidos in vitro foram considerados como duas subpopulações de forma a realizar comparações acerca das diferenças de variação anual genética e do coeficiente de endogamia. A tendência anual do coeficiente de endogamia (F) foi superior para a população geral, sendo detectado efeito quadrático. No entanto, a média de F para a sub- população de embriões foi maior do que na população geral e das doadoras. Foi observado ganho genético anual superior para a idade ao primeiro parto e para a produção de leite (305 dias) entre embriões produzidos in vitro do que entre doadoras ou entre a população geral. O terceiro estudo examinou os efeitos do coeficiente de endogamia da doadora, do reprodutor (usado na fertilização in vitro) e dos embriões sobre resultados de produção in vitro de embriões na raça Guzerá. Foi detectado efeito da endogamia da doadora e dos embriões sobre as características estudadas. O quarto (e último) estudo foi elaborado para comparar a adequação de modelos mistos lineares e generalizados sob método de Máxima Verossimilhança Restrita (REML) e sua adequação a variáveis discretas. Quatro modelos hierárquicos assumindo diferentes distribuições para dados de contagem encontrados no banco. Inferência foi realizada com base em diagnósticos de resíduo e comparação de razões entre componentes de variância para os modelos em cada variável. Modelos Poisson superaram tanto o modelo linear (com e sem transformação da variável) quanto binomial negativo à qualidade do ajuste e capacidade preditiva, apesar de claras diferenças observadas na distribuição das variáveis. Entre os modelos testados, a pior qualidade de ajuste foi obtida para o modelo linear mediante transformação logarítmica (Log10 X +1) da variável resposta.

EFICÁCIA ADAPTATIVA DE MULHERES E HOMENS INSERIDOS NUM PROGRAMA DE FERTILIZAÇÃO IN VITRO

Este estudo teve como objetivos: caracterizar uma amostra de pacientes inseridos num programa de fertilização in vitro; avaliar a eficácia adaptativa de homens e mulheres inseridos neste programa; analisar dois casos clínicos representativos da amostra, quanto aos micro e macro fatores indicadores de adaptação psicológica. Os dados foram coletados num Centro de Reprodução Humana, tendo sido selecionados 65 homens e 65 mulheres, num período de março a outubro de 2002 e caracterizados segundo dados sócio-econômico-culturais. Foram extraídos 57 pares, sendo 57 homens e 57 mulheres diagnosticados pela Escala Diagnóstica Adaptativa Operacionalizada representando a maior concentração de casos com diagnóstico médico de mulheres inférteis e homens férteis, dos quais foram realizados os diagnósticos adaptativos. Depois, foram selecionados dois casos um homem e uma mulher, representativos deste grupo, que foram estudados em análise qualitativa, clínica, seguindo-se os pressupostos da teoria da adaptação. Os homens encontravam-se em faixa etária de 23 a 56 anos e as mulheres de 23 a 45 anos. Em relação ao diagnóstico, houve as seguintes concentrações: os homens (64,9 %) foram classificados no Grupo 2 Adaptação Ineficaz Leve e as mulheres (52,6 %) no Grupo 4 Adaptação Ineficaz Severa. Durante o diagnóstico, verificou-se que 20,2 % dos sujeitos estavam em crise, sendo 8,8 % dos homens e 31,6 % das mulheres. No estudo de caso representativo sendo H = Grupo 2 em condição adaptativa melhor do que a sua parceira que se encontrava no Grupo 4, verificou-se, que neste homem sua adequação nos setores era: Afetivo-Relacional - resposta pouco adequada; Produtividade - resposta adequada; Sócio-Cultural - resposta adequada, e Orgânico - resposta adequada. Sua parceira, entretanto, com diagnóstico do Grupo 4 Adaptação Ineficaz Severa sem crise, apresentou resposta pouquíssima adequada no setor Afetivo-Relacional, resposta pouco adequada no setor Produtividade, resposta pouco adequada no Sócio-Cultural e resposta pouco adequada no Orgânico. Concluímos que os homens desta amostra possuíam melhor eficácia adaptativa em relação às mulheres durante o processo de fertilização in vitro. Levantamos a necessidade de acompanhamento psicológico preventivo ante possível entrada em crise e de intervenções específicas para o momento de crise.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Immunohistochemical localization of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in bovine oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha. regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for PA was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for RA in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immurloreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Allele-Specific Expression of the MAOA Gene and X Chromosome Inactivation in In Vitro Produced Bovine Embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8-to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. MoL Reprod. Dev. 77: 615-621, 2010. (C) 2010 Wiley-Liss, Inc.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL +/- 1.13 vs 22.12 mg/dL +/- 0.86; mean +/- SEM), neither the number of COCs recovered (8.8 +/- 1.0 vs 9.2 +/- 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes. (C) 2011 Elsevier Inc. All rights reserved.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Non-random fertilization in mice correlates with the MHC and something else

One evolutionary explanation for the success of sexual reproduction assumes that sex is an advantage in the coevolutionary arms race between pathogens and hosts. Accordingly, an important criterion in mate choice and maternal selection thereafter could be the allelic specificity at polymorphic loci involved in parasite-host interactions, e.g. the MHC (major histocompatibility complex). The MHC has been found to influence mate choice and selective abortions in mice and humans. However, it could also influence the fertilization process itself, i.e. (i) the oocyte's choice for the fertilizing sperm, and (ii) the outcome of the second meiotic division after the sperm has entered the egg. We tested both hypotheses in an in vitro fertilization experiment with two inbred mouse strains congenic for their MHC. The genotypes of the resulting blastocysts were determined by polymerase chain reaction. We found nonrandom MHC combinations in the blastocysts which may result from both possible choice mechanisms. The outcome changed significantly over time, indicating that a choice for MHC combinations during fertilization may be influenced by one or several external factors.

The In Vitro Mass-Produced Model Mycorrhizal Fungus, Rhizophagus irregularis, Significantly Increases Yields of the Globally Important Food Security Crop Cassava.

The arbuscular mycorrhizal symbiosis is formed between arbuscular mycorrhizal fungi (AMF) and plant roots. The fungi provide the plant with inorganic phosphate (P). The symbiosis can result in increased plant growth. Although most global food crops naturally form this symbiosis, very few studies have shown that their practical application can lead to large-scale increases in food production. Application of AMF to crops in the tropics is potentially effective for improving yields. However, a main problem of using AMF on a large-scale is producing cheap inoculum in a clean sterile carrier and sufficiently concentrated to cheaply transport. Recently, mass-produced in vitro inoculum of the model mycorrhizal fungus Rhizophagus irregularis became available, potentially making its use viable in tropical agriculture. One of the most globally important food plants in the tropics is cassava. We evaluated the effect of in vitro mass-produced R. irregularis inoculum on the yield of cassava crops at two locations in Colombia. A significant effect of R. irregularis inoculation on yield occurred at both sites. At one site, yield increases were observed irrespective of P fertilization. At the other site, inoculation with AMF and 50% of the normally applied P gave the highest yield. Despite that AMF inoculation resulted in greater food production, economic analyses revealed that AMF inoculation did not give greater return on investment than with conventional cultivation. However, the amount of AMF inoculum used was double the recommended dose and was calculated with European, not Colombian, inoculum prices. R. irregularis can also be manipulated genetically in vitro, leading to improved plant growth. We conclude that application of in vitro R. irregularis is currently a way of increasing cassava yields, that there is a strong potential for it to be economically profitable and that there is enormous potential to improve this efficiency further in the future.