Este estudo teve por objetivo avaliar os efeitos de dois diluentes comerciais, Dilutris® (SEMENCON – Produtos Agropecuários Ltda., Porto Alegre, RS, Brasil) e TQC Holding Plus® (Nutricell – Nutrientes Celulares Ltda., Campinas, SP, Brasil), na qualidade do sêmen, nas taxas de concepção utilizando inseminação artificial cervical (IAC) e por laparoscopia (IAL) e na reação inflamatória uterina após a inseminação laparoscópica. Para os testes in vitro do sêmen foram coletados seis ejaculados de dois carneiros. Cada ejaculado foi dividido em cinco frações de 0,20 mL, e acrescentado a cada fração o volume de diluidor equivalente a concentração da dose inseminante: para IAC (100x106 espermatozóides) e para IAL (40x106 espermatozóides), formando quatro tratamentos T1, T2, T3 e T4, respectivamente: Dilutris®/cervical; Dilutris®/laparoscopia; Holding®/cervical; Holding®/laparoscopia. Foram avaliadas a motilidade/ vigor, testes de termo-resistência e hiposmótico de cada fração. A motilidade e o vigor não diferiram entre os tratamentos e foram semelhantes aos verificados no sêmen fresco. No teste hiposmótico, os valores verificados no T4 e T1 foram semelhantes e não diferiram dos observados no sêmen fresco. Os valores observados nos tratamentos T2 e T3 não diferiram entre si, mas foram significativamente inferiores (P<0,05) aos verificados no T4. Os resultados do teste de TTR verificados em todos os tratamentos utilizados, foram superiores ao valor mínimo de 30% preconizado pelo CBRA. O T1 apresentou valor significativamente inferior (P<0,05) ao dos demais tratamentos. Para a inseminação artificial, 208 fêmeas tiveram o estro sincronizado com esponjas vaginais contendo 60 mg de MAP por 13 dias e aplicação de 400 UI de eCG no momento da retirada. Foram inseminadas pela via cervical (IAC) ou por laparoscopia (IAL), 54 horas e 60 horas, respectivamente, após a retirada do dispositivo intra-vaginal, com sêmen diluído com Dilutris®, Holding® ou solução fisiológica (Grupo controle). Independentemente do diluente utilizado, os índices obtidos na IAL foram superiores (P<0,05) aos obtidos pela IAC. Na IAC a taxa de gestação obtida no grupo inseminado com o diluente TQC Holding Plus® (45,6%), foi semelhante à verificada no grupo controle (48,9%). Já no grupo que foi inseminado com Dilutris®, a taxa de gestação (29%) foi inferior à obtida no grupo controle (P<0,05). Com relação aos aspectos histológicos, verificou-se um infiltrado inflamatório de neutrófilos de grau discreto a acentuado nos úteros inseminados por laparoscopia com Dilutris®, de ausente a moderado nos úteros inseminados com TQC Holding Plus®, e no grupo controle não houve presença de infiltrado inflamatório de neutrófilos. Podese concluir que na avaliação in vitro, o meio de manutenção de embriões TQC Holding Plus® não causou efeitos negativos na qualidade do sêmen, enquanto que o meio diluidor Dilutris® foi menos efetivo na manutenção da qualidade do sêmen. O local de deposição do sêmen teve influência significativa no índice de fertilidade (P<0,05). Por laparoscopia as taxas de gestação foram semelhantes independentemente do diluidor utilizado. Um maior número de avaliações uterinas são necessárias para um melhor conhecimento da reação inflamatória intra-uterina em ovinos inseminados por laparoscopia. / This study aims to assess the effects of two commercial diluents, Dilutris® (SEMENCON – Produtos Agropecuários Ltda., Porto Alegre, RS, Brazil) and TQC Holding Plus® (Nutricell – Nutrientes Celulares Ltda., Campinas, SP, Brazil), on semen quality, conception rates using Cervical Artificial Insemination (CAI) and Laparoscopic Artificial Insemination (LAI), and uterine inflammatory reactions after the laparoscopic insemination. For the in vitro tests with the semen, six ejaculates of two rams were collected. Each ejaculate was divided into five fractions of 0.20 mL, and we added to each fraction the volume of extender equivalent to the concentration of the inseminant dose: for CAI (100x106 spermatozoa) and for LAI (40x106 spermatozoa), forming four treatments T1, T2, T3, and T4, respectively: Dilutris®/cervical; Dilutris®/laparoscopy; Holding®/cervical; Holding®/laparoscopy. The motility/vigor, thermo-resistance and hypoosmotic tests of each fraction was evaluated. The motility and vigor were not different between the treatments and were similar to those verified in fresh semen. In the hypoosmotic test, the values verified in T4 and T1 were similar and were not different from those observed in fresh semen. The values observed in T2 and T3 treatments have not differed between them, but were significantly lower (P<0.05) to those verified in T4. Results of the TTR test verified in all used treatments were superior to the minimum value of 30% preconized by CBRA. T1 presented a significantly lower value (P<0.05) compared to the other treatments. For artificial insemination, 208 female had their estrus synchronized with vaginal sponges containing 60 mg of MAP for 13 days and the application of 400 UI of eCG at the moment of removal. They were inseminated by cervical via (CAI) or by laparoscopy (LAI), 54 and 60 hours, respectively, after the removal of the intravaginal device, with semen diluted with Dilutris®, Holding® or physiological solution (control group). Independently of the extender used, the obtained rates in the LAI were superior (P<0.05) to those obtained with the CAI. In CAI the pregnancy rate obtained in the inseminated group with the TQC Holding Plus® diluent (45.6%) was similar to the one verified in the control group (48.9%). In the group inseminated with Dilutris®, the pregnancy rate (29%) was inferior to the obtained in the control group (P<0.05). Related to the histological aspects, we verified a discreet to accentuated degree of neutrophils inflammatory infiltrate in the uterus inseminated by laparoscopy with Dilutris®, from absent to moderate in uterus inseminated with TQC Holding Plus®, and in the control group there was not inflammatory infiltrate of neutrophils. We can conclude that in the in vitro evaluation, the embryos maintenance means TQC Holding Plus® has not caused negative effects in the quality of the semen, while the Dilutris® extender was less effective to maintain the quality of the semen. The semen deposition site has a significant impact in the fertility rate (P<0.05). By laparoscopy the pregnancy rates were similar independently of the extender used. A larger number of uterine analyses are required for a better knowledge of the intrauterine inflammatory reaction in sheep inseminated by laparoscopy.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-11T13:50:55Z No. of bitstreams: 1 Bartira Pastor Andrade Sousa.pdf: 687607 bytes, checksum: 33d9827ebaf5d1b383b9c07059174be5 (MD5) / Made available in DSpace on 2016-08-11T13:50:55Z (GMT). No. of bitstreams: 1 Bartira Pastor Andrade Sousa.pdf: 687607 bytes, checksum: 33d9827ebaf5d1b383b9c07059174be5 (MD5) Previous issue date: 2008-08-29 / The aim of the present study was to assess the in vitro and in vivo viability of sperm cells following the addition of diluent in the refrigeration process of sheep semen and the fertilization of oocytes following the insemination of superovulated ewes. In Experiment I, three ejaculates from each of three Dorper breeders were used, collected with an artificial vagina during three repetitions with three-day intervals. The macroscopic and microscopic characteristics of the semen were analyzed before and after the pooling, analyzing sperm concentration, DNA integrity and acrosome integrity as well. The pooled semen was divided into five equal parts. Dilution was performed (1:3, semen:diluent) to establish the diluent groups: Equimix (DI), Laiciphos Green Ovine (DII), FR 4 (DIII), Equimix-Yolk [Equimix with 20% egg yolk (DIV)] and Tris-Yolk (DV; control). Each group was subdivided in quadruplicate, refrigerated and kept at 4 °C until the evaluations (MIP, vigor, DNA integrity and acrosome integrity), corresponding to 0, 12, 24, 36 and 48 hours. In the in vitro assessments, DI exhibited the greatest drop in MIP at 12 h in comparison to the other groups (p<0.05). At 24 h, DII, DIV and DV exhibited the highest MIP (p<0.05), with no significant differences between one another (p>0.05). At 48 h, DII and DV were superior to the other groups (p<0.05). Regarding vigor, DII and DV had higher values (p<0.05) than DI and DIII at 12 hours and DIV had the highest values at 24 hours (p<0.05). In all groups, there was total preservation of DNA integrity and a high number of spermatozoids with intact acrosomes for all the intervals studied. In Experiment II, the same collection method and processing of the ejaculates from the three Dorper breeders were performed. A pool was formed of the threeejaculates of each animal, divided into two equal parts at a 1:3 proportion in order to establish the experimental groups: Equimix-Yolk (DI) and Tris-Yolk (DII; control). Each group was subdivided into two aliquots: fresh – F (F-DI and F-DII), which was used immediately; and refrigerated – R (R-DI and R-DII), which was kept at 4 °C for a storage time of 24 hours. Laparoscopic inseminations were preformed with an inseminate volume of 0.25 mL per uterine horn, which was when the ovary evaluations were performed. Thirty-nine embryo harvesting procedures were performed, 19 using refrigerated semen and (R-DI and R-DII) and 20 using fresh semen (F-DI and F-DII). In the in vivo test, a general rate of 71.0% (237/334) of fertilized structures was obtained, 59.3% (198/334) of which were viable embryos. There was no significant variation (p>0.05) between the types of semen and diluents. Among the total number of embryos, 86.4% exhibited quality Grades I and II, with the refrigerated semen of R-DI obtaining the best percentage (100%)(p<0.05). The ovarian status at the time of insemination affected fertilization, as better results were obtained for the fresh semen of F-DI when the ovary was ovulating. For F-DII, this status exhibited a smaller number of fertilized structures (p<0.05). At the end of the experiments, it was concluded that both the Laiciphos Green Ovine and Tris-Yolk can be used in the conservation of semen at 4 °C for 48 hours, whereas Equimix added with 20% egg yolk is recommended for use in semen storage (4 °C) of up to 24 hours. The refrigeration of ovine semen at 4 °C for 24 hours is viable for use inembryo transference programs. Equimix added with 20% egg yolk resulted in a fertilization rate and embryo quality similar to the traditional Tris-Yolk. / Neste estudo objetivou-se avaliar a viabilidade in vitro e in vivo das células espermáticas após a adição de diluente no processo de refrigeração do sêmen ovino, e na fertilização de oócitos após inseminação de fêmeas ovinas superovuladas. No Experimento I foram utilizados três ejaculados de cada um dos três reprodutores Dorper, coletados com vagina artificial a intervalo de 3 dias, em três repetições. As características macro e microscópicas do sêmen foram analisadas antes e após a formação de um pool, além da concentração espermática, integridade do DNA e do acrossoma. O pool foi dividido em cinco partes iguais, procedeu-se com a diluição (1:3, sêmen:diluente) para constituir os respectivos grupos de diluentes: Equimix (DI), Laiciphos Green Ovine (DII), FR 4 (DIII), Equimix-Gema – Equimix com 20% gema de ovo (DIV) e Tris-Gema (DV; controle). Cada grupo foi subdividido em quadruplicata, refrigerado e mantido a 4 °C até as avaliações (MIP, vigor, integridade do DNA e do acrossoma) correspondendo a 0, 12, 24, 36 e 48 horas. Nas avaliações in vitro do sêmen o DI apresentou maior queda de MIP às 12 h em relação aos demais grupos (p<0,05). Às 24 h os grupos DII, DIV e DV apresentaram a melhor MIP (p<0,05), não divergindo (p>0,05) entre si, enquanto que às 48 h o DII e o DV foram superiores (p<0.05) aos demais. Com relação ao vigor, os grupos DII e o DV apresentaram valores superiores (p<0,05) ao DI e DIII a partir das 12 horas e ao DIV a partir das 24 horas (p<0,05). Em todos os grupos de diluentes houve a preservação total da integridade do DNA e alto índice de espermatozóides com acrossoma intacto, para todos os intervalos avaliados.Para o Experimento II foi utilizada a mesma técnica para colheita e processamento dos ejaculados de três reprodutores Dorper. Formou-se um pool com três ejaculados de cada animal, dividiu-se em duas partes iguais diluídas na proporção 1:3 para constituir os respectivos grupos experimentais: Equimix-Gema (DI) e Tris-Gema (DII; controle). Cada grupo foi subdividido em outras duas alíquotas: fresco – F (F-DI e F-DII), que foi usado imediatamente, e refrigerado – R (R-DI e R-DII), mantido a 4 °C por 24 horas de armazenamento. Foram realizadas inseminações laparoscópicas, com volume inseminante de 0,25 mL por corno uterino, momento em que foram realizadas as avaliações dos ovários. Foram realizados 39 procedimentos de colheitas de embriões, em 19 foram utilizados sêmen refrigerado (R-DI e R-DII) e em 20 sêmen fresco (F-DI e F-DII). No teste in vivo obteve-se uma taxa geral de estruturas fertilizadas de 71,0% (237/334), sendo 59,3% (198/334) de embriões viáveis, o que não variou significativamente (p>0,05) entre os tipos de sêmen e de diluentes. No total dos embriões, 86,4% apresentaram qualidade de grau I e II, sendo o sêmen refrigerado do R-DI o de melhor percentual (100%) (p<0,05). O “status” ovariano no momento da inseminação interferiu na fertilização, observado-se melhores resultados para o sêmen fresco do F-DI quando o ovário se encontrava em ovulação. Para o F-DII, este status foi o que apresentou menor quantidade de estruturas fertilizadas (p<0,05). Ao final dos experimentos pode-se concluir que: o diluente Laiciphos Green Ovine, da mesma forma que o Tris-gema, pode ser utilizado na conservação do sêmen a 4 °C por 48 horas; enquanto o Equimix, acrescido de 20% de gema de ovo, recomenda-se que seja utilizado no armazenamento do sêmen (4 °C) por até 24 horas. É viável a refrigeração do sêmen ovino a 4°C por 24 horas para a utilização em programas de transferência de embriões; e que o diluidor Equimix, acrescido de 20% gema de ovo, resultou em taxa de fertilização e qualidade embrionária similares ao tradicional Tris-Gema.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objectives were to improve the cost effectiveness of the technique of artificial insemination by transcervical fixed time (TAI) in sheep, testing the effect of dose reduction and use of the route submucosis (SMV) in the application of equine chorionic gonadotropin (eCG) on the efficiency of the synchronization protocol and fertility to artificial insemination (AI) and measure the stress level of the sheep as a result of the application of biotech. Were used six groups of twenty animals, where each group received doses of 200, 300 and 400 IU of eCG intramuscularly (IM) and SMV. Estrus was detected and made 48 hours after insemination by transcervical. Of the 120 treated ewes, 87.5% in estrus. The percentage of intra-uterine deposition of semen was 88.3%. Pregnancy rate ranged from 20 to 70% among the treatments, obtaining an average of 46.66%. The mean cortisol results were significant at 1.15, 2.86 and 0.52 μg/dL according to the three collections, being higher after the animal off the rack at the end of IA. The through SMV can be used to implement eCG at doses of 300 and 400 IU induction protocols and synchronizing estrus and ovulation in sheep associated TAI. The dose reduction to 200 IU eCG intramuscularly decreases the costs of maintaining the efficiency of estrus synchronization protocols and TAI showing better pregnancy rate and prolificacy in sheep, but should not be used route SMV. In general the procedures listed by the transcervical AI will cause stress in sheep. Although the technique of transcervical AI cause stress in animals produces satisfactory results in fertility multiparous ewes Santa Ines. / Os objetivos deste trabalho foram melhorar a relação custo benefício da aplicação da técnica de inseminação artificial pela via transcervical em tempo fixo (IATF) em ovinos, testando o efeito da redução da dose e da utilização da via submucosa vulvar (SMV) na aplicação da gonadotrofina coriônica eqüina (eCG) sobre a eficiência do protocolo de sincronização e fertilidade à inseminação artificial (IA), e mensurar o nível de estresse das ovelhas em conseqüência da aplicação desta biotécnica. Foram utilizados seis grupos de vinte animais, onde cada grupo receberam dosagens de 200, 300 e 400 UI da eCG pela via intramuscular (IM) e SMV. O estro foi detectado e 48 horas após feita as inseminações pela via transcervical. Das 120 ovelhas tratadas, 87,5% manifestaram estro. O percentual de deposição intra-uterina de sêmen foi de 88,3%. A taxa de gestação variou de 20 a 70% entre os tratamentos, obtendo-se média de 46,66%. A média dos resultados do cortisol foram significativos com 1,15, 2,86 e 0,52 μg/dL de acordo com os três momentos de coleta, sendo maior após o animal descer do cavalete ao término da IA. A via SMV pode ser utilizada para aplicação da eCG em doses de 300 e 400 UI em protocolos de indução e sincronização de estro e da ovulação em ovinos associados à IATF. A redução da dose da eCG para 200 UI pela via IM diminui os custos mantendo a eficiência dos protocolos de sincronização de estro e IATF apresentando melhor taxa de gestação e prolificidade em ovinos, porém não deve ser utilizada pela via SMV. De um modo geral os procedimentos relacionados á IA pela via transcervical provocam estresse nas ovelhas. Apesar da técnica de IA transcervical causar estresse nos animais, apresenta resultados satisfatórios de fertilidade em ovelhas multíparas da raça Santa Inês.

Made available in DSpace on 2014-08-20T14:37:56Z (GMT). No. of bitstreams: 1 dissertacao_viviane_rabassa.pdf: 236038 bytes, checksum: 5cfc0f0e2ed580d8304a3d1d8257390c (MD5) Previous issue date: 2007-02-05 / The artificial insemination (AI) with frozen semen in sheep is limited by the high costs of the laparoscopic technique and the variability of the results obtained from transcervical technique. This technique requires attachment of cervix, a procedure that can cause located injuries, further, the extensive uterine manipulation, which can lead to an exacerbated endometrial prostaglandin synthesis, could modify the uterine environment at the moment of conception. The objective of this study was to compare the efficiency of transcervical AI, through the technique of cervix attachment and retraction, in comparison to laparoscopic AI, as well as, to determine the effect of flunixin meglumine application at the moment of AI, on pregnancy rate of ewes, using frozen semen. In fixed-time inseminated ewes, during seasonal anestrous, there was no difference in the pregnancy rate between the transcervical and laparoscopic techniques, which was 40%. In respect to flunixin meglumine, the experiment was carried out during the breeding season and was performed using AI with estrus detection. There was no difference in the pregnancy rate between ewes receiving or not flunixin meglumine (p>0.05), which was 68.9% for the control group and 60.0% for the group that received flunixin meglumine at the moment of transcervical AI. These results were probably due to the minimum manipulation exerted on the cervix of these females, which don t cause injuries that could lead to exacerbated production of embryotoxic substances. Thus, when the transcervical AI technique is carried out through trained inseminators and without extreme manipulation of cervix, it becomes a good alternative for costs reduction in sheep AI, allowing the use of frozen semen in commercial applications. / A inseminação artificial (IA) com sêmen congelado em ovinos é limitada pelos altos custos da técnica por laparoscopia e pelos resultados variáveis obtidos com a IA transcervical. A IA por via transcervical requer pinçamento da cérvix, um procedimento que pode causar lesões localizadas, além de extensa manipulação uterina, podendo levar a uma síntese exagerada de prostaglandina pelo endométrio, alterando o ambiente uterino no momento da concepção. O objetivo deste estudo foi comparar a eficiência da IA transcervical, através da técnica de fixação e tracionamento da cérvix, em relação a IA por laparoscopia, bem como, determinar o efeito da aplicação de flunixin meglumine sobre a taxa de prenhez de ovelhas, inseminadas com sêmen congelado. Em ovelhas inseminadas em tempo-fixo, durante o período de anestro estacional, não houve diferença entre as técnicas transcervical e laparoscópica, quanto à taxa de prenhez (40%). Com relação ao uso de flunixin meglumine, o experimento foi realizado durante a estação reprodutiva da espécie ovina, efetuando-se a IA com observação de cio. Neste estudo não observou-se diferença quanto à taxa de prenhez entre ovelhas recebendo ou não flunixin meglumine (p>0,05), sendo de 68,9% no grupo controle e 60,0% no grupo em que foi aplicado flunixin meglumine uma hora antes da IA transcervical. Estes resultados se devem, provavelmente, à manipulação mínima exercida sobre a cérvix destas fêmeas, não causando lesões que pudessem levar à produção exacerbada de substâncias embriotóxicas. Assim, quando a técnica de IA transcervical é realizada por inseminadores treinados e sem manipulação excessiva da cérvix, esta se torna uma adequada alternativa de redução de custos na IA de ovinos, permitindo a utilização de sêmen congelado na ovinocultura.

MSCAGR (Animal Science) / Department of Animal Science / See the attached abstract below

MSCAGR (Animal Science) / Department of Animal Science / This study determined the effects of in vitro culture media (Ham’s F10 and TCM199) and assisted hatching techniques (laser or mechanical) on mice embryo survival following cryopreservation. Pure strain C57BL/6 (B6) female (50) and strain BALB /c (C) Male (25) mice were crossed to produce F1 generation of females which were injected for follicular growth and super ovulation at 6 weeks of age and from which embryos were produced 21 h later through in vivo fertilization. Embryos were randomly divided into Petri dishes with different culture media, and the development of embryos was assessed until the morula stage. At the morula stage, selected embryos were assisted to hatch using different techniques, and then cryopreserved in liquid nitrogen using the slow freezing method for a period of 1 week. After 1 week of cryopreservation, the embryos were thawed and cultured in the two different in vitro culture media for 72 hours. Thereafter, the numbers of embryos hatched or survived were recorded after 24 h, 48 h and 72 h. Data was analyzed using ANOVA in Minitab Software Version 16 (2010). Significant difference in embryo quality development was observed between in vitro culture media and stage of embryo development (P<0.05). In the TCM-199 in vitro culture medium, embryo quality development yielded 72, 69 and 69% from day 1 to day 3, while in Ham’s F10 embryo quality development yielded 68, 63 and 60% respectively. Relative to the control (18.1%) assisted hatching improved hatchability significantly (P<0.05) in the order laser (23.6%)>, mechanical (20.8%). There was significant (P<0.01) interaction between assisted hatching techniques and evaluation time, whereby laser assisted hatching was most successful at 48 h (42.0%) while mechanical assisted hatching was most successful at 72 h (36.8%). Cryopreservation reduced the embryo survival compared to fresh embryos. In conclusion laser was the best assisted hatching technique, while TCM-199 was the better medium for in vitro culture of embryo. / NRF

This diploma thesis aims to describe policy design of assisted human reproduction in the Czech Republic in the frame of two examined groups: 1) women undergoing this procedure, 2) service providers. The goals are also to identify the policy framework of assisted human reproduction in the political environment. The theory background is supported by the theory of social construction of the target audience written by the authors Ingram and Schneider and the analysis of the framework by Rein and Schon. The legislation of assisted human reproduction is covered mostly in the period of 2011 - 2021. The analytical part of this work is based on systematic expert surveys, questionnaire surveys and analysis of stenographic records. The benefits and drawbacks arising from the legislations, political power and social constructions in relation to both examined groups are formulated in the paper's results. Furthermore, two competing policy frameworks are identified during the debate of amendments to the Act on Specific health Services and the Act on Public Health Insurance.

Philosophiae Doctor - PhD / Infertility is currently a serious problem that is escalating, not only in South Africa, but also worldwide. In Cape Town, a culturally diverse, urban community of approximately 1000 couples are referred to the Groote Schuur Hospital Infertility Clinic annually. Although infertility is primarily regarded as a medical condition, the treatments have emotional effects on infertile couples due to the recurring highs and lows that often accompany treatments. This study aimed to qualitatively explore and understand the emotional and psychological experiences of infertility and its treatments (specifically In Vitro Fertilisation and Embryo Transfer). Social constructionism is based on the premise that realities are not constructed in a vacuum but rather undergo a process whereby the subjective and inter-subjective experiences over time and through cultural processes come to be regarded as truths. These truths become internalised and function as lenses through which we see ourselves, compose and invent ourselves accordingly, making sense of what would otherwise have been chaotic and meaningless experiences. Additional aims were to examine women's experiences of infertility care whilst undergoing treatment and describe their experiences of coping with infertility and In Vitro Fertilisation and Embryo Transfer (IVF-ET). Semi-structured, in-depth individual interviews were conducted with 21 women presenting with primary infertility at a fertility clinic. This study utilised an ethnographic case study design. The results of the study suggested that women perceived themselves as not conforming to a dominant belief system that promotes motherhood as the most important role for women. The women described their 'failure' to fulfill socio-cultural expectations as emotionally turbulent. Some of the psychological responses to infertility included feelings of disappointment, shock, denial, devastation, anger, frustration, sadness, inadequacy, poor self-image and self-esteem. The women's personal accounts of their experiences of In Vitro Fertilisation and Embryo Transfer (IVF-ET) revealed that they found the treatment to be highly stressful, with emotional bouts of anxiety, nervousness, excitement and optimism. A psychological synopsis of infertility and IVF-ET is infertility and IVF-ET presents. The fmdings in this study suggest the need for the incorporation of presented. This diagrammatic representation shows the intensity of the emotional rollercoaster that psychosocial intervention into infertility management. Greater attention to the psychological and emotional repercussions of infertility treatment could lead to a more personalised approach which, in turn, would optimise patient satisfaction and also prepare couples for the demands of the program by informing them about better ways of coping.

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Η ενδοκυττάρια έγχυση σπέρματος (ICSI) εισήχθη ως μέθοδος υποβοηθούμενης αναπαραγωγής (ΑRT) κυρίως για την αντιμετώπιση της ανδρικής στειρότητας. Όμως, λόγω των υψηλών ποσοστών επιτυχίας, και παρά τις αυξανόμενες ανησυχίες για τους κινδύνους από τη σημαντική ανθρώπινη παρεμβολή στο γονιδίωμα, το επιγονιδίωμα και την ανάπτυξη των παιδιών, προτιμάται ακόμα και σε περιπτώσεις όπου δεν απαιτείται ιατρικά. Από τις λίγες σήμερα συστηματικές μελέτες παιδιών από ART, καταγράφεται αυξημένο ποσοστό προδιάθεσης τους σε ασθένειες που σχετίζονται με κακό καρδιομεταβολικό πρότυπο στην ενήλικη ζωή. Στόχος της εργασίας ήταν η διερεύνηση της δυνατότητας χρήσης της μεταβολομικής ανάλυσης για τον πρώϊμο και έγκυρο προσδιορισμό σχετικών διαταραχών σε δείγματα πλάσματος προεφηβικών κοριτσιών και αγοριών από ΙCSI, που επιλέχτηκαν από ένα συστηματικά χαρακτηρισμένο σύνολο παιδιών μελέτης της Α’ Παιδιατρικής Κλινικής, Νοσοκομείου «Αγία Σοφία», Ιατρικής Σχολής, ΕΚΠΑ. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Τα μεταβολικά πρότυπα πλάσματος (α) 10 κοριτσιών από ΙCSI και 10 από φυσιολογική γονιμοποίηση (NC) και (β) 16 αγοριών από ΙCSI και 16 από NC ποσοτικοποιήθηκαν με χρωματογραφία αερίων – φασματομετρία μάζας (GC-MS). Μετά από την ταυτοποίηση κορυφών και την κατάλληλη κανονικοποίηση των προτύπων, 86 πρότυπα 70 μεταβολιτών στα κορίτσια και 92 πρότυπα 80 μεταβολιτών στα αγόρια αναλύθηκαν ξεχωριστά, και συγκριτικά με αλγορίθμους πολυπαραμετρικής στατιστικής ανάλυσης των λογισμικών TM4-MeV (v.4.9.0), και ΧLSTAT (v.2013.4.03). Οι διαφορές στο πρότυπο σύστασης του πλάσματος σε μικρού μεγέθους μεταβολίτες μεταξύ των ΙCSI και ΝC ομάδων σε κορίτσια και αγόρια και μεταξύ των δύο φύλων οπτικοποιήθηκαν σε κατάλληλα ανακατασκευασμένο από τη βιβλιογραφία και σχετικές βάσεις δεδομένων μεταβολικό δίκτυο πολλών ιστών. ΑΠΟΤΕΛΕΣΜΑΤΑ: Στα κορίτσια, ο αλγόριθμος μερικών ελαχίστων τετραγώνων-διακριτής ανάλυσης (PLS-DA) κατέδειξε σαφή διαχωρισμό των μεταβολικών πρoτύπων μεταξύ των ομάδων ΙCSI και NC. Ο διαχωρισμός αυξάνεται με το συνυπολογισμό των βιοχημικών μετρήσεων. Στα αγόρια, η PLS-DA των μεταβολικών ή και βιοχημικών προτύπων κατέδειξε επίσης διαχωρισμό, αν και μικρότερο, σε σχέση με κορίτσια. Η ανάλυση σημαντικότητας για μικροσυστοιχίες (SAM), που ενδείκνυται για την ανάλυση ομικών δεδομένων, ανέδειξε 37 από τους 70 μεταβολίτες που αναλύθηκαν στα κορίτσια με σημαντικά διαφορετική συγκέντρωση μεταξύ των ΙCSI και ΝC ομάδων, με 34 από αυτούς να αυξάνονται στην ICSI ομάδα. Οι 34 μεταβολίτες αφορούν κύρια σε σάκχαρα, αλκοόλες και οξέα σακχάρων, οργανικά οξέα και λιπίδια, που έχουν συνδεθεί με αντίσταση στην ινσουλίνη, μεταβολικό σύνδρομο, ή/και την παχυσαρκία. Η ίδια ανάλυση στα αγόρια ανέδειξε 25 από τους 80 μεταβολίτες που αναλύθηκαν με χαρακτηριστική διαφορά μεταξύ των ομάδων ICSI και NC, εκ των οποίων 9 με σημαντικά μικρότερη συγκέντρωση στην ομάδα ΙCSI. Χαρακτηριστικά αναφέρεται ότι στους 9 μεταβολίτες ανήκουν οι 4 με την πλέον διαφορετική συγκέντρωση μεταξύ των ICSI και NC ομάδων, που είναι η σορβιτόλη, και τα αρωματικά αμινοξέα τρυπτοφάνη, φαινυλαλανίνη και τυροσίνη. Συγκριτική ανάλυση του μεταβολικού προτύπου των δύο φύλων στην NC ομάδα κατέδειξε μια σαφή διαφοροποίηση, η οποία φαίνεται να αποτελεί κύρια αιτία της παρατηρούμενης φυλο-ειδικής μεταβολικής διαφοροποίησης μεταξύ των ομάδων ICSI και ΝC. ΣΥΜΠΕΡΑΣΜΑΤΑ: Η πολυπαραμετρική ανάλυση της σύστασης του πλάσματος σε μικρού μοριακού βάρους μεταβολίτες επέτρεψε τον προσδιορισμό μεταβολικών διαφορών μεταξύ των ομάδων ICSI και NC, που υποστηρίζουν την προδιάθεση των παιδιών από ICSI σε αντίσταση στην ινσουλίνη, με διακριτούς όμως μεταβολικούς και βιοχημικούς δείκτες μεταξύ των δύο φύλων. Τα ευρήματα αυτά πρέπει να επιβεβαιωθούν σε ένα ευρύτερο σύνολο παιδιών και των δύο φύλων. Καταδεικνύουν όμως την αξία της μεταβολομικής να παρέχει μία υψηλής ευκρίνειας προοπτική της μεταβολικής κατάστασης, οδηγώντας στον προσδιορισμό χαρακτηριστικών μεταβολικών προτύπων ακόμα και σε πολύπλοκες καταστάσεις φυσιολογίας. / The intracytoplasmic sperm injection (ICSI) method was introduced in artificial reproduction technology (ART) mainly to treat male infertility. However, due to its high success rates and despite the growing concerns concerning the risk that the significant human intervention associated with this method may have to the genome, epigenome and development of the offspring, the use of ICSI has gradually increased in the recent years, even when it is not medically required. Based on the few currently available systematic studies of ART conceived children, the latter are considered of higher risk for cardio-metabolic diseases as adults. The goal of the present study is to investigate whether metabolomic analysis of the blood plasma could contribute to the early and accurate determination of relevant predisposition in ICSI conceived prepubertal girls and boys, specifically selected from a systematically characterized group of children, participated in a study of the First Department of Pediatrics of the “Agia Sophia” Hospital, Medical School, University of Athens. MATERIALS AND METHODS: The blood plasma metabolic profiles of (a) 10 ICSI- and 10 naturally conceived (NC) girls and (b) 16 ICSI and 16 NC boys were acquired using gas chromatography-mass spectrometry. After peak identification and appropriate normalization, 86 profiles of 70 metabolites in girls and 92 profiles of 80 metabolites in boys were analyzed separately and comparatively using multivariate statistical analysis algorithms of TM4-MeV (v.4.9.0) and XLSTAT (v.2013.4.03) software. The differences in the plasma metabolite concentration profiles between the ICSI and NC groups in girls and boys were visualized in an inter-tissue metabolic network that was reconstructed based on relevant literature and metabolic databases. RESULTS: For the girls, the algorithm of partial least squares-discriminant analysis (PLS-DA) indicated a clear differentiation of the metabolic profiles between the ICSI and NC groups. The discrimination is more pronounced, when biochemical data are also considered. For the boys also, PLS-DA indicated separation between the metabolomic profiles of the two groups analyzed individually or in combination with the biochemical data, but not as explicit as in girls. Significance analysis for microarrays (SAM) determined 37 out of the 70 analyzed metabolites in the plasma profiles of the girls with significantly different concentration between the ICSI and the NC groups; 34 of these were of higher concentration in the ICSI group. The 34 metabolites include mainly sugars, sugar alcohols and acids, organic acids and lipids that have been associated with insulin resistance, metabolic syndrome and/or obesity. The same analysis in the plasma profiles of the boys determined 25 out of the 80 analyzed metabolites with significant difference between the ICSI and NC groups; nine of these were of significantly lower concentration in the ICSI group. It is underlined that the four most discriminatory metabolites between the ICSI and NC groups, i.e. sorbitol and the aromatic amino acids tryptophan, phenylalanine and tyrosine, are among the nine negatively significant. Comparative analysis of the metabolic profiles between the two sexes within the NC group indicated an unequivocal differentiation, which is considered to be the main cause of the observed sex-specific metabolic differences between the ICSI and NC groups. CONCLUSIONS: The multivariate statistical analysis of blood plasma metabolite profiles enabled the determination of sex-specific metabolic differences between the ICSI and NC groups; these differences support increased predisposition to insulin resistance for the ICSI offspring, with clearly different, however, metabolic and biochemical markers in the two sexes. These findings need to be confirmed in a wider group of children. They demonstrate, however, the value of metabolomics to provide a high-resolution perspective of the metabolic state, leading to the determination of characteristic metabolic profiles even in complex physiological conditions.