Dissertations / Theses on the topic 'In vitro fertilization-embryo transfer'

Cette thèse porte sur l'étude du comportement hydrodynamique d'un embryon lors de la procédure de transfert suivant la fécondation in-vitro. Un couple sur six fait l'expérience de problèmes d'infertilité. Aujourd'hui 5 millions de nourrissons sont nés depuis la première fécondation in-vitro en 1978. En 2009, 1.5 millions de cycles de Procréation Médicalement Assistée étaient débutés, donnant ainsi naissance à350 000 nourrissons de par le monde. Le nombre de cycle est en constante augmentation de 5 à 10 % par an et le nombre de cycle de PMA pourrait être proche de 4 millions à l'horizon 2020. Bien que l'étape de fertilisation soit maintenant bien maitrisée avec 80% de réussite, l'étape finale du transfert d'embryon dans la cavité intra-utérine reste une étape critique puisque seulement 25% des cycles mènent à une grossesse viable. Bien que chaque cycle soit couteux, aucun protocole spécifique, optimisé, et indépendant de l'opérateur n'a encore été mis au point. Dans cette thèse, nous nous proposons de démontrer dans un premier temps l'intérêt et la faisabilité d'une approche de bio ingénierie. En effet, bien que l'issue de transfert dépende de nombreux facteurs chimiques et physiologiques, cette étape cruciale peut aussi être étudiée d'un point de vue mécanique des fluides. Cette étape peut être décomposée en plusieurs sous-étapes : l'introduction du cathéter dans la cavité intra utérine, l'injection du fluide medium contenant un ou plusieurs embryons, et le retrait du cathéter. On peut dégager plusieurs paramètres d'importance comme la viscosité des fluides, la vitesse d'injection, la vitesse de retrait du cathéter, le schéma de chargement du cathéter, et les géométries de la cavité et du cathéter. Dans une deuxième partie, nous nous intéressons à la structure des écoulements de fluides intra-uterins au moment de l'injection. L'influence des paramètres constitutifs d'importance est étudiée grâce à un code de calcul résolvant les équations de Navier-Stokes dans une géométrie tri-dimensionnelle idéalisée. Une étude des trajectographies potentielles des embryons est également réalisée et mis en relation directe avec les zones d'implantation optimales et à risques. A l'issue de ces calculs, nous sommes en mesure de proposer des recommandations à l'usage des cliniciens pratiquant le transfert d'embryon. La dernière partie de la thèse est une ouverture vers les méthodes numériquesnécessaires à l'appréhension des phénomènes d'interaction fluide/structure à l'échelle de l'embryon. L'embryon est en effet soumis à des contraintes potentiellement destructrices au moment du transfert qu'il ne nous est pas possible de définir précisément _à l'_échelle de l'utérus. Dans l'optique du développement d'un modèle mécanique d'un blastocyste pour déterminer les paramètres procéduraux minimisant les contraintes, nous présentons l'implémentation de deux méthodes numériques de type Eulerienne-Eulerienne. La première est une méthode level-set dans un code en volumes finis et bénéficiant de raffinement de maillage automatique. La seconde concerne une méthode phase-field basée sur un formalisme éléments finis de type Galerkin discontinu
This thesis focuses on the study of the hydrodynamic behavior of an embryo during the transfer process following the in vitro fertilization. Worldwide, one in six couples experiences infertility problems. Today, 5 millions babies are born from an in-vitro fertilization since the first one in 1978. In 2009, 1.5 millions Assisted Reproductive Technology cycles have been started, resulting in 350 000 births. The total number of cycles per year is constantly increasing (from 5 to 10 %), and the number of ART cycles is believed to reach 4 millions per year in 2020. Although the fertilization step is now fairly mastered with a 80% success rate, the final stage consisting in the embryo transfer into the uterine cavity remains a critical step, since only 25% of the cycles lead to a live birth. Even though every cycle is expensive, no specific, optimized and operator-independent protocol has been developed yet. In this thesis, we first demonstrate the interest and the feasibility of a bio-engineering approach. Indeed, although the issue of the transfer depends on numerous chemical and physiological factors, this crucial step can also be studied from a fluid mechanical point of view. This step can be divided in several sub-steps : introduction of the catheter in the intra-uterine cavity, injection of the medium fluid containing one or several embryos, and the withdrawal of the catheter. One can identify several important parameters such as fluids viscosity, injections speeds, catheter withdrawal speed, catheter loading scheme and the geometries of the uterine cavity and the catheter. In a second part, we focus on the fluid ow patterns inside the uterine cavity during the injection. The influence of the system parameters is studied thanks to a computational solving of the Navier-Stokes equations in an idealized three-dimensional uterine cavity. A study of the potential trajectories of the embryos is also conducted and confronted against the location of optimal implantation zones but also risky zones. As the outcome of these computations, we are able to propose recommendations for physicians practicing embryo transfers. In the last part of the thesis, we discuss numerical methods for the fluid{structure interaction study of embryo transfer. The embryo is indeed submitted to potentially destructive stress constraints at injection time that we are not capable of defining precisely at the scale of the uterine cavity. With the aim of developing a mechanical model for the blastocyst to determine system parameters minimizing the constraints, we present the implementation of two Eulerian numerical methods. The first one is a fluid-structure level set method in a finite volume code benefiting from an automatic mesh refinement feature. The second one addresses a phase field method based on a Discontinuous Galerkin finite element formalism

The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low.
Master of Science

Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Tot i que l’origen del seu nom no és del tot clar, a mi m’agrada pensar que l’etimologia de l’hormona progesterona ve donat perquè és la hormona pro gestare, és a dir, que afavoreix la gestació. Sabem que aquesta hormona es secreta en grans quantitats després de la ovulació i, donat el cas, durant tot l’embaràs. Tanmateix, és la responsable de preparar l’endometri (la part interna de l’úter) per rebre un embrió i aconseguir així una gestació. Quan fem un tractament de reproducció assistida, en concret una fecundació in vitro, i no aconseguim un embaràs en el primer intent, moltes vegades tenim embrions congelats per poder fer un nou intent o en cas de desig d’un segon fill/a. En aquestes ocasions, hem de decidir com preparem aquest endometri per rebre l’embrió i poder aconseguir l’embaràs. La importància i necessitat de la progesterona en la preparació de l’endometri és inqüestionable. Tot i així, en un primer estudi vam trobar que les dones amb valors en sang de progesterona inferiors a 10.6ng/mL el dia abans de transferir un embrió, presentaven més avortaments (26.6% vs 9.5%) i menys nascuts vius (47.5% vs 62.3%) respecte aquelles amb valors més elevats (Gaggiotti-Marre, 2019). Aquesta tendència vam veure que es mantenia tant quan preparàvem l’endometri amb tractament mèdic hormonal com quan seguíem el cicle natural de la pacient. És a dir, tant si la progesterona la donàvem nosaltres (administració vaginal cada 8 hores) com si la fabrica la dona de forma natural (Gaggiotti-Marre, 2020). I això, per què pot passar? Vam descobrir que certs factors eren determinants dels valors de progesterona en sang: edat, pes, un cicle previ de tractament amb ja valors baixos de progesterona, i el temps des de la última administració del tractament (González-Foruria, 2020), amb la qual cosa aquests factors poden ajudar a preveure quines pacients tenen més risc de tenir uns valors baixos de progesterona i pitjors resultats reproductius. Ens queda aleshores trobar una solució a aquesta troballa que fa que les parelles tinguin més avortaments i menys nascuts vius. Vam dissenyar un estudi prospectiu pel qual les dones amb valors baixos de progesterona el dia abans de transferir l’embrió, rebien una dosi diària de progesterona amb una formulació subcutània de recent aparició al mercat. De les 453 pacients incloses en l’estudi, un 37.7% tenien valors baixos de progesterona. De les que van rebre el tractament addicional, el 98.2% van arribar a valors òptims, obtenint els mateixos resultats reproductius (nascuts vius i avortaments) que aquelles pacients que inicialment ja tenien valors elevats (Álvarez, 2021). En conclusió, la detecció de valors de progesterona en sang inferiors a 10.6ng/mL el dia abans de la transferència d’embrions congelats es relaciona amb menors nascuts vius i més avortaments, però és una troballa corregible si es detecta amb temps. Això ens permet oferir un tractament individualitzat i continuar el camí cap a una medicina personalitzada i no un tractament ‘one size fits all’.
A pesar de que el origen de su nombre no es del todo claro, a mí me gusta pensar que la etimología de la hormona progesterona deriva de que es la hormona pro gestare, es decir, que favorece la gestación. Sabemos que esta hormona se secreta en grandes cantidades después de la ovulación y, dado el caso, durante la gestación. A su vez, es la responsable de preparar el endometrio (parte interna del útero) para recibir un embrión y lograr así un embarazo. Cuando realizamos un tratamiento de reproducción asistida, en concreto una fecundación in vitro, y no conseguimos una gestación en el primer intento o por una causa médica, en muchas ocasiones tenemos embriones congelados que podemos usar para un nuevo intento o en caso de desear otro hijo/a. En estas ocasiones, hay que decidir cómo se prepara el endometrio para recibir el embrión y conseguir la gestación. La importancia y necesidad de la progesterona en la preparación del endometrio es incuestionable. Aun así, en un primer estudio hemos encontrado que las mujeres con valores en sangre de progesterona inferiores a 10.6ng/mL el día antes de la transferencia de un embrión, presentaban más abortos (26.6% vs 9.5%) y menos nacidos vivos (47.5% vs 62.3%) respecto a aquellas con valores más elevados (Gaggiotti-Marre, 2019). Esta tendencia se mantenía tanto cuando preparábamos el endometrio con tratamiento médico hormonal como cuando seguíamos el ciclo natural de la paciente. Es decir, tanto si la progesterona la dábamos nosotros (administración vaginal cada 8 horas) como si la fabrica la mujer de forma natural (Gaggiotti-Marre, 2020). Y esto, por qué puede pasar? Descubrimos que ciertos factores eran determinantes de los valores de progesterona en sangre: edad, peso, un ciclo previo de tratamiento ya con valores bajos de progesterona, y el tiempo transcurrido desde la última administración del tratamiento (González-Foruria, 2020), con la cual cosa estos factores pueden ayudar a prever qué pacientes tiene un mayor riesgo de tener unos niveles bajos de progesterona y unos peores resultados reproductivos. Nos queda entonces encontrar una solución a este hallazgo que hace que las parejas tengan más abortos y menos nacidos vivos. Diseñamos un estudio prospectivo por el cual las mujeres con valores bajos de progesterona el día antes de transferir un embrión recibían una dosis diaria de progesterona con formulación subcutánea de reciente aparición en el mercado. De las 453 pacientes incluidas en el estudio, un 37.7% presentaban valores bajos de progesterona. De las que recibieron el tratamiento adicional, el 98.2% llegaron a valores óptimos, obteniendo los mismos resultados reproductivos (nacidos vivos y abortos) que aquellas pacientes que inicialmente ya presentaban unos valores elevados (Álvarez, 2021). En conclusión, la detección de valores de progesterona en sangre inferiores a 10.6ng/mL el día antes de la transferencia de embriones congelados se relaciones con menores tasas de nacidos vivos y más abortos, pero es un hallazgos corregible si se detecta con tiempo. Esto nos permite ofrecer un tratamiento individualizado y así continual el camino hacia una medicina personalizada y no un tratamiento ‘one size fits all’.
Although its origin is not clear, I like to think that the etymology of the hormone progesterone comes from it being the hormone pro gestare, which favors the gestation. We know that this hormone is secreted in great quantities after ovulation, and specially in case of a pregnancy. Also, it is responsible for preparing the endometrium (inner lining of the uterus) to receive an embryo and accomplish a viable pregnancy. When we perform an assisted reproduction treatment, specifically an in vitro fertilization and we don’t achieve a pregnancy on the first trial, or for other medical reasons, we may have frozen embryos that we can transfer. In these cases, we need to decide how to prepare the endometrium to receive these embryos and achieve a pregnancy. The importance and need of progesterone for the endometrial preparation is unquestionable. Even though, on a first publication we found that women with serum progesterone levels below 10.6ng/mL the day prior to embryo transfer, presented higher miscarriage rates (26.6% vs 9.5%) and lower live birth rates (47.5% vs 62.3%) compared to those with higher values (Gaggiotti-Marre, 2019). This tendency was maintained both with medical preparation of the endometrium and without any treatment (natural cycle) (Gaggiotti-Marre, 2020). Y why does this happen? We found that certain factors were determinant of the serum progesterone levels: age, weight, a previous cycle with low serum progesterone, and the time from the last administered dose of progesterone (González-Foruria, 2020). This indicates that these factors can help detect those patients at a higher risk for low serum progesterone levels and detrimental reproductive outcomes. We need then to find a solution for this finding that makes that couples have more miscarriages and fewer living children. We designed a prospective study in which women with low serum progesterone levels on the day prior to frozen embryo transfer received an additional daily dose of subcutaneous progesterone. Of the 453 women included in the study, 37.7% had low serum progesterone levels. From those who received an additional dosage, 98.2% reached optimal levels, with similar reproductive outcomes to those with original optimal levels. (Álvarez, 2021). In conclusion, the detection of low serum progesterone levels, below 10.6ng/mL the day prior to frozen embryo transfer, is related to lower live birth rates and higher miscarriage rates, but this finding is correctable when detected in time. This allows us to offer an individualized treatment and continue working towards a personalized medicine in stead of a ‘one size fits all’ approach.
Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologia

This study was undertaken to evaluate fertilization and early embryo development of in vitro matured (IVM) horse oocytes following transfer with homologous sperm to the oviduct of estrous ewes. A total of 1023 follicles (5.1 per ovary) were found after processing 202 slaughterhouse ovaries by aspiration and subsequent slicing. Most follicles (79%) were less than 20-mm in diameter. Six hundred sixty-seven oocytes were recovered (3.3 per ovary; recovery rate, 65%). About two-thirds of oocytes were recovered by slicing, which yielded twice the number of oocytes as aspiration. Sixty four percent cumulus oocyte complexes (COCs) recovered by each method were grade A and the overall distribution of oocytes by grade was not affected by the method of recovery. Oocytes underwent IVM for an average of 41-h and were subjected to either in vitro fertilization (IVF) or xenogenous gamete intrafallopian transfer (XGIFT). At the onset of IVM, 83% COCs had compact cumulus investment. At the end of IVM, 78% COCs showed cumulus expansion. The expansion score was not improved with increasing the IVM duration from 32.3 to 50.3 h. Five (15%) IVF oocytes showed changes indicative of fertilization and two cleaved to 3 and 4-cell stages. Oviducts of 16 ewes were use for XGIFT, which involved surgical transfer of an average of 13 oocytes with 40x103 capacitated spermatozoa per oocyte. Of 259 oocytes transferred, 36 (14%) were recovered between 2 to 7 d post XGIFT and 13 (36%) showed cleavage ranging from the 2-cell to hatching blastocyst stage. The ovarian status of ewes and ligation of the uterotubal junction (UTJ) at the time of XGIFT, or the duration gametes were allowed to reside in the uterine tube, did not affect the recovery and cleavage rate. However, the most advanced stage embryos were recovered from ewes ovulating shortly after XGIFT. Fertilization following XGIFT was further demonstrated by the detection of ZFY loci in one embryo. This study demonstrated, for the first time, that horse embryos could be produced in a non-equine species. However, further studies focusing on the establishment of pregnancy in the mare using such embryos and improvement of the recovery and fertilization rates following XGIFT are recommended for use of XGIFT in horse assisted reproduction.
Master of Science

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Aiming to study the effect of different estrus synchronization protocols on the pregnancy rate in Bos taurus indicus x Bos taurus taurus recipient cattles, transferred with in vitro produced embryos, this study used 1,933 recipients (3,649 treatments) divided into 6 groups. In the first group, recipients received 2.0 mg of estradiol benzoate (EB) and 500 mg of cloprostenol, in addiction to an intravaginal device (1.9 g of progesterone) that remained for 8 days. Upon withdrawal of the intravaginal device each recipient received a single dose of 0.5 mg of estradiol cypionate (EC), 500 mg of cloprostenol and 400 IU of equine chorionic gonadotropin (eCG). In the second group, recipients received the same treatment as the first group, but without the 500 mg dose of cloprostenol at the placement of the progesterone intravaginal device. In the third group, recipients received at the time of intravaginal progesterone device placement a single dose of 500 mg cloprostenol and 2.0 mg of EB, and the device remained for 9 days. Two days before the intravaginal device removal (day 7) females received a single dose of 500 mg cloprostenol, and at the time of device removal, received a single dose of 0.5 mg of EC and 400 IU of eCG. In the fourth group, the recipients received the same treatment as the third group, but without cloprostenol in the intravaginal progesterone device placement. In the fifth group, recipients received 2.0 mg of EB, and an intravaginal progesterone device that remained for 8 days. Upon withdrawal of the intravaginal device, each recipient received 0.5 mg of EC, 500 mg of cloprostenol and 300 IU of eCG. In the sixth group, recipie nts received 2.0 mg of EB and an intravaginal progesterone device for 9 days. Two days before the withdrawal, (day 7), received 500 mg of cloprostenol and at the time of device removal received 0.5 mg of EC and 300 IU of eCG. All recipients that had corpus luteum were transferred on average 10 days after implant removal, in other words, about 8 days after estrus, and evaluated by ultrasonography 58 days after embryo transfer to pregnancy diagnosis. Data were subjected to descriptive statistics analysis (means, standard deviations and frequency distribution) and qualitative data were arranged in contingency tables and analyzed by chi-square at 5% of probability. Fourth group showed the best recovery rates (84.9%). However, the number of treatments performed (n=86) in group 4 was reduced in comparison with other protocols. Recipients who received Prostaglandin F2α (PGF2α) 48 hours before device removal showed better recovery rates than others and protocol 4 females had better pregnancy rates. The recipients that were in estrus longer than 91 days before device placing had worse recovery rates than recipients in estrus earlier (p<0.05), although did not influence pregnancy rate (p>0.05) . Despite some recipients peculiarities who presented estrus less than 16 days, the interval form estrus day to device placement did not influence positively these assessments (p>0.05). The uterus classified as normal in protocol 4 showed the best percentages of recovering and pregnancy rates (p<0.05) compared to the values of other protocols. However, comparing all protocols, the uterus classified as flaccid showed better recovery responses and classification did not influence uterine pregnancy rate. As the ovarian activity, the presence of corpus luteum influenced recipients recovery rates (p<0.05), whereas the follicles presence only interfered with the pregnancy rate of protocol 4 animals (p<0.05). The number of uses of the device did not influence the recipients recovery pregnancy rates (p>0.05). The reproductive status and protocol number in the history did not affect the recipients recovery rate (p>0.05). However, recipients who did not received PGF2α before intravaginal device placing, had better results than those who received (p<0.05). The recipients which were transferred with expanded blastocyst had better pregnancy rates than they which were transferred with blastocyst (p<0.05). No differences were found in the cow and heifer categories in recipient donor estrus synchrony in relation to pregnancy rate (p>0.05). No influence of the time trial on the pregnancy rate (p>0.05). The protocols which employed PGF2α 48 hours before the withdrawal had better recipients recovery rates than the protocols that applications were made on intravaginal device removal (p<0.05). Protocol 4 recipients had higher pregnancy rates, although it was a group of low numbers of animals. The used protocols interfered with the recipients recovery rate, and the applications of PGF2α 48 hours before intravaginal device removal, resulted in improved estrus synchronization responses, so the answer was more pronounced when females were cyclical in occasion of the beginning of synchronization; intervals from the last estrus to intravaginal device placement over 90 days (anestrus) influence negatively the responses to estrus and ovulation synchronization protocols. However, the classes of interval from estrus to intravaginal device placement (CLAPROT), the covariates showed no marked effect on the recipients recovery response; The female categories (cows and heifers) did not influence the responses to estrus synchronization treatments, although heifers in the pre-puberty are less responsive to PGF2α application in the beginning of the protocol. Regardless of the females category, the presence of the corpus luteum and flaccid uterine tone at the time of intravaginal device placement proved to be positively related to the recipients recovery response; Reusing intravaginal device has no influence on the recovery and pregnancy rates in embryo recipients; Females with histories of prior use of synchronization protocols with the use of PGF2α become less responsive to new synchronization protocols, while not presenting the same behavior with respect to protocols with progesterone associated with PGF2α.
Com o objetivo de estudar o efeito de diferentes protocolos de sincronização de estro (Uso de cloprostenol no momento da colocação do implante intravaginal e sua permanência por período de oito e nove dias) sobre a taxa de prenhez em receptoras bovinas Bos taurus indicus x Bos taurus taurus, inovuladas com embriões de PIV, o presente estudo utilizou 1933 receptoras (3.649 tratamentos) divididas em 6 protocolos. No protocolo 1, as receptoras receberam 2,0 mg de Benzoato de estradiol (BE) e 500 μg de cloprostenol, e um dispositivo intravaginal (1,9 g de Progesterona) que permaneceu por 8 dias. No momento da retirada do dispositivo intravaginal cada receptora recebeu uma dose única de 0,5 mg de Cipionato de estradiol (CE), 500 μg de Cloprostenol e 400 UI de gonadotrofina coriônica eqüina (eCG). No protocolo 2, as receptoras receberam o mesmo tratamento que o primeiro grupo, porém sem a dose de 500 μg de Cloprostenol na colocação do dispositivo intravaginal de progesterona. No protocolo 3, as receptoras receberam no momento da colocação do dispositivo intravaginal de progesterona uma dose única de 500 μg de Cloprostenol e 2,0 mg de BE, sendo que o dispositivo permaneceu por 9 dias. Dois dias antes da retirada do dispositivo intravaginal (dia 7) as fêmeas receberam uma dose única de 500 μg de Cloprostenol, e no momento da retirada do implante, receberam uma dose única de 0,5 mg de CE e 400 UI de eCG. No protocolo 4, as receptoras receberam o mesmo tratamento que o protocolo 3, porém sem Cloprostenol na colocação do dispositivo intravaginal de progesterona. No protocolo 5, as receptoras receberam 2,0 mg de Benzoato de estradiol, e um dispositivo intravaginal de progesterona por 9 dias. Dois dias antes da retirada, no dia 7, receberam 500 μg de Cloprostenol e no momento da retirada do implante 0,5mg de CE e 300 UI de eCG. No protocolo 6, as receptoras receberam 2,0 mg de BE, e um dispositivo intravaginal de progesterona que permaneceu por 8 dias. No momento da retirada do dispositivo intravaginal, cada receptora recebeu 0,5 mg de CE, 500 μg de Cloprostenol e 300 UI de eCG. Todas as receptoras que apresentaram corpo lúteo foram inovuladas em média 10 dias após a retirada do dispositivo, ou seja, por volta de 8 dias após estro; e avaliadas por meio de ultrassonografia aos 58 dias após inovulação para o diagnóstico de gestação. Os dados foram submetidos a análises estatísticas descritivas (distribuição de freqüência) e os dados qualitativos foram arranjados em tabelas de contingência e analisados pelo teste de qui-quadrado a 5 % de probabilidade de erro. As receptoras do quarto protocolo apresentaram as melhores (p<0,05) taxas de aproveitamento (84,9%). No entanto, o número de tratamentos realizados (n=86) para o protocolo 4 foi reduzido em relação aos demais protocolos, mais estudos tornam-se necessários para confirmar a eficácia desse protocolo. Receptoras que receberam PGF2α 48 horas antes da retirada do dispositivo apresentaram melhores índices de aproveitamento de receptoras (p<0,05) e as fêmeas do protocolo 4 apresentaram melhores índices de prenhez (p<0,05). As receptoras que apresentaram estro em período superior a 91 dias antes da colocação do dispositivo apresentaram piores taxas de aproveitamento que receptoras que apresentaram estro mais recente (p<0,05). Apesar de algumas particularidades das receptoras que apresentaram estro em período inferior a 16 dias, o intervalo dia do estro a colocação do implante não influenciou positivamente nessas avaliações (p>0,05). O útero classificado como normal no protocolo 4 foi o que apresentou melhores valores percentuais de taxa de aproveitamento e de prenhez em relação aos valores dos demais protocolos (p<0,05). Entretanto, comparando todos os protocolos, o útero classificado como flácido apresentou melhores respostas de aproveitamento de receptoras (p>0,05) e a classificação uterina não influenciou a taxa de prenhez (p>0,05). Quanto a atividade ovariana, a presença do CL influenciou na taxa de aproveitamento de receptoras (p<0,05), já a presença de folículos só interferiu na taxa de prenhez dos animais do protocolo 4 (p<0,05). O número de utilização do dispositivo não influenciou na taxa de aproveitamento de receptoras e na taxa de prenhez (p>0,05). O status reprodutivo e o número de protocolo no histórico não interferiram na taxa de aproveitamento de receptoras (p>0,05). No entanto, receptoras que não receberam PGF2α antes da colocação do dispositivo intravaginal, apresentaram melhores resultados que as receptoras que receberam PGF2α (p<0,05). As receptoras que foram inovuladas com blastocisto expandido apresentaram melhores taxas de prenhez do que as receptoras que foram inovuladas com blastocisto (p<0,05). Não houve diferença nas categorias vacas e novilhas na sincronia do estro receptora doadora, em relação à taxa de prenhez (P>0,05). Não houve influência da época experimental sobre a taxa de prenhez (p>0,05). Os protocolos que empregaram PGF2α 48 horas antes da retirada apresentaram melhores taxas de aproveitamento de receptoras do que os protocolos em que as aplicações foram feitas no momento da retirada do dispositivo intravaginal (p<0,05). As receptoras do protocolo 4 apresentaram melhores taxas de prenhez, embora tenha sido um grupo de baixo número de animais. Os protocolos utilizados interferiram na taxa de aproveitamento de receptoras, sendo que, as aplicações de PGF2α 48 horas antes da retirada do dispositivo intravaginal, resultaram em melhores respostas de sincronização de estro, sendo a resposta mais acentuada quando as fêmeas estavam cíclicas na ocasião do início das sincronizações; Os intervalos do ultimo estro à colocação do dispositivo intravaginal superiores a 90 dias (anestro) influenciam negativamente as respostas aos protocolos de sincronização de estro de ovulaç ão. No entanto, as classes de intervalo do estro à colocação do dispositivo intravaginal (CLAPROT), as co-variáveis estudadas não apresentaram efeito marcante sobre a resposta de aproveitamento de receptoras; As categorias de fêmeas (vacas e novilhas) não influenciam a respostas aos tratamentos de sincronização de estro (p>0,05), embora novilhas na fase pré-puberal são menos responsivas a aplicação de PGF2α no início do protocolo. Independente da categoria de fêmeas, a presença do corpo lúteo e tonicidade uterina flácida no momento da colocação do dispositivo intravaginal mostraram-se positivamente relacionado à resposta de aproveitamento de receptoras; A reutilização de dispositivo intr avaginal não apresenta influencia sobre a taxa de aproveitamento e prenhez em receptoras de embriões; Fêmeas com históricos prévios de uso de protocolos de sincronização com uso de PGF2α a apresentam-se menos responsíveis a novos protocolos de sincronização, embora não apresentam o mesmo comportamento com relação aos protocolos com progestagenos associado a PGF2α.

Abstract The main goal of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment is a healthy mother and a healthy child. The most important complication following IVF/ICSI arises from the increased risk of multiple pregnancies. An elective single embryo transfer (eSET) with the freezing of spare embryos and subsequent treatment with frozen embryo transfer (FET) is the only way to avoid this complication. For this reason, the number of children born after FET is steadily rising. The aim of this study was to provide more detailed evidence on the safety of FET, particularly focusing on serum hormone profiles during the first trimester weeks of singleton pregnancies after IVF/ICSI fresh embryo transfer (ET), after FET during a natural menstrual cycle, and after spontaneous conception. Another part of this study compared the perinatal outcomes, congenital anomalies (CAs), and morbidity of singletons born after FET and IVF/ICSI fresh ET. The reference group was those born after spontaneously conceived (SC) pregnancies. In the clinical prospective study, the maternal serum estradiol and progesterone levels in pregnancies after fresh ET (n=39) were higher during early pregnancy weeks than in FET (n=30) and SC pregnancies (n=41), while the hormonal profiles after FET did not differ from SC pregnancies. In the large register study, FET children (n=1830) were found to have a reduced risk for adverse perinatal outcomes, such as preterm birth, a low birthweight, and being small for their gestational age compared with children born after fresh ET (n=2942). However, FET children have an increased risk for being large for their gestational age. The major CAs and morbidity until three years of age did not differ between groups. When compared with SC children (n =31 243), the perinatal outcome was worse and the rates of CAs and morbidity were higher in FET children. The FET cycle seemed to provide a better physiological environment for early fetal development than fresh ET. Further, FET protects against some of the adverse perinatal outcomes of children when compared with fresh ET, but not when it comes to the major CAs and early somatic health. This study provides further evidence of the safety of FET in comparison with fresh ET. This information should further encourage clinicians to implement eSET combined with cryopreservation in their IVF/ICSI program
Tiivistelmä Koeputkihedelmöityshoidon (in vitro fertilization, IVF ja intracytoplasmic sperm injection, ICSI) tavoitteena on terve äiti ja terve lapsi. Monisikiöraskaus on hoidon komplikaatio, koska siihen liittyy selkeästi kohonnut riski äidille ja lapselle. Yhden alkion siirto, jäljelle jääneiden alkioiden pakastus ja myöhemmin tehtävä pakastetun alkion siirto (PAS) ovat lisänneet IVF/ICSI-hoitojen turvallisuutta ja tehokkuutta. Täten PAS:sta syntyneiden lasten määrä kasvaa. Tutkimuksen tavoitteena on lisätä PAS-hoitojen turvallisuutta tarkastelemalla veren steroidihormonien muutoksia alkuraskaudessa naisilta, jotka olivat tulleet raskaiksi IVF/ICSI-tuorealkion siirroista, luonnollisen kuukautiskierron aikana tehdystä PAS:sta ja luonnollisesti. Lisäksi tutkimuksessa verrattiin PAS- ja IVF/ICSI-tuorealkion siirrosta alkunsa saaneiden lasten terveyttä kolmeen ikävuoteen asti. Viiteryhmän muodostivat luonnollisesti alkunsa saaneet lapset. Kliinisessä prospektiivisessa tutkimuksessa havaittiin naisilla, joilla oli tuorealkion siirrosta alkanut raskaus (n=39), merkittävästi koholla olevat seerumin estradioli- ja progesteronipitoisuudet 7-8 raskausviikolle asti verrattuna naisiin, joilla raskaudet olivat alkaneet PAS:sta (n=30) tai luonnollisesti (n=41). Vastaavasti PAS-raskauksissa hormonipitoisuudet eivät eronneet merkittävästi luonnolliseen raskauteen verrattuna. Laajassa rekisteritutkimuksessa havaittiin PAS-lapsilla (n=1830) olevan pienempi riski ennenaikaisuuteen ja pienipainoisuuteen kuin tuorealkiolapsilla (n=2942). Kuitenkin PAS-lapsilla oli lisääntynyt riski syntyä isokokoisina raskausviikkoihin nähden. Synnynnäisten epämuodostumien ja eri sairauksien esiintyvyyksissä ei ollut eroja. Luonnollisesti alkunsa saaneisiin lapsiin (n= 31 243) verrattaessa, PAS-lapsilla oli vastasyntyneisyyskaudelta lähtien enemmän terveyteen liittyviä ongelmia. Tutkimus osoitti PAS-raskaudessa sikiön kehittyvän alkuviikkoina luonnollisemmassa ympäristössä kuin tuorealkion siirtoraskaudessa. Vaikka suurin osa PAS- ja tuorealkiolapsista oli terveitä, tuorealkiolapsilla oli vastasyntyneisyyskaudella enemmän ongelmia kuin PAS-lapsilla. Muita terveyseroja lasten välillä ei todettu. Tutkimus antaa lisänäyttöä PAS hoidon turvallisuudesta. Alkion pakastamisella voidaan välttää koeputkihedelmöityshoidon riskejä pyrkimällä mahdollisimman usein yhden alkion siirtoon

Abstract Fertility declines with advancing age and the number of couples seeking infertility treatment at an older age is constantly increasing. A top quality embryo is believed to have the highest potential for implantation and development into a child. A better understanding of the relative importance of patient and treatment characteristics and of embryo quality could help to optimise the existing therapeutic schemes and the safety of in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI). In this work, databases of five Finnish infertility clinics were studied retrospectively. Data on treatments performed in the years 1994–2005 were collected. A total of 19,000 treatment cycles were analysed. Special attention was paid to the relative significance of the transfer of top quality embryos with regards to pregnancy, miscarriage, live birth and cost of treatment in the general IVF/ICSI patient population and in groups with expected poor outcome. The results showed that the transfer of a top quality embryo is associated with a better chance of pregnancy and live birth. However, it does not diminish the probability of miscarriage. Both low and high BMI increase the miscarriage rate. Advancing age and a positive history of previous miscarriages are also associated with a higher probability of miscarriage. In addition, the need for hormonal substitution in cases of frozen-embryo transfer is a risk factor of miscarriage, probably because of suboptimal endometrial function. Since the transfer of several embryos leads to multiple pregnancies, which are associated with a high risk of maternal and fetal complications, elective single embryo transfer (eSET) of a top quality embryo allows all additional good quality embryos to be frozen and transferred later in frozen-thawed embryo transfer cycles. The present work demonstrates that eSET is a safe treatment strategy at least until the age of 40. However, it might not be performed in women with fewer than four collected oocytes, since the prognosis might remain poor even if the response is improved in a following cycle. When eSET is applied routinely and on a large scale, it diminishes treatment costs while increasing the number of deliveries occurring at term, making IVF/ICSI at the same time safer and more affordable even to patients without access to reimbursed IVF treatment.

Introducción Los buenos resultados reproductivos obtenidos tras la transferencia de embriones criopreservados en fecundación in vitro (FIV) han provocado un aumento significativo de la proporción de ciclos segmentados que se realizan actualmente en todo el mundo. A pesar de esto, la evidencia científica disponible indica que esta estrategia sólo parece indiscutible en ciertas situaciones clínicas (prevención del Sindrome de Hiperestimulación Ovárica, presencia o sospecha de anormalidades endometriales al momento de la recuperación ovocitaria y/o necesidad de otras intervenciones tales como la realización de Diagnóstico Genético Preimplantacional (PGT) o estudios de receptividad endometrial). Sin embargo, no hay evidencia publicada que analice si la edad de la mujer constituye un factor a tener en cuenta a la hora de decidir segmentar un ciclo de FIV. Por otro lado, una vez hemos decidido segmentar, surgen otras interrogantes como cuándo es el mejor momento para realizar la primera criotransferencia embrionaria. Este trabajo busca responder estas dos preguntas. Materiales y métodos Se plantean dos estudios de cohortes retrospectivos: 1. Comparación de resultados reproductivos de la primera transferencia en fresco (1412 ciclos) vs. ciclo segmentado (470 ciclos) en ciclos autólogos, estratificando en tres grupos de edad materna (menores de 35 años, entre 35 y 38 años y mayores de 38 años). 2. Comparación de resultados reproductivos de pacientes sometidas a una FIV segmentada que hayan realizado la primera criotransferencia de embriones durante el primer ciclo menstrual tras la punción folicular (263 ciclos) versus aquellas que la hayan realizado en ciclos posteriores (249 ciclos). Estas comparaciones se llevaron a cabo mediante un análisis univariado (Chi-cuadrado) y multivariado (regresión logística) para ajustar por factores que potencialmente puedan generar confusión. Resultados No se observaron diferencias estadísticamente significativas en la tasa de nacido vivo en mujeres de más de 35 años, independientemente del número de ovocitos recuperados. La tasa de aborto fue significativamente más baja en los ciclos segmentados de mujeres entre 35 y 38 años, sin impacto sobre la tasa de nacido vivo. Tampoco observamos diferencias significativas del momento en el cual se realiza la primera criotransferencia de un ciclo segmentado en la tasa de nacido vivo, embarazo clínico ni aborto. Conclusiones Las pacientes de menos de 35 años de edad se beneficiarían de la segmentación del ciclo de FIV para optimizar su tasa de nacido vivo. Las pacientes entre 35 y 38 años de edad se beneficiarían de la segmentación del ciclo de FIV para disminuir su tasa de aborto. El tiempo transcurrido entre la estimulación ovárica controlada y la primera criotransferencia de embriones en la estrategia freeze-all, no influye en las tasas de nacido vivo, embarazo clínico ni aborto. Esta nueva información permite al clínico tener en cuenta las preferencias de los pacientes al momento de decidir cuándo es el mejor momento para transferir en esta población de pacientes.
Introduction The promising reproductive results of frozen embryo transfers (FET) in in-vitro fertilisation (IVF) have been currently associated with a significant increase in the proportion of “freeze-all” cycles worldwide. Despite this, available evidence only supports its use in certain clinical scenarios (Ovarian Hyperstimulation Syndrome prevention, evidence of endometrial pathology and need for preimplantation genetic screening or endometrial receptivity assays). However, no published trials have assessed if the maternal age is an independent factor to take into account when deciding to undergo a freeze-all strategy. On the other hand, once we’ve decided to perform an elective freezing of all the embryos, we come across other relevant clinical questions such as when is the best moment to perform the first FET. This thesis aims to provide an answer to these questions. Materials and methods This thesis includes two independent studies: 1. Comparison of reproductive results of the first embryo transfer in autologous fresh (1412 cycles) versus freeze-all cycles (470 cycles, stratifying patients into three age groups (under 35 years, between 35 and 38 years, and over 38 years). 2. Comparison of reproductive results of patients undergoing a “freeze-all” IVF cycle who have received their first FET during the first menstrual cycle after egg retrieval (263 cycles) versus those who received it in subsequent cycles (249 cycles). We performed a univariate (Chi-square) analysis of the results and a multivariate logistic regression to adjust for potential confounders. Results We observed no statistically significant differences in live birth rate in women older than 35 years of age, independently of the number of retrieved oocytes. The pregnancy loss rate was significantly lower in the 35-38 years group that underwent a freeze-all cycle. Our results showed no significant impact of timing of the first FET on live birth, clinical pregnancy or pregnancy loss rates in women undergoing a freeze-all strategy. Conclusions Women under 35 years of age would benefit from a freeze-all strategy to optimize their live birth rate. Women between 35 and 38 years of age would benefit from a freeze-all strategy to decrease their pregnancy loss rate. Time elapsed between egg retrieval and the first FET in women undergoing a freeze-all strategy has no impact on live birth, clinical pregnancy or pregnancy loss rates. This information allows clinicians to take patients’ preferences into account when deciding the best moment to perform the embryo transfer in this population of patients.

Objectives: To determine the effect of female and male cigarette smoking, caffeine and alcohol consumption, stress and indicators of dietary status on the clinical outcomes of NF treatment. Design: Prospective cohort study. Setting: PIVET Medical Centre, Perth, Western Australia. Patients: Of 351 couples who commenced IVF treatment at PIVET Medical Centre between January 1997 and August 1998, 281 females and 247 males participated in this study, generating participation rates of 80.1% and 70.4%, respectively. Main Outcome Measures: Multivariate methods of data analyses were used to control for patient and treatment variables in the examination of the effect of lifestyle factors on the following clinical outcomes: 1) number of oocytes retrieved by transvaginal oocyte aspiration (oocyte production), 2) fertilisation, measured as the number of oocytes fertilised weighted by the number of oocytes inseminated, 3) B-hCG pregnancy, 16 days post-embryo transfer, and 4) <12 week pregnancy loss following confirmation of B-hCG pregnancy. As a measure of ovarian reserve, serum basal FSH levels were also investigated as a dependent variable. Lifestyle factors included years of cigarette smoking (smoke years), tobacco, alcohol, caffeine and fruit and vegetable consumption, and stress from daily living and NF treatment. Results: Daily stress, tobacco consumption and smoke years were the female lifestyle factors shown to have a significant effect on NF treatment. Oocyte production decreased with increasing levels of daily stress (P=0.039). However, female patients with high daily stress levels experienced higher than average rates of fertilisation in vitro (P=0.0059) and pregnancy (P--0.0207). Smoke years had an adverse effect on ovarian reserve (P=0.035), which in turn, compromised oocyte production.Female smoke years was negatively associated with rates of fertilisation (P<0.0001), and this effect was exacerbated by cigarette smoking at the time of treatment (P=0.0187). Of the male lifestyle factors, caffeine, alcohol and fruit and vegetable consumption and IVF stress affected fertilisation in vitro. Fertilisation increased with alcohol consumption (P<0.0001), and with fruit and vegetable consumption (P<0.0001). A significant interaction term between these two factors (P=0.0144) implied a threshold of benefit from the combined effect of the consumption of alcohol and fruit and vegetables. Caffeine consumption negated the beneficial effect of alcohol consumption, as shown by a significant interaction term between alcohol consumption and caffeine consumption (P=0.0007). Male stress from NF treatment had an adverse effect on rates of fertilisation in vitro (P<0.0001). Cigarette smoking by the male partner increased the likelihood of the female partner experiencing a <12 week pregnancy loss (P=0.0084). Conclusions: In meeting with its principal objective, this study has demonstrated that specific lifestyle factors impact on the clinical outcomes of IVF treatment. It confirms the findings from former studies, namely the adverse effect of female smoking on ovarian reserve, and daily stress on ovulation. Moreover, this study has identified numerous new and unexpected relationships. Of note, the positive effect of male alcohol consumption on fertilisation in vitro and the elevated risk of early pregnancy loss associated with male smoking. This study has paved the way for future research into the identification of specific mechanisms of effect, including those suggested.

Objectives: To determine the effect of female and male cigarette smoking, caffeine and alcohol consumption, stress and indicators of dietary status on the clinical outcomes of NF treatment. Design: Prospective cohort study. Setting: PIVET Medical Centre, Perth, Western Australia. Patients: Of 351 couples who commenced IVF treatment at PIVET Medical Centre between January 1997 and August 1998, 281 females and 247 males participated in this study, generating participation rates of 80.1% and 70.4%, respectively. Main Outcome Measures: Multivariate methods of data analyses were used to control for patient and treatment variables in the examination of the effect of lifestyle factors on the following clinical outcomes: 1) number of oocytes retrieved by transvaginal oocyte aspiration (oocyte production), 2) fertilisation, measured as the number of oocytes fertilised weighted by the number of oocytes inseminated, 3) B-hCG pregnancy, 16 days post-embryo transfer, and 4) <12 week pregnancy loss following confirmation of B-hCG pregnancy. As a measure of ovarian reserve, serum basal FSH levels were also investigated as a dependent variable. Lifestyle factors included years of cigarette smoking (smoke years), tobacco, alcohol, caffeine and fruit and vegetable consumption, and stress from daily living and NF treatment. Results: Daily stress, tobacco consumption and smoke years were the female lifestyle factors shown to have a significant effect on NF treatment. Oocyte production decreased with increasing levels of daily stress (P=0.039). However, female patients with high daily stress levels experienced higher than average rates of fertilisation in vitro (P=0.0059) and pregnancy (P--0.0207). Smoke years had an adverse effect on ovarian reserve (P=0.035), which in turn, compromised oocyte production.
Female smoke years was negatively associated with rates of fertilisation (P<0.0001), and this effect was exacerbated by cigarette smoking at the time of treatment (P=0.0187). Of the male lifestyle factors, caffeine, alcohol and fruit and vegetable consumption and IVF stress affected fertilisation in vitro. Fertilisation increased with alcohol consumption (P<0.0001), and with fruit and vegetable consumption (P<0.0001). A significant interaction term between these two factors (P=0.0144) implied a threshold of benefit from the combined effect of the consumption of alcohol and fruit and vegetables. Caffeine consumption negated the beneficial effect of alcohol consumption, as shown by a significant interaction term between alcohol consumption and caffeine consumption (P=0.0007). Male stress from NF treatment had an adverse effect on rates of fertilisation in vitro (P<0.0001). Cigarette smoking by the male partner increased the likelihood of the female partner experiencing a <12 week pregnancy loss (P=0.0084). Conclusions: In meeting with its principal objective, this study has demonstrated that specific lifestyle factors impact on the clinical outcomes of IVF treatment. It confirms the findings from former studies, namely the adverse effect of female smoking on ovarian reserve, and daily stress on ovulation. Moreover, this study has identified numerous new and unexpected relationships. Of note, the positive effect of male alcohol consumption on fertilisation in vitro and the elevated risk of early pregnancy loss associated with male smoking. This study has paved the way for future research into the identification of specific mechanisms of effect, including those suggested.

INTRODUÇÃO: Sabe-se que as técnicas de reprodução assistida estão associadas com potenciais riscos, principalmente relacionados às gestações múltiplas, em torno de 20 a 25%, em consequência da estimulação ovariana associada à transferência de vários embriões. As gestações múltiplas apresentam maior incidência de complicações maternas e neonatais, além de aumentarem consideravelmente a proporção de partos cesarianos. Frente a esta realidade, as gestações múltiplas podem ser evitadas pela transferência eletiva de embrião único (eSET, do inglês elective Single Embryo Transfer), uma prática que vem crescendo em todo mundo. Apesar dos diversos estudos publicados, o grande desafio ainda é comprovar os benefícios da eSET aplicada corretamente, impactando na incidência das complicações sem comprometimento do sucesso do tratamento. O objetivo deste estudo foi comparar as taxas de sucesso cumulativas da transferência eletiva de até dois embriões transferidos um a um (eSET), versus a taxa de sucesso da transferência eletiva de dois embriões em um único evento (DET, do inglês Double Embryo Transfer), em casais inférteis de bom prognóstico. MÉTODOS: Estudo retrospectivo avaliou 610 casais inférteis de bom prognóstico submetidos às TRA, divididos em dois grupos: grupo SET: pacientes submetidas à transferência eletiva de um único embrião de boa qualidade e que possuíam ao menos um embrião excedente congelado (grupo SET, n=237), possibilitando uma segunda transferência eletiva de um embrião descongelado em caso de não ocorrência de gestação; e grupo DET: pacientes submetidas a transferência eletiva de dois embriões de boa qualidade e que possuíam ao menos um embrião excedente (grupo DET, n=373). RESULTADOS: As taxas de gestação clínica acumulada após a transferência de dois embriões foram semelhantes (DET: 46.6% e SET acumulado: 45,9%; p=0,898). Por outro lado, a taxa de gestação múltipla foi significantemente inferior no grupo que recebeu transferência de dois embriões um a um versus a transferência eletiva de dois embriões em um único evento (DET: 32,2% e SET acumulado: 6,1%; p < 0,001). CONCLUSÕES: A política de SET deve ser estimulada para casais de bom prognostico, já que resulta em taxas de gestação clínica acumulada semelhantes a DET, evita gestações múltiplas e consequentemente as complicações materno-fetais, levando a reduzido custo indireto do tratamento quando considera-se os gastos obstétricos e neonatais
INTRODUCTION: It is known that Assisted Reproductive Techniques are associated with potential risks, mainly related to multiple pregnancies, which are around 20 to 25% due to ovarian stimulation associated with high number of embryos transferred. Multiple pregnancies lead to higher incidence of complications for mother and newborns, as well as a significant increase in the proportion of cesarean deliveries. This way, iatrogenic multiple pregnancies can be avoided by the elective single embryo transfer (eSET), a growing practice worldwide. Despite the several published studies, adequately applied eSET, which impact on the incidence of complications without compromising treatment success, is still a challenge. The aim of this study was to compare the cumulative success rates of elective transfer of two embryos when transferred one by one (eSET), versus the success rates of elective double embryos transfer (DET) in a single procedure, in a good prognosis population. METHODS: This retrospective study evaluated 610 good prognosis infertile couples undergoing ART, split into two groups: SET group included those receiving elective single good quality embryo transfer and having at least one spared good quality embryo cryopreserved (SET group, n=237). For those who did not become pregnant, they could receive a second frozen-thawed elective embryo transfer; and DET group (n=373) who received elective transfer of two good quality embryos in the first transfer and had at least one spared good quality embryo cryopreserved. RESULTS: The accumulated clinical pregnancy outcomes after a transfer of two embryos were similar between groups (DET: 46.6% vs accumulated SET: 45.9%; p=0.898). On the other hand, the multiple pregnancy rate was significantly lower in the group receiving transfer of two embryos, one by one, compared to DET in a single procedure (DET: 32.2% vs accumulatedSET: 6.1%; p < 0.001). CONCLUSIONS: The SET policy should be stimulated for good prognosis couples, as it maintain the accumulated clinical pregnancy rates, avoid multiples pregnancies and consequently the maternal and neonates complication and indirect costs of treatment when considering obstetrics spends are reduced

Introducción: La fecundación in vitro (FIV) se asocia a peores resultados maternos y perinatales, existiendo controversia sobre la causas. El advenimiento de nuevos estudios en el tema justifican la investigación de la seguridad de la FIV. El objetivo principal de esta tesis es evaluar los efectos de las técnicas de reproducción asistida sobre los resultados maternos y perinatales en pacientes sometidas a FIV autóloga (FIV AO) con transferencia de embriones en fresco (TEF) y congelados (TEC) y FIV con donación de ovocitos (FIV DO). Material y métodos: A fin de poder alcanzar el objetivo principal, esta tesis ha sido dividida en tres capítulos. Capítulo 1: Se realizó un estudio de cohortes retrospectivo utilizando datos del Registro Nacional de Actividad en Reproducción Humana Asistida de España entre el año 2010 y 2015, analizando la edad gestacional parto y tasa de parto prematuro (PP) de dos poblaciones diferentes: FIV AO y FIV DO. Capítulo 2: Revisión sistemática y metanálisis de estudios que compararon los resultados maternos y perinatales de embarazos únicos obtenidos por FIV con TEF versus TEC. Las variables incluyeron: PP, bajo peso al nacer (BPN), muy bajo peso al nacer (MBPN), pequeño para la edad gestacional (PEG), grande para la edad gestacional (GEG), macrosomía, preeclampsia (PE), preeclampsia severa (PES), diabetes gestacional (DG), desprendimiento prematuro de placenta normoinserta (DPPNI), placenta previa, hemorragia postparto y ruptura prematura de membranas (RPM). Capítulo 3: Revisión sistemática y metanálisis de estudios que compararon los resultados maternos y perinatales de embarazos únicos obtenidos por FIV DO versus FIV AO. Las variables incluyeron síndrome hipertensivo del embarazo (SHE), hipertensión inducida por el embarazo (PIH), PE, PES, PP, PP temprano, BPN, MBPN, cesárea, DG, RPM, placenta previa, DPPNI, y hemorragia postparto. Resultados: Capítulo 1: Se analizaron 41022 FIV AO (28754 TEF y 12268 TEC) observándose una mayor proporción de PP en TEF comparado con el grupo TEC (P .01). En el grupo de FIV DO, con un total de 19941 casos (12394 TEF y 7547 TEC) no se observaron diferencias en cuanto al parto prematuro en ninguno de los grupos al comparar TEF versus TEC. Capítulo 2: 35 estudios se incluyeron en el análisis. TEC se asoció con un riesgo menor para PP (RR 0.89; IC95% 0.82, 0.97), BPN (RR 0.73; IC95% 0.69, 0.78), MBPN (RR 0.63; IC95% 0.60, 0.66), PEG (RR 0.63; IC95% 0.60, 0.66); y un riesgo mayor para GEG (RR 1.53; IC95% 1.48, 1.58), macrosomía (RR 1.72; IC95% 1.65, 1.78), PE (RR 1.20; IC95% 1.06, 1.35) y PES (RR 1.96; IC95% 1.33, 2.88). No hubo diferencias estadísticamente significativas para el riesgo de DG y RPM. Capítulo 3: 23 estudios fueron incluidos en el análisis. FIV DO se asocia con un riesgo mayor de SHE (OR 2.63, 2.17-3.18), PE (OR 2.64; 2.29-3.04), PES (OR 3.22; 2.30-4.49), PP (OR 1.57; 1.33-1.86), BPN (OR 1.25, 1.20-1.30). No hubo diferencias estadísticamente significativas en cuanto al riesgo de PP ni BPN luego de ajustar por PE. Conclusiones: Los embarazos obtenidos por FIV AO con TEC presentan un menor riesgo de PP y BPN comparado con FIV AO con TEF. Los embarazos obtenidos por FIV AO con TEC presentan mayor riesgo de SHE y PE comparado con FIV AO con TEF. Los embarazos obtenidos por FIV DO presentan mayor riesgo de PP, BPN y SHE y PE comparado con FIV AO.
Introduction: In vitro fertilization (IVF) is associated with adverse maternal and perinatal outcomes and there is controversy about the causes. The publication of new studies on the subject justify the investigation of the safety of IVF. The main objective of this thesis is to evaluate the effects of assisted reproduction techniques on maternal and perinatal outcomes in patients undergoing autologous IVF (IVF AO) with fresh embryo transfer (ET) and frozen embryo transfer (FET); and IVF with oocyte donation (IVF OD). Methods: In order to achieve the main objective, this thesis has been divided into three chapters. Chapter 1: A retrospective cohort study was conducted using data from the National Registry of Activity in Assisted Human Reproduction of Spain between 2010 to 2015, analyzing the gestational age at delivery and the preterm birth (PB) rate of two different populations: IVF AO and IVF OD. Chapter 2: Systematic review and meta-analysis of studies comparing maternal and perinatal outcomes of singleton pregnancies after IVF with fresh ET versus FET. Outcomes included PB, low birth weight (LBW), very low birth weight (VLBW), small for gestational age (SGA), large for gestational age (LGA), macrosomia, preeclampsia (PE), severe PE, gestational diabetes (GD), placental abruption, placenta previa, postpartum hemorrhage and premature rupture of membranes (PROM). Chapter 3: Systematic review and meta-analysis of studies comparing maternal and perinatal outcomes of singleton pregnancies obtained by IVF OD versus IVF AO. Outcomes included hypertensive disorders of pregnancy (HDP), PE, PES, pregnancy induced hypertension (PIH), PB, early PB, LBW, VLBW, cesarean section, GD, PROM, placenta previa, placental abruption, and postpartum hemorrhage. Results: Chapter 1: 41022 IVF AO cases (28754 fresh ET and 12268 FET) were analyzed, with a higher proportion of PB in fresh ET group compared to FET group (P .01). In the IVF OD group, with a total of 19941 cases (12394 fresh ET and 7547 FET), no differences were observed between fresh ET and FET. Chapter 2: 35 studies were included in the analysis. FET was associated with a lower risk of PB (RR 0.89, 95%CI 0.82, 0.97), LBW (RR 0.73, 95%CI 0.69, 0.78), VLBW (RR 0.63, 95%CI 0.60, 0.66) and SGA (RR 0.63, 95%CI 0.60, 0.66); and a higher risk of LGA (RR 1.53; 95%CI 1.48, 1.58), macrosomía (RR 1.72; 95%CI 1.65, 1.78), PE (RR 1.20, 95%CI 1.06, 1.35) and severe PE (RR 1.96, 95%CI 1.33, 2.88). There were no statistically significant differences for the risk of GD and PROM. Chapter 3: 23 studies were included. IVF-OD is associated with a higher risk of hypertensive disorders in pregnancy (OR 2.63, 2.17-3.18), preeclampsia (OR 2.64; 2.29-3.04), severe preeclampsia (OR 3.22; 2.30-4.49), pregnancy induced hypertension (OR 2.16; 1.79-2.62), preterm birth (OR 1.57; 1.33-1.86), low birth weight (OR 1.25, 1.20-1.30). There was no significant difference in the risk of preterm birth or low birth weight when adjusted for preeclampsia.. Conclusions: Pregnancies after IVF AO and FET have a lower risk of PB and LBW compared to IVF AO and fresh ET. Pregnancies after IVF AO and FET have a higher risk of PE compared with IVF AO and fresh ET. Pregnancies after IVF OD have a higher risk of PB, LBW and HDP and PE compared to IVF AO.

Objetivo: Avaliar as evidências disponíveis comparando a eficácia da estimulação ovariana (EO) com uso de citrato de clomifeno (CC) e/ ou letrozol (LTZ) para reduzir o consumo de FSH, em relação à estimulação ovariana padrão (EOP). Métodos: Realizamos uma revisão sistematizada e meta-análise de ensaios clínicos randomizados (ECRs) que compararam os desfechos reprodutivos na fertilização in vitro. As buscas foram realizadas em onze bancos de dados eletrônicos e avaliamos manualmente a lista de referência dos estudos incluídos e revisões similares. Nós estratificamos os resultados separando os estudos baseados no agente oral utilizado (CC ou LTZ) e nas características da mulher incluída (em que se espera e em que não se espera má resposta ovariana). Os desfechos avaliados foram risco relativo (RR) para nascimento vivo, gravidez clínica, aborto, e taxa de cancelamento de ciclo, Peto Odds Ratio (OR) para síndrome de hiperestímulo ovariano (SHO), e diferença média (MD) para número de óocitos captados e consumo de FSH (ampolas). Resultados: Foram incluídos 22 estudos nesta revisão. Considerando o grupo de mulheres em que se espera má resposta, a evidência sugere que o uso de CC durante a estimulação ovariana resulta em similares taxas de nascidos vivos (RR= 0,9, IC95% = 0,6 a 1,2, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,8 a 1,4, evidência de moderada qualidade); o uso de LTZ não causa alteração significativa no número de oócitos captados (MD= -0,4, IC95% = -0,9 a +0,1, evidência de alta qualidade). Considerando os estudos que avaliaram mulheres em que não se esperava má resposta, a evidência sugere que o uso de CC reduz o número de oócitos captados (MD= -4,6, IC95%= -6,1 a -3,0, evidência de alta qualidade) e o risco de SHO (Peto OR= 0,2, IC95%= 0,1 a 0,3, evidência de moderada qualidade), enquanto os resultados são semelhantes para taxas de nascidos vivos (RR= 0,9, IC 95% = 0,7 a 1,1, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,9 a 1,2, evidência de alta qualidade). Para os demais desfechos a qualidade das evidências foi baixa ou muito baixa. Conclusões: A utilização de CC em mulheres em que se espera má resposta tem a vantagem de alcançar resultados reprodutivos semelhantes com redução dos custos. Para as demais mulheres, o uso do CC tem a vantagem adicional de reduzir o risco de SHO, mas também reduz o número de oócitos captados. Mais estudos seriam necessários para avaliar o efeito do LTZ com o mesmo propósito. Estudos futuros devem ter como objetivo estudar a taxa de gravidez cumulativa por oócito captado, insatisfação da paciente e aceitação para repetir o ciclo se não engravidar, que são dados importantes para a tomada de decisões clínicas.
Objective: To assess the available evidence comparing effectiveness of ovarian stimulation (OS) using clomiphene citrate (CC) and/or letrozole (LTZ) for reducing FSH consumption compared with standard OS. Methods: We performed a systematic review and meta-analysis of randomized controlled trials (RCTs) that compared the reproductive outcomes following in vitro fertilization. We searched eleven electronic databases and hand-searched the reference list of included studies and related reviews. We stratified the results separating the studies depending on the oral agent (CC or LTZ) and on the characteristics of the included women (expected poor ovarian response or other women). When combining the results of included studies, we assessed the relative risk (RR) for live birth, clinical pregnancy, miscarriage, and cycle cancelation, Peto Odds Ratio (OR) for OHSS, and mean difference (MD) for the number of oocytes retrieved and FSH consumption. Results: A total of 22 studies were included in this review. Considering women with expected poor ovarian response, the available evidence suggests that using CC for reducing FSH consumption during OS provide similar live birth (RR=0.9, 95%CI=0.6-1.2, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.8-1.4, moderate quality evidence); the use of LTZ doesn\'t cause a relevant change on the number of oocytes retrieved (MD=-0.4, 95%CI= -0.9 to +0.1, high quality evidence). Considering the studies evaluating other women, the available evidence suggests that using CC for reducing FSH consumption during OS reduces the number of oocytes retrieved (MD=-4.6, 95%CI=-6.1 to -3.0, high quality evidence) and the risk of OHSS (Peto OR=0.2, 95%CI=0.1-0.3, moderate quality evidence), while results in similar live birth (RR=0.9, 95%CI=0.7-1.1, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.9-1.2, high quality evidence). The quality of the evidence was low or very low for the other outcomes. Conclusion: The use of CC for reducing FSH consumption in women with expected poor ovarian response has the advantage of providing similar reproductive outcomes with reduced costs. For the other women, the use of CC for reducing FSH consumption has the additional advantage of reducing OHSS, but also reduces the total number of oocytes retrieved. More studies are necessary to evaluate the effect of LTZ for the same purpose. Future studies should aim on cumulative pregnancy per oocyte retrieval, patient dissatisfaction and agreement to repeat the cycle if not pregnant; which are important outcomes for clinical decisions.

A produção in vitro (PIV) de embriões bovinos é uma das biotécnicas de reprodução que mais evoluíram nas últimas duas décadas. No Brasil, a associação com a técnica de aspiração folicular guiada por ultra-som determinou uma significativa disseminação do uso comercial da PIV. A produção em larga escala de embriões PIV associada à dificuldade em criopreservá-los, determinou que um número significativo desses embriões passasse a ser descartado nas atividades de campo, gerando prejuízos aos produtores e técnicos envolvidos no processo. Os experimentos realizados no âmbito desta tese tiveram como objetivo identificar e propor soluções para um aproveitamento mais eficiente dos ovócitos e embriões PIV. Em virtude da qualidade dos embriões poder ser influenciada pelo sistema de produção in vitro, foi realizado um experimento (Capítulo 1) para determinar a capacidade dos embriões derivados de dois sistemas de PIV em resistir à vitrificação. Entretanto, nas condições testadas, não foram verificadas influências significativas do efeito do sistema de PIV. No segundo experimento (Capítulo 2), buscou-se identificar a influência da solução crioprotetora sobre a taxa de sobrevivência embrionária in vitro e in vivo. Os resultados obtidos revelaram um efeito significativo da solução de vitrificação sobre a sobrevivência in vitro, por outro lado as taxas de prenhez foram semelhantes. Estes resultados também comprovaram a viabilidade do método de transferência direta desenvolvido neste experimento. Finalmente, em um terceiro grupo de experimentos (Capítulo 3), foi determinada a viabilidade da vitrificação de ovócitos imaturos usando duas estratégias de resfriamento. Não foram observadas diferenças entre os tratamentos. Entretanto, a obtenção de produtos nascidos de embriões vitrificados, derivados de ovócitos imaturos vitrificados, confirma a viabilidade de criopreservação desses ovócitos como alternativa para programação das atividades de PIV.
The in vitro production (IVP) of bovine embryos has achieved an important development during the last two decades. In Brazil the large scale use of the follicular aspiration guided by ultrasonography has determined a significant improvement in the in vitro embryo production as a commercial tool. The difficulties to cryopreserve bovine oocytes and IVP embryos are at the moment the greatest barrier impairing the efficient application of this reproductive biotechnology which results in the disposal of large numbers of bovine IVP embryos. Considering these factors, this Thesis aimed to highlight new approaches to reduce embryo disposal and improve the pregnancies rates after the cryopreservation of bovine immature oocytes and IVP blastocysts. The first experiment (Chapter 1) was designed to observe the cryotolorance of embryos produced by two different IVP systems. The tested in-vitro systems did not show differences in the cryotolerance by in vitro production system. A second experiment (Chapter 2), was carried out to determine in vitro and in vivo embryo survival rates of IVP blastocysts cryopreserved using different vitrifications solutions. The results showed a significant vitrification solution effect on in vitro survival, however, when in vivo, the pregnancy rates were not different. This experiment also verified the feasibility of the direct transfer method for vitrified embryos. Finally, in a third experiment (Chapter 3) the viability of the vitrification of immature oocytes was determined using two cooling strategies. Differences among the treatments were not observed. However, calves born from the transfer of vitrified embryos, derived from vitrified immature oocytes, highlighted that the increase in cryopreservation efficiency of immature oocytes may be an important alternative to improve the IVP commercial application.

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The variability of successful transfers of embryos produced in vitro is still one of the obstacle to its expansion, where some of the problems are related to early embryonic mortality. The aim of this work was to evaluate the application of flunixin meglumine and recombinant bovine somatotropin and observe the effect of variables such as embryonic development, embryo synchrony with the recipient, corpus luteum size at the time of transfer and pregnancy rate. In the first experiment 55 recipient heifers were randomly in the three different groups: G1 control group (n=15 animals); G2 group receiving 500mg of recombinant bovine somatotropin (bST)/animal/subcutaneous (n=20 animals) and G3 group receiving 500mg flunixin meglumine/animal/intramuscular (n=20 animals). Pregnancy rates for G1 53,33% (8/15), G2 60% (12/20), G3 55% (11/20) with no statistically significant difference between groups (P> 0,05). In experiment II 134 heifers were used as recipients of embryos produced in vitro. The pregnancy rate was evaluated according to the degree of development of the structure transferred embryo synchrony with the receiver, and corpus luteum size. Embryos (blastocyst, expanded blastocyst, ecloded blastocyst) showed better pregnancy rates than less development younger embryos (morula, early blastocyst), 57,14% and 25% respectively (P<0,05). Synchrony with the recipient embryo -1 (68,42%), 0 (88,88%), +1 (41,5%) for P<0,05 and size of the corpus luteum large 46,83% CL1, CL2 average 55,88%, 42,85% CL3 small (P> 0,05). In conclusion, under experiment conditions described, the application of flunixin meglumine, recombinant bovine somatotropin and was not efficient to increase the pregnancy rate, but it is note that the pregnancy rate varied in the degree of development of the embryo and the embryo synchrony with the recipient.
A variabilidade do sucesso das transferências de embriões produzidos in vitro ainda é um dos entraves para sua expansão, onde alguns dos problemas são relacionados à mortalidade embrionária precoce. O objetivo com estes trabalhos foi avaliar a aplicação da flunixina meglumina e da somatotropina recombinante bovina e observar o efeito de algumas variáveis como grau de desenvolvimento do embrião, sincronia do embrião com a receptora e classificação do corpo lúteo da receptora no momento da transferência sobre a taxa de prenhez. No experimento I foram utilizadas 55 novilhas receptoras de embrião agrupadas aleatoriamente: G1 grupo controle (n=15animais); G2 grupo que recebeu 500mg de somatotropina recombinante bovina/animal/por via subcutâneo (n=20 animais) e G3 grupo 500mg de flunixina meglumina/animal/via intramuscular (n=20 animais). As taxas de prenhez para os grupos foram G1 53,33% (8/15), G2 60% (12/20), G3 55% (11/20) não havendo diferença significativa entre os grupos (P>0,05). No experimento II foram utilizadas 134 novilhas como receptoras de embriões produzidos in vitro. A taxa de prenhez foi avaliada segundo grau de desenvolvimento da estrutura transferida, sincronia do embrião com a receptora e a classificação corpo lúteo. Embriões desenvolvidos (Blastocisto, Blastocisto expandido) apresentaram melhores índices de prenhez que embriões jovens (Mórula, Blastocisto inicial), 57,14% e 25% respectivamente (P<0,05). Sincronia do embrião com a receptora que apresentou melhores taxas de prenhez foram: sincronia 0 (88,88%) e sincronia - 1 (68,42 %) em relação a sincronia + 1 (41,5 % ) para P<0,05 e classificação do corpo lúteo não houve diferença com CL1 grande 46,83%, CL2 médio 55,88%, CL3 pequeno 42,85% (P>0,05). Nas condições deste experimento a aplicação da flunixina meglumina e da somatotropina recombinantes bovina não foi eficiente para aumentar a taxa de prenhez, porém deve-se atentar que a taxa de prenhez foi dependente do grau de desenvolvimento do embrião e sincronia da receptora com o embrião.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Os objetivos do estudo foram: avaliar o efeito antioxidante do Trolox® 1) no meio de manutenção de embriões sobre as taxas de concepção (25 e 46 dias) e a perda gestacional de receptoras bovinas Holandesas repetidoras de serviço, em dois períodos (abril-junho/setembro-novembro) e 2) no meio de transporte de oócitos bovinos aspirados de ovários de abatedouro sobre as taxas de clivagem, blastocisto e eclosão in vitro. No Experimento 1, doadoras de embriões foram superovuladas e submetidas à lavagem uterina. Os embriões colhidos foram divididos em dois grupos, deixados em meio com ou sem antioxidante, por 2 a 6h. No Experimento 2, folículos foram aspirados e os complexos cumulus-oócito grau I e II foram divididos em quatro grupos: Controle 8h, Antioxidante 8h, Controle 20h e Antioxidante 20h. Então, foram mantidos em uma transportadora de oócitos (37ºC) por 8 ou 20 horas. Após a maturação, fecundação e cultivo in vitro, as taxas de clivagem (D3), blastocisto (D6, 7 e 9) e eclosão (D11) foram avaliadas. Alíquotas de 200 μL do meio de transporte foram retiradas em cada momento experimental (0, 8 e 20h) para realização dos testes de capacidade antioxidante total (CAT). No Experimento 1, não houve efeito de tratamento sobre as variáveis avaliadas. Foi verificado efeito de período experimental (P=0,001), categoria da doadora (P<0,05), qualidade embrionária (P=0,049) e intervalo Divisão dos Grupos-Inovulação (P<0,0001). No Experimento 2, não houve efeito de tratamento sobre as taxas analisadas. No entanto, houve redução das taxas de clivagem e blastocisto (D7 e 9) nos grupos 20 horas. Ainda, no D6 foram obtidas taxas de blastocisto semelhantes nos grupos Antioxidante 8 e 20h. A análise da CAT evidenciou que o Trolox® auxiliou o combate às ROS.
The objectives of the present study were to evaluate the effect of the addition of an antioxidant (Trolox®) 1) to a holding media for bovine in vivo produced embryos, on conception rates 25 and 46 days of pregnancy and pregnancy loss, in Holstein bovine recipients at 4th or more service, during two periods of the year (April-June/September-November) and 2) to a transport media for bovine oocytes aspirated from slaughter house ovaries, on in vitro cleavage, blastocyst and hatching rates. In Experiment 1, donor cows were superovulated and submitted to uterine flushings. The recovered embryos were divided into two groups, kept in holding media with or without antioxidant, for 2-6h. In Experiment 2, follicles were aspirated and cumulus-oocyte complexes grade I and II were divided into four groups: Control 8h, Antioxidant 8h, Control 20h and Antioxidant 20h. Oocytes were kept in a transportable machine (37ºC) for 8 or 20 hours. After in vitro maturation, fertilization and culture, cleavage (D3), blastocyst (D6, 7 and 9) and hatching rates (D11) were evaluated. Samples (200 μL) of the transport media were collected in each experimental moment (0, 8 e 20h) for total antioxidant capacity assay (TACA). In Experiment 1, no effect of treatment was observed. Effects of experimental period (P=0.001), donor category (P<0.05), embryo quality (P=0.049) and interval Group division-embryo transfer (P<0.0001) were detected. In Experiment 2, no effect of treatment was found on the analyzed rates. However, reduction on cleavage and blastocyst (D7 e 9) rates was detected on Groups 20h. Also, on D6 similar blastocyst rates were observed in Groups Antioxidant 8 and 20h. The analysis of TACA evidenced that Trolox® collaborated with the combat against the ROS.

La ICSI porcina es una herramienta con gran potencial aplicativo en diversos campos, entre los que destacan la producción de animales transgénicos, y la recuperación de razas en peligro de extinción. Aunque en la actualidad existen referencias de obtención de descendencia viva, el rendimiento es inferior al de otras especies, posiblemente debido al desconocimiento de las condiciones idóneas, y la dificultad de los cigotos para alcanzar el estadío de blastocisto in vitro. El presente trabajo se llevó a cabo para determinar diferentes factores que podrían afectar al rendimiento de la técnica, estudiando el efecto de 1) la secuencia de cultivo de los zigotos recién inyectados; 2) modificaciones en el sistema de MIV tradicional, y por último 3) la activación exógena del ovocito mediante la inyección de inositol trifosfato con el espermatozoide. El objetivo global de este estudio fue el de incrementar el rendimiento final de la ICSI en la especie porcina.
ICSI in pigs is a tool with an important applicable potential in diverse fields. One of this is the production of transgenic animals, and the conservation of endangered species. Even though there are some cases of living offspring, its output is still quite low comparing to other species, possibly due to unknown factors referring to ideal conditions for the development, and to the difficulty of the zygotes to reach the blastocyst stage in vitro. The goal of this study was to evaluate different factors affecting the ICSI performance. This was done by studying 1) the sequence of culture of the injected oocytes; 2) In vitro maturation (IVM) modifications, through meiotic inhibitors, such as roscovitine, and changes in IVM duration time, and finally 3) the exogenous oocyte activation through inositol triphosphate (InsP3) injection together with the sperm. The main objective of this study was to increase the final performance of ICSI in pigs.

Um Projeto Temático de Pesquisa foi desenvolvido no Laboratório de Poluição Ambiental do Departamento de Patologia da Faculdade de Medicina da Universidade de São Paulo com o objetivo de avaliar os efeitos da exposição aguda/crônica ao ar ambiente de um grande centro urbano sobre a saúde. Dentro deste projeto, uma linha de pesquisa foi dedicada ao estudo dos efeitos dessa exposição sobre a saúde reprodutiva feminina. Evidências de estudos epidemiológicos e experimentais implicam os fatores ambientais na infertilidade humana e resultado obstétrico adverso. Contudo, poucos estudos foram conduzidos até o presente para avaliar um possível efeito da exposição à poluição ambiental particulada sobre a saúde reprodutiva feminina. Portanto, o objetivo dos projetos da minha linha de pesquisa é fornecer dados que possam demonstrar os possíveis efeitos da exposição pré-concepcional de curta duração às partículas de exaustão do diesel (PED) e à poluição ambiental particulada sobre a função ovariana, o desenvolvimento embrionário inicial e resultado gestacional utilizando um modelo experimental e um epidemiológico. O objetivo do primeiro projeto desta tese foi avaliar os efeitos de dois meios de cultura comerciais no desenvolvimento de oócitos de camundongo fertilizados in vitro até o estágio de blastocisto. Zigotos obtidos de fêmeas de camundongo de 8 semanas de idade submetidas à indução da ovulação foram cultivados in vitro até o estágio de blastocisto em meio simples otimizado enriquecido com potássio (KSOM) ou meio G1/G2. A porcentagem de zigotos que se desenvolveu até o estágio de blastocisto 96 e 120 horas após a inseminação e que sofreu eclosão parcial ou completa no quinto dia de cultivo foi significativamente maior no grupo KSOM. O número médio de células da massa celular interna (MCI) foi 11,7 ± 4,0 e 9,2 ± 5,2 para os zigotos cultivados nos grupos KSOM e G1/G2, respectivamente, mostrando um número significativamente maior de células MCI em blastocistos derivados da cultura no meio KSOM. Concluímos que o meio KSOM comercialmente disponível é superior ao meio seqüencial G1/G2 para o cultivo de zigotos até o estágio de blastocisto no modelo de fertilização in vitro (FIV) em camundongos. No segundo projeto que compõe esta tese, o objetivo foi avaliar os efeitos da exposição de curta duração às PED sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais utilizando o modelo de FIV em camundongo. A instilação intranasal de água destilada (grupo controle), de PED nativas (grupo PED-N) ou de PED ácidoextraídas (grupo PED-AE), realizada uma vez ao dia por três dias, iniciada no primeiro dia de administração de gonadotrofinas, foi realizada em fêmeas de camundongo com oito semanas de idade. Os pontos de avaliação reprodutivos analisados incluíram a resposta ovariana a estimulação, taxa de fertilização, desenvolvimento embrionário, taxas de formação e de eclosão dos blastocistos, contagem celular total e proporção da alocação celular à MCI e trofoectoderma (TE), e a morfologia da MCI. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariado para a exposição às PED-N e PED-AE na coloração diferencial de blastocistos e na morfologia da MCI, mas não para a FIV ou desenvolvimento embrionário, foi observado. A contagem celular da MCI e a razão MCI/TE em blastocistos produzidos no grupo controle foram significativamente maiores do que em blastocistos produzidos nos grupos PED-N e PED-AE. O número total de células dos blastocistos foi similar entre os grupos. O escore que representa a morfologia da massa celular interna foi significativamente maior no grupo controle quando comparado àquele encontrado nos grupos PED-N e PEDAE. Baseando-se nesses resultados, nosso estudo sugere que a exposição de curta duração às PED pode afetar negativamente o processo reprodutivo através do distúrbio da especificação das linhagens celulares do embrião em estágio de blastocisto. Finalmente, a exposição a toxinas ambientais pode ser inevitável durante o período pré-concepcional em grandes centros urbanos e seus efeitos são desconhecidos. Portanto, o propósito do terceiro projeto que compõe esta tese foi avaliar os potenciais efeitos da exposição de curta duração à poluição ambiental particulada durante a fase folicular sobre os resultados clínicos, laboratoriais e gestacionais de casais submetidos à fertilização in vitro e transferência de embriões (FIVETE). Trezentos e quarenta e oito mulheres submetidas ao seu primeiro ciclo de FIVETE foram avaliadas retrospectivamente neste estudo coorte, casocontrole casado. A exposição ao material particulado ambiental (MP) durante a fase folicular de cada paciente foi estimada baseando-se em dados da poluição ambiental (1997-2006) categorizados em período Q1-3 ( 56,72 g/m3) e Q4 (>56,72 g/m3). Desse grupo, 177 pacientes que engravidaram (casos) foram comparadas com 354 mulheres que conceberam espontaneamente (controles). Os principais pontos de avaliação incluíram a resposta ovariana às gonadotrofinas, o número de oócitos recuperados e as taxas de fertilização, de clivagem, de qualidade embrionária, de implantação, de gestação, de abortamento e de nascidos vivos. Nenhum efeito da exposição a níveis elevados de MP durante a fase folicular foi observado nos resultados clínico e laboratorial, na transferência embrionária ou no sucesso dos ciclos de tratamento das pacientes submetidas à FIVETE. Mulheres expostas ao período Q4 durante a fase folicular do ciclo de concepção apresentaram um risco significativamente maior de abortamento, independentemente do método de concepção (razão de chance: 2,58; intervalo de confiança de 95%: 1,63 4,07), quando comparado àquele de mulheres expostas ao período Q1-3. O risco de perda da gestação aumentou 3% por unidade de aumento do valor médio do MP na fase folicular (p= 0,000). Os resultados apresentados aqui fornecem evidências para uma relação causal entre a breve exposição a níveis elevados de MP ambiente durante o período pré-concepcional e a perda gestacional inicial, independentemente do método de concepção e está associada a um aumento de 2,6 vezes no risco de abortamento. Apesar da ausência de efeitos dessa exposição sobre os resultados clínicos e laboratoriais e sobre o sucesso do tratamento, a FIVETE foi incapaz de reduzir esse risco
A thematic research project to evaluate the health effects of acute/chronic exposure to ambient air in a large urban center was developed at the Air Pollution Laboratory in the Department of Pathology at the University of São Paulo School of Medicine. Within this project a specific research line was committed to the study of the effects of this exposure on female reproductive health. Evidence from epidemiological and experimental studies implied environmental factors as possible contributors to human infertility and poor obstetric outcome. However, very few studies evaluating a possible effect of exposure to particulate air pollution on female reproductive health have been conducted so far. Thus, the aim of the projects in my research line was to provide data that could show the possible effects of short-term preconceptional exposure to diesel exhaust particles and particulate air pollution on ovarian function, early embryo development and pregnancy outcome using experimental and epidemiological models. The objective of the first project was to examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. One-cell embryos obtained from eight-week old superovulated mice were cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 media. The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination and that partially or completely hatched by day five of culture was significantly higher in the KSOM group. The mean number of inner cell mass (ICM) cells was 11.7 ± 4.0 and 9.2 ± 5.2 for zygotes cultured in KSOM and G1/G2 groups respectively, revealing a significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. I concluded that commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model. In the second project the objective was to evaluate the effects of short-term exposure to diesel exhaust particles on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the mouse IVF model. Intranasal instillation of distilled water (control group), native diesel exhaust particles (N-DEP group) or acid-extracted diesel exhaust particles (AE-DEP) once a day, for three days starting on the first day of gonadotrophin administration was performed on eight-week old female mice. Reproductive endpoints evaluated included ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to ICM and trophectoderm (TE), and ICM morphology. Ovarian response was not affected by the exposure protocol. A multivariate effect for exposure to NDEP and AE-DEP on blastocyst differential staining and ICM morphology but not on IVF or embryo development was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the control group were significantly higher than in blastocysts produced in N-DEP and AE-DEP groups. The total cell count was similar among groups. The score that represents ICM morphology was significantly higher in the control group when compared to that found in N-DEP and AE-DEP groups. Based on these results this study suggests that short-term exposure to DEP may negatively affect the reproductive process by disrupting the lineage specification at the blastocyst stage. Finally, exposure to environmental toxins may be unavoidable during the preconceptional period in large urban centers and its effects are unknown. Thus, the purpose of the third project was to assess the potential effects of short-term exposure to particulate air pollution during the follicular phase on clinical, laboratory, and pregnancy outcomes for couples undergoing IVF/ET. Three hundred forty-eight patients undergoing their first IVF/ET cycle were evaluated in this retrospective cohort-matched casecontrolled single-center study. Exposure to ambient particulate matter (PM) during the follicular phase for each patient was estimated based on air pollution data (1997-2006) categorized in Q1-3 ( 56.72 g/m3) and Q4 (>56.72 g/m3) periods. From this group 177 women who became pregnant (cases) were compared with 354 who had conceived spontaneously (controls). Main outcome measures included response to gonadotrophins, number of oocytes retrieved, fertilization, cleavage, embryo quality, implantation, pregnancy, miscarriage, and live birth rates. No effects of follicular phase exposure to high levels of PM on clinical, laboratory, embryo transfer or treatment outcome were found in women undergoing IVF/ET. Women exposed to Q4 level PM during the follicular phase of the conception cycle had significantly higher risk of miscarriage, regardless of the method of conception (odds ratio, 2.58; 95% confidence interval: 1.63-4.07) when compared to women exposed to Q1-3 level PM. The risk of miscarriage increased 3% per unit increase in follicular phase PM average level (p=0.000). The results presented here provide evidence of a causal role for brief exposure to high levels of ambient PM during the preconceptional period in early pregnancy loss, regardless of the method of conception, with a 2.6-fold increase in risk of miscarriage. Despite the absence of effects of this exposure on clinical, laboratory, and treatment outcome, IVF/ET was unable to reduce this risk

Orientador: Paulo Henrique Franceschini
Banca: Antônio Cláudio Tedesco
Banca: Francisco Guilherme Leite
Resumo: Os objetivos do estudo foram: avaliar o efeito antioxidante do Trolox® 1) no meio de manutenção de embriões sobre as taxas de concepção (25 e 46 dias) e a perda gestacional de receptoras bovinas Holandesas repetidoras de serviço, em dois períodos (abril-junho/setembro-novembro) e 2) no meio de transporte de oócitos bovinos aspirados de ovários de abatedouro sobre as taxas de clivagem, blastocisto e eclosão in vitro. No Experimento 1, doadoras de embriões foram superovuladas e submetidas à lavagem uterina. Os embriões colhidos foram divididos em dois grupos, deixados em meio com ou sem antioxidante, por 2 a 6h. No Experimento 2, folículos foram aspirados e os complexos cumulus-oócito grau I e II foram divididos em quatro grupos: Controle 8h, Antioxidante 8h, Controle 20h e Antioxidante 20h. Então, foram mantidos em uma transportadora de oócitos (37ºC) por 8 ou 20 horas. Após a maturação, fecundação e cultivo in vitro, as taxas de clivagem (D3), blastocisto (D6, 7 e 9) e eclosão (D11) foram avaliadas. Alíquotas de 200 μL do meio de transporte foram retiradas em cada momento experimental (0, 8 e 20h) para realização dos testes de capacidade antioxidante total (CAT). No Experimento 1, não houve efeito de tratamento sobre as variáveis avaliadas. Foi verificado efeito de período experimental (P=0,001), categoria da doadora (P<0,05), qualidade embrionária (P=0,049) e intervalo Divisão dos Grupos-Inovulação (P<0,0001). No Experimento 2, não houve efeito de tratamento sobre as taxas analisadas. No entanto, houve redução das taxas de clivagem e blastocisto (D7 e 9) nos grupos 20 horas. Ainda, no D6 foram obtidas taxas de blastocisto semelhantes nos grupos Antioxidante 8 e 20h. A análise da CAT evidenciou que o Trolox® auxiliou o combate às ROS.
Abstract: The objectives of the present study were to evaluate the effect of the addition of an antioxidant (Trolox®) 1) to a holding media for bovine in vivo produced embryos, on conception rates 25 and 46 days of pregnancy and pregnancy loss, in Holstein bovine recipients at 4th or more service, during two periods of the year (April-June/September-November) and 2) to a transport media for bovine oocytes aspirated from slaughter house ovaries, on in vitro cleavage, blastocyst and hatching rates. In Experiment 1, donor cows were superovulated and submitted to uterine flushings. The recovered embryos were divided into two groups, kept in holding media with or without antioxidant, for 2-6h. In Experiment 2, follicles were aspirated and cumulus-oocyte complexes grade I and II were divided into four groups: Control 8h, Antioxidant 8h, Control 20h and Antioxidant 20h. Oocytes were kept in a transportable machine (37ºC) for 8 or 20 hours. After in vitro maturation, fertilization and culture, cleavage (D3), blastocyst (D6, 7 and 9) and hatching rates (D11) were evaluated. Samples (200 μL) of the transport media were collected in each experimental moment (0, 8 e 20h) for total antioxidant capacity assay (TACA). In Experiment 1, no effect of treatment was observed. Effects of experimental period (P=0.001), donor category (P<0.05), embryo quality (P=0.049) and interval Group division-embryo transfer (P<0.0001) were detected. In Experiment 2, no effect of treatment was found on the analyzed rates. However, reduction on cleavage and blastocyst (D7 e 9) rates was detected on Groups 20h. Also, on D6 similar blastocyst rates were observed in Groups Antioxidant 8 and 20h. The analysis of TACA evidenced that Trolox® collaborated with the combat against the ROS.
Mestre

Muitas das técnicas utilizadas para gerar animais transgênicos são caras e laboriosas. Neste contexto, a transferência gênica mediada por espermatozóides (TGME) pode ser uma alternativa para a produção em larga escala de animais transgênicos. Estudos de SMGT têm seu foco no número de cópias de DNA incorporada pelo espermatozóide. Por isso, há pouca informação disponível sobre como as moléculas de DNA se comportam durante o processo de fecundação e quais os efeitos dos protocolos de TGME sobre a célula espermática. Neste sentido, com o objetivo de avaliar a existência de sitio de integração preferencial das moléculas exógenas de DNA no genoma hospedeiro, utilizamos a hibridização in situ para acompanhar a veiculação do transgene durante o processo de fecundação. Foram avaliadas as membranas acrossomais, plasmática e potencial de membrana mitocondrial de espermatozóides submetidos a TGME. Para isso, o sêmen de três diferentes touros foram submetidos ao gradiente de Percoll 45-90%. As células viáveis foram incubadas com o vetor recombinante pCX-EGFP (0, 250, 500 ou 1000ng/106 células) seguidas ou não de eletroporação (300v, 35µF e 0,25ms). Os espermatozóides tratados foram utilizados para a produção in vitro de embriões. Os embriões foram cultivados por sete dias até o estágio de blastocisto. Espermatozóides e embriões produzidos in vitro foram submetidos ao ensaio de hibridização in situ, com metodologia descrita Whyte et al. (2000). O potencial de membrana mitocondrial (PMM), integridade de membrana acrossomal (MA) e plasmática (MP) foram avaliados por citometria de fluxo (Guava Technologies, Hayward, CA, USA) utilizando as sondas fluorescentes JC1, FITC-PSA e PI (Molecular Probes), respectivamente. Os dados foram analisados pelo teste paramétrico ANOVA (teste LSD) usando o programa estatístico SAS for Windows, com nível de significância de 5%. A hibridização in situ não foi possível em espermatozóides bovinos, pois não houve hibridação da sonda controle. Blastocistos oriundos de espermatozóides incubados com DNA exógeno apresentaram integração de forma difusa, embriões oriundos de espermatozóides eletroporados apresentaram integração pontual. As diferentes concentrações de DNA não exerceram efeitos deletérios nas MP ou PMM, a adição de 500ng de DNA causou aumento de lesão na MA (p<0,05). A eletroporação não afeta a MP e MA, mais apresenta grande efeito no PMM causando redução da função mitocondrial. Este estudo conclui que maiores esforços são necessários para elucidar o comportamento das moléculas exógenas de DNA durante o processo de fecundação e quais são os efeitos da TGME sobre a célula espermática.
Most techniques used to produce transgenic animals are laborious and expensive. In this manner, sperm mediated gene transfer (SMGT) may be a viable alternative for long-scale production of transgenic animals. Many SMGT studies have focused the DNA internalization and number of DNA copies incorporated by spermatozoa. However, limited data is available about how foreign DNA molecules behave during fertilization and the direct effects of the SMGT technique on sperm cells. Hence, in order to monitor the existence of preferential integration sites by the exogenous DNA at the host genome, in situ hybridization was used to track the transgene conveyance during in vitro fertilization. In addition, acrosome and plasmatic membrane integrity and mitochondrial membrane potential of sperm cells subjected to SMGT were assessed. Briefly, thawed semen from three different bulls was submitted to a 45- 90% Percoll gradient. Viable cells were incubated with recombinant PCX-EGFP vector (0, 250, 500 or 1000ng/106 sperm cells) or incubated and electroporated (300V, 35µF and 0.25ms). Treated sperm cells were then used for in vitro production of embryos. Embryos were in vitro cultured for 7 days until blastocyst stage. Treated spermatozoa and in vitro produced blastocysts were submitted to in situ hybridization assay, as described by Whyte et al. (2000). The mitochondrial membrane potential (MMP), acrosomal membrane (AM) and plasmatic membrane (PM) integrity were assessed by flow cytometry (Guava Technologies, Hayward, CA, USA) using JC1, FITC-PSA and PI probes (Molecular Probes), respectively. Data were analyzed by parametric ANOVA (LSD test) using SAS for Windows software, at a 5% level. The transgene was not observed at the bovine spermatozoa because the control probe could not be hybridized. In situ hybridization revealed that blastocysts produced from incubated sperm cells had a diffuse foreign DNA integration while blastocysts produced from electroporated sperm cells had a punctual DNA integration. No deleterious effects of exogenous DNA concentrations on PM or MMP were observed. However, the addition of 500ng of exogenous DNA caused sperm AM injury (P<0.05). Electroporation did not affect PM or AM integrity, but it had a great effect on MMP, which may cause a reduction of mitochondrial function. This study suggest that more efforts are needed to elucidate the behavior of exogenous DNA during fertilization and the effects of SMGT in bovine sperm cells.

Uvod: Višeplodne trudnoće se javljaju u 1,5% svih trudnoća nakon spontane koncepcije, dok nakon postupaka vantelesne oplodnje ovaj postotak u Evropi iznosi preko 20% uz velike varijacije među zemljama. U našoj sredini, stopa višeplodnih trudnoća nakon postupaka vantelesne oplodnje iznosi daleko iznad 30%. Pojava hipertenzivnog sindroma u trudnoći, gestacijskog dijabetesa, operativnog završavanja trudnoće, prevremenog porođaja, male porođajne telesne mase, neuroloških sekvela kod rođene dece i gotovo svih drugih komplikacija po majku i plod, kao i celokupno opterećenje zdravstvenog sistema višestruko su veći kod višeplodnih u odnosu na jednoplodne trudnoće i udeo navednih komplikacija raste sa brojem plodova. Sa druge strane deca iz postupaka vantelesne oplodnje čine i do 4,5% sve živorođene dece u pojedinim zemljama, što uz činjenicu da infertilitet pogađa 16-18% parova u našoj sredini daje ovoj pojavi posebnu dimenziju i činije i društvenim problemom. Perinatalni ishodi trudnoća iz postupaka vantelesne oplodnje su u velikoj meri kompromitovani visokom stopom multiplih trudnoća, koje se danas smatraju komplikacijom, a ne uspehom postupaka vantelesne oplodnje. Jednoplodne trudnoće iz postupaka vantelesne oplodnje u većim studijama pokazuju diskretno slabije perinatalne ishode u odnosu na one spontano začete, dok kod višeplodnih trudnoća ova korelacija nije jasno izražena i dokumentovana, uz prisutnu dilemu da li je višeplodnost sama po sebi ili način koncepcije glavni problem u zapaženoj pojavi. Cilj rada: Uporediti perinatalne ishode višeplodnih trudnoća nastalih postupcima vantelesne oplodnje i spontano začetih kao i perinatalne ishode jednoplodnih i višeplodnih trudnoća iz postupaka vantelesne oplodnje. Pored navdenog cilj rada je i ukazati sveobuhvatnost navedenog problema i na moguća rešenja za smanjenje njihove učestalosti. Materijal i metode: Kombinacijom retrospektivne opservacione studije i prospektivne longitudinalne kohortne studije u periodu analizom perinatalnih ishoda pacijentkinja porođenih na Klinici za ginekologiju i akušerstvo Kliničkog centra Vojvodine u periodu od od 01.01.2008. do 31.12.2010. godine, studija je analizirala i poredila perinatalne ishode kod 174 spontano začete višeplodne trudnoće, 163 višeplodne trudnoće nastale postupkom vantelesne oplodnje, kao i 155 jednoplodnih trudnoća začete postupkom vantelesne oplodnje. Analizirani parametric bili su telesna masa novorođenčeta, dostignuta gestacijska starost, vrednosti Apgar skora, učestalost hipertenzivnog sindroma kod majke i brojni drugi parametri perinatalnog ishoda. Uzeti od strane obučenih kliničara i unošeni u posebno dizajniranu bazu podataka, rezultati su statistički analizirani u program JMP ver 9.0 (SAS publisher) uz korišćenje ANOVA analize za testiranje statističke značajnosti između srednjih vrednosti kontinuiranih varijabli, dok je statistička značajnost razlike učestalosti kategorijskih varijabli je određivana Pearsonovim χ2 testom. Rezultati: Jednoplodne ART trudnoće uz prosečnu starost od 33,5 godine, prosečnu gestacijsku starost na porođaju od 38,26 gn, udeo prevremenih porođaja od 12,9%, prosečnu telesnu masu od 3258 g, AS u prvom minutu od 8,35 i u petom minutu od 9,2, stopu carskog reza od 65,81%, udeo GDM-a od 7,1%, anemije od 41,94% i preeklampsije od 4,52%, ima sve relevantne parametre perinatalnog ishoda statistički značajno (p<0.0001) superiornije od kako ART tako i non ART blizanačkih trudnoća. ART blizanačke trudnoće pokazale su prosečnu starost majke od 32,9 godina, prosečnu gestacijsku starost na porođaju od 35,6 gn, udeo prevremenih porođaja od 58,27%, prosečnu telesnu masu od 2374 g, AS u prvom minutu od 7,45 i u petom minutu od 8,65, stopu carskog reza od 83,7%, udeo GDM-a od 15,11%, anemije od 78,42% i preeklampsije od 12,23%, dok su non ART blizanačke trudnoće pokazale prosečnu starost majke od 28,8 godina, prosečnu gestacijsku starost na porođaju od 36,08 gn, udeo prevremenih porođaja od 49,71%, prosečnu telesnu masu od 2433 g, AS u prvom minutu od 7,75 i u petom minutu od 8,75, stopu carskog reza od 58,33%, udeo GDM-a od 7,02%, anemije od 67,84% i preeklampsije od 11,11%. Pored godina majke i udela carskog reza koji su bili viši u ART blizanačkim trudnoćama (<0.0001), kao i blago veće pojavi poremećaja količine plodove vode (p=0,033), gotovo svi ostali pokazatelji toka i ishoda trudnoće bili su komparabilni u navedenim grupama. Diskusija i zaključak: Studija je pokazala da su tok i ishod višeplodnih trudnoća nastalih spontano i postupcima vantelesne oplodnje ekvivalentni u gotovo svim pokazateljima uz sličnu prosečnu telesnu masu i gestacijsku starost novorođenčadi, kao i da su svi navedeni parametri ovih višeplodnih trudnoća bez obzira na način koncepcije upadljivo i podjednako lošiji u poređenju sa jednoplodnim trudnoćama iz postupka vantelesne oplodnje. Izuzimajući višeplodnost kao factor rizika deca iz postupaka vantelesne oplodnje su generalno zdrava. Sama višeplodnost, a ne način koncepcije predstavljaju problem, koje se sa pravom smatra najvećom komplikacijom vantelesne oplodnje. Dodatna analiza iskustava drugih zdravstvenih sistema ukazuje da jedino široka i sveobuhvatna implementacija strategije vraćanja samo jednog embriona (Single embryo transfer – SET) može da dovede do smanjivanje stope multiplih trudnoća nakon postupaka vantelesne oplodnje, i sledstvenih komplikacija, a bez ugrožavanja samog uspeha vantelesne oplodnje. Iskustva drugih zdravstvenih sistema ukazuju da je uspešna implementacija SET-a jedino moguća uz angažovanje celog društva, zajedno sa brojnim legislativnim merama iz domena nadzora, kontrole i finansiranja postupaka vantelesne oplodnje. Obim i način finansiranja postupaka vantelesne oplodnje od strane države (uz više besplatnih pokušaja za infertilne parove) uz obaveznu upotrebu SET-a, i sistema krioprezervacije na osnovu primera iz prakse predstavlja ključ u borbi za smanjenje problema višeplodnih trudnoća nakon postupaka vantelesne oplodnje.
Introduction: Multiple pregnancies occur in 1.5% of all pregnancies after spontaneous conception and in more than 20 % of all pregnancies concieved after assisted reproductive technologies in Europe, with large variations between countries. In our setting, the rate of multiple pregnancies after the ART is well above 30%. The occurrence of hypertensive syndrome in pregnancy, gestational diabetes, operative delivery, premature birth, low birth weight, neurological and developmental impairment in children, and almost all the other complications for the mother and fetus, as well as the entire burden of the health system are several times higher in multiple pregnancies compared with singleton pregnancies. Incidence of  forementioned complications rises with number of fetuses. On the other hand, children from in vitro fertilization procedures make up 4.5% of all live births in some countries, which together with the fact that infertility affects aproximately 16-18% of couples in our country gives an extra dimension to this phenomenon and makes it not just medical but wider social problem. Perinatal outcomes of pregnancies after assisted reproductive technologies (ART) are greatly compromised by the high rate of multiple pregnancies, which are now considered to be a complication rather than success of ART procedures. ART Singleton pregnancies have, in larger studies, show discretely lower perinatal outcomes compared with those conceived spontaneously, while for the multiple pregnancies, this correlation is not clearly expressed and documented. There remains dilemma whether multiplicity itself or the way of conception (ART vs. non ART) constitutes a major problem in the observed differences regarding perinatal outcome of ART pregnancies. Objective: To compare the perinatal outcomes of multiple pregnancies conceived by In vitro fertilization (IVF) and spontaneously and perinatal outcomes of IVF conceived singleton and multiple pregnancies. Additional aim of this thesis is to point out the complexity of this problem and offer possible solutions. Materials and Methods: Design of a study was a combination of retrospective and prospective observational longitudinal cohort study. Analysis included pregnancies which had delivery at the Department of Gynecology and Obstetrics, Clinical Center of Vojvodina in the period from 1.01.2008. to 31.12.2010. The study analyzed and compared the perinatal outcomes in 174 spontaneous conceived multiple pregnancies, 163 multiple pregnancies resulting from IVF procedures, and 155 singleton pregnancies conceived by IVF procedure. Analyzed parameters were newborns birth weight, gestational age at delivery, the value of the Apgar score, occurrence of hypertensive syndrome in pregnancy, gestational diabetes, as well as numerous parameters of perinatal outcome. Taken by trained clinicians and were entered into a specially designed database, the results were statistically analyzed in JMP ver 9.0 software (SAS publisher) using ANOVA analysis to test the statistical significance between the mean values of continuous variables, while the statistical significance of the difference in frequency of categorical variables was assessed by Pearsons χ2 test. Results: ART singleton pregnancies had an average mothers age of 33.5 years, the average gestational age at birth of 38.26 gestational weeks (gw), preterm delivery rate of 12.9%, average birth weight 3258 g, Apgar score (AS) in the first minute 8.35, and in the fifth minute 9.2, cesarean section rate 65.81%, Gestational diabetes (GDM) in 7.1% pregnancies, anemia occurred in 41.94% of pregnancies, while preeclampsia was observed in 4.52% of all pregnancies. All relevant parameters of perinatal outcome were significantly (p<0.0001) superior to both ART and non-ART twin pregnancies. ART twin pregnancy showed the average mothers age of 32.9 years, the average gestational age at birth of 35.6 gw, the preterm delivery rate 58.27%, the average body weight newborns 2374 g, AS in the first minute of 7.45, and in the fifth minute of 8.65, the cesarean section rate of 83.7%, GDM in 15.11% of all pregnancies, anemia occurred in 78.42% and preeclampsia in 12.23% of pregnancies, while the non-ART twin pregnancy showed an average mothers age of 28.8 years, the average gestational age at birth of 36.08 gw, the preterm delivery rate of 49.71%, the average body weight of 2433 g, AS in the first minute of 7.75 in the fifth minute 8.75, the caesarian section rate of 58.33%, GDM-a occurred in 7.02%, anemia in 67.84% and preeclampsia in 11.11% of pregnancies. Except for maternal age and the caesarean section rate, which were significantly higher in ART twin pregnancies (p<0.0001), as well as small increase in proportion of amniotic fluid volume disorders (p = 0.033), almost all other parameters of perinatal outcome of were comparable in these groups. Discussion and Conclusion: The study showed that the course and outcome of multiple pregnancies conceived spontaneous and after IVF procedures are equivalent in almost all parameters with similar average body weight and gestational age at birth, and that all these parameters of multiple pregnancies regardless of the conception mode are equally worse compared with singleton pregnancies from IVF procedures. With the exception of multiplicity as a risk factor children from in vitro fertilization procedures are generally healthy. Multiplicity itself and not the mode of conception presented a problem, which is rightly considered the major complication of IVF today. Additional analysis of the experiences of other health system indicates that only a broad and comprehensive implementation of strategy to return only one embryo (SET–single embryo transfer) can lead to a reduction of the rate of multiple pregnancies after IVF procedures, and the accompanying complications, without compromising IVF success. The experience of other health systems indicate that a successful implementation of SET is only possible with the involvement of the whole society, along with a number of legislative measures in the field of monitoring, control and reimbursement of assisted reproduction procedures. The scope and funding of an IVF procedures (with more free attempts for infertile couples, reimbursed by public health) with mandatory use of SET, and good cryopreservation programs are, based on examples in other countries who had successfully dealt with his problem, is the key in reducing the problem of multiple pregnancies after IVF procedures.

Within many communities and religions, including the LDS community, there is some controversy surrounding the use of stem cells – particularly embryonic stem cells (ESC). Much of this controversy arises from confusion and misconceptions about what stem cells actually are, where they come from , and when life begins. The theology of the Church of Jesus Christ of Latter-day Saints has interesting implications for the last of these considerations, and it becomes less a question of “when does life begin” and more an exploration of “when does personhood begin” or “when does the spirit enter the body.” With no official Church stance, statements from Church leaders vary on this topic, and this first section of the thesis explores the philosophical and practical meaning of personhood with a biological background intended for those not familiar with the origin or uses of stem cells.The second portion of the thesis explores possible cloning technologies. Recent events and advances address the possibility of cloning endangered and extinct species. The ethics of these types of cloning have considerations uniquely different from the type of cloning commonly practiced. Cloning of cheetahs (and other endangered or vulnerable species) may be ethically appropriate, given certain constraints. However, the ethics of cloning extinct species varies; for example, cloning mammoths and Neanderthals is more ethically problematic than conservation cloning, and requires more attention. Cloning Neanderthals in particular is likely unethical and such a project should not be undertaken. It is important to discuss and plan for the constraints necessary to mitigate the harms of conservation and extinct cloning, and it is imperative that scientific and public discourse enlighten and guide actions in the sphere of cloning.

Clinical Assisted Reproductive Technology (ART) practices seek to make improvements in embryo quality and resultant procedural success rates. There is a significant variance in live birth rates among clinics nationwide. The goal of this thesis is make comparisons of embryo quality among clinics and understand these differences. This analysis focuses on the stage between egg retrieval and embryo transfer. Because the currently accepted embryo scoring methods are not directly proportional to performance, a new scoring methodology is proposed and applied. Data provided by the Society for Assisted Reproductive Technology (SART) consisting of 36,836 patient cycles from 40 anonymous clinics nationwide is considered. After necessary reductions are made, the data is anatomized to link each embryo transferred to an implantation probability. A score is generated for each morphology grouping based on the average implantation rate of that group. This score is used as the basis for clinic comparisons. Top-performing clinics (in terms of live birth rates in patients agedold) are then shown to both produce embryos of higher score and achieve better results from embryos of identical morphology.

Introduction: Light microscopy has remained the primary tool for the assessment of embryo quality and the selection of embryos for transfer in clinical IVF practice. Recent studies have suggested that metabolomic profiling of embryo culture media can distinguish human embryos with better implantation potential. We therefore undertook the following study to further assess the usefulness of metabolomic profiling “spent” embryo culture medium using Raman spectroscopy. Methods: Patients undergoing IVF+/-ICSI treatment from the UBC Centre for Reproductive Health were recruited for study. Demographic and clinical information was collected. As part of routine clinical procedures, embryos were individually cultured in G1 media from Day 1-3 and in G2 media from Day 4-6. G1 and G2 culture medium (vitrolife, Englewood, CO) introduced specifically for cleavage embryo and blastocyst culture respectively. Embryo-free G1 and G2 droplets were placed alongside the embryo-containing ones as controls. For the study, fresh droplets of spent and control culture media were individually collected on Day 3 and 6 and prepared for assessment by Raman spectroscopy. The assessment score under light microscopy of the corresponding embryo and its fate were recorded for comparison and correlation. To validate the detection limits of Raman spectroscopy a wide range of glucose and glycine concentrations between 0 and 500 mM in distilled water were analyzed. Results: A total of 300 embryos/spent media droplets from 54 patients aged 27-43 years (mean age ± SD: 36.33 ± 3.26) were evaluated. Of 111 embryos transferred, 19 implanted and led to a pregnancy: 7 (12.96%) single and 6 (11.1%) twin pregnancies. Irrespective of pregnancy, there were no systematic differences between the Raman spectra generated from spent media of Day 3 and Day 6 embryos, or between spent media and control media. Conclusions: In contrast to published reports, our study does not show that metabolomic profiling of spent embryo culture media by Raman spectroscopy can differentiate embryos with better implantation potential or add value to light microscopic assessment as in clinical practice.

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: CHAPTER 1 In this chapter the aim is to outline the different chapters under section A. Against this background, we will conduct a literature review of relevant studies performed, and evaluate their comments regarding identifying embryo markers which can be utilized to improve overall ART outcome. We will evaluate the embryo marker sHLA-G in detail, using a prospective randomized study as well as a retrospective multi-centre study. The role of the morphology and genetic profile of an oocyte, zygote and embryo and subsequent blastocyst formation will be evaluated with the help of WGA/CGH. The work will then be summarized and conclusions will be made as well as possible suggestions for future directions will be indicated. In section B the methodology of the studies explaining the role of the candidate is illustrated. CHAPTER 2 In this chapter the impact of the oocyte/zygote and the embryo on implantation/pregnancy rate was discussed. The morphologic characteristics of the oocyte, the cumulus–oocyte-complex (COC), the zona pellucida, the perivitelline space, cytoplasm and meiotic spindle and the polar body and its appearance were discussed in detail. The morphologic characteristics of embryo fragmentation and its effect on embryo development, ploidy and blastocyst formation were also studied. Embryo markers to predict pregnancy outcome were researched based on the international literature. The pronuclear morphology and early cleavage were highlighted as non-invasive embryo markers to predict outcome. A non-invasive biochemical marker, soluble Human Leucocyte-Antigen-G (sHLA-G), that is expressed by developing embryos was researched. The value of blastocyst transfer and the improved ongoing pregnancy rate compared to cleavage stage embryos were highlighted based on a recent meta-analysis. A detailed discussion on sHLA-G as well as Array-CGH and the future of these tests followed. CHAPTER 3 In this chapter the aim was to compare pregnancy and implantation rates when embryos are selected based on a single Day 3 (D 3) morphology score vs. a GES score plus sHLA-G expression. This was a prospective randomized study (n=214) undergoing fresh ICSI cycles. Embryos were selected for transfer based on either Day 3 morphology score (Group A) or GES-scoring plus sHLA-G expression (Group B). The following results were reported: Clinical [35/107 (33%) vs. 52/107 (49%)] and ongoing pregnancy [20/107 (19%) vs. 52/107 (49%)] rates were significantly different between Group A and Group B (p<0.05). Implantation rates were not significantly different between Group A [52/353 (15%)] and Group B [73/417 (18%)] (p<0.05). The number of pregnancies lost during the first trimester was nearly 12 times higher in Group A [25/52 (48%)]. It was concluded that the miscarriage rate was significantly lower in Group B than Group A and the pregnancy results were superior when embryos were selected based on GES plus sHLA-G expression. CHAPTER 4 Several studies have reported an association between the presence of soluble human leukocyte antigen-G (sHLA-G) in human embryo culture supernatants (ES) with implantation and pregnancy outcome in vitro. However, the actual presence role during implantation and effect on implantation and pregnancy outcome are still controversial. A retrospective multi-centre study was performed on 2040 ICSI patients in six different centers. All embryos were individually cultured and a chemiluminescence enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of sHLA-G in culture medium surrounding embryos. In all centers, a positive sHLA-G result was associated with an increase in odds of multiple clinical implantations (OR: 1.48, 95% CI: 1.07 to 2.05, p-value: 0.0170), and an increased odds of multiple on-going pregnancies (OR: 1.66, 95% CI: 1.10, 2.51, p-value: 0.0170). Data from this multi-centre study emphasize that sHLA-G expression is a valuable non-invasive embryo marker to assist in improving pregnancy outcome with the theoretical potential to reduce multiple pregnancies. A combination of sHLA-G expression and extended embryo culture to the blastocyst stage might provide future tools by which to select single embryos for transfer and reduce the risk of multiple gestational, without compromising their pregnancy rates. CHAPTER 5 In this chapter the ploidy status of first and second polar bodies and Day 3 blastomere, embryo morphology and biochemical (sHLA-G) characteristics were correlated with blastocyst development and subsequent pregnancy outcome. All oocytes/zygotes and embryos were individually cultured to the blastocyst stage. PB-I, PB-II and blastomeres underwent whole genome amplification (WGA) and comparative genome hybridization (CGH) and complete karyotyping. Each embryo‟s culture medium supernatant was collected and analyzed for sHLA-G expression on Day 2. The following results were reported: Fifty seven mature (MII) donor oocytes were obtained, 33/57 (57.9%) were aneuploid, 21/57 (36.8%) were euploid and 3/57 (5%) were “inconclusive”. No correlation was found between CGH status of PB-I, PB-II and the GES-score. Furthermore, no correlation was established between PB-I CGH results and blastocyst morphology grade. There was a significant correlation between PB-I CGH and blastomere CGH results. Euploid and aneuploid PB-I developed into 58% and 67% blastocysts, respectively. Kappa statistics (>0.7) revealed a positive correlation between the ploidy of PB-I, PB-II and the blastomeres. It was concluded that following ICSI and sequential genetic karyotyping of the oocyte/zygote and subsequent blastomeres, the majority of oocytes fertilized and subsequent zygotes developed into blastocysts, despite their ploidy status. We therefore conclude that blastocyst development is not associated with ploidy. CHAPTER 6 Identifying a developmentally competent embryo to transfer that has the highest probability to develop into a live baby has been an issue of debate and continues research. The aim of this chapter is to discuss the morphological, biochemical and genetic features of an embryo that has been shown to be predictive of implantation and pregnancy outcome in ART using most current evidence. A literature search was performed looking at the correlation between pronuclear morphology, early cleavage, cleavage stage embryos, blastocyst development, the presence of sHLA-G, CGH, embryo development and implantation/pregnancy rates in ART. Based on the available literature, a combination of observations could assist the scientist with embryo selection. The pronuclear stage morphology, the early embryo division, cleavage embryo stage and quality of the day 3 embryos provides limited guidance. However, choosing a blastocyst with a positive sHLA-G result on Day 5 is the optimal combination to make the final selection before embryo transfer or freezing. This non-invasive approach should improve pregnancy outcome and reduce multiple pregnancy rates. As far as the use of the more invasive technology such as aCGH is concerned, more research on pregnancy outcome is needed. CHAPTER 7 A combination of observations for embryo selection, starting with oocyte grading, pronuclear stage morphology, early zygote cleaving and cleavage-stage embryo morphology/quality on Day-3, however, ultimately using extended embryo culture and choosing a blastocyst on Day 5 with positive sHLA-G values available, will assist the scientist in making the final decision before selecting an embryo for transfer or cryopreservation. The use of aCGH (for chromosomal analysis) is invasive and is still considered experimental. Finally we conclude that despite all the above mentioned parameters to select an embryo for transfer that will develop into a live baby, more extensive research and international corroboration is needed in order to improve and standardize embryo selection criteria.
AFRIKAANSE OPSOMMING: HOOFSTUK 1 Die doel in hierdie hoofstuk is om die verskillende hoofstukke onder Afdeling A uiteen te sit. Daar word beplan om „n literatuur oorsig te doen van toepaslike studies rakend embriomerkers wat swangerskap-uitkoms in in vitro bevrugting kan verbeter. Verder sal die embriomerker sHLA-G deeglike bestudeer word met behulp van „n prospektiewe gerandomiseerde studie, asook „n retrospektiewe multisentrum studie. Die rol van embrio morfologie en die genetiese profiel van die ovum, sigoot asook die embrio en die daaropvolgende blastosist vorming sal geëvalueer word met behulp van WGA/CGH. Alle bevindings sal daarna opgesom word, gevolg deur „n sinvolle gevolgtrekking en laastens sal voorstelle gemaak word vir toekomstige navorsing op die gebied. In Afdeling B sal die metodiek van die studies verduidelik word, asook „n beskrywing gegee word van die kandidaat se rol gedurende die navorsings projekte in hierdie tesis. HOOFSTUK 2 In hierdie hoofstuk word die impak van die oösiet en die embrio op die inplanting/swangerskap-koers bespreek. Die morfologiese eienskappe van die oösiet, die kumulus-oösiet kompleks, die sona pellucida, die perivitelline spasie, sitoplasma en meiotiese spoel, die poolliggaam en die se voorkoms word breedvoerig bespreek. Die morfologiese eienskappe van die embrio, fragmentasie en die invloed daarvan op die embrio, ploïdie, en blastosistvorming word bespreek. Embriomerkers om swangerskapsuitkoms te voorspel, gebaseer op internasionale literatuur, is ook nagevors. Die pronukleêre morfologie en vroeë deling word as nie-indringende embriomerkers uitgelig om swangerskapsuitkoms te voorspel. „n Biochemiese, nie-indringende merker wat deur ontwikkelende embrios uitgedruk word, oplosbare menslike leukosiet antigeen-G (sHLA-G), word bespreek. Die waarde van blastosist oordrag en die verbeterde koers van voortgaande swangerskappe in vergelyking met verdelende embrios, is ook uitgelig, gebaseer op „n onlangse metanalise. „n Breedvoerige bespreking van sHLA-G asook “Array-CGH” en die toekoms van hierdie toetse word behandel. HOOFSTUK 3 Die doel van hierdie hoofstuk is om swangerskap en inplantingskoerse te vergelyk wanneer embrios geselekteer word op „n enkel Dag 3 (D 3) morfologie beoordeling, teenoor „n kumulatiewe GES-telling plus sHLA-G uitdrukking. Hierdie was „n prospektiewe ewekansige studie (n=214) waar pasiënte ICSI-siklusse ondergaan het. Embrios is geselekteer vir terugplasing gebaseer op óf Dag 3 morfologie telling (Groep A), óf „n kumulatiewe GES-telling plus sHLA-G uitdrukking (Groep B). Die volgende resultate is gerapporteer: kliniese swangerskappe [35/107 (33%) vs 52/107 (49%)] en voortgaande swangerskappe [20/107 (19%) vs. 52/107 (49%)] se sukses koerse is beduidend verskillend tussen Groep A en Groep B (p<0.05). Inplantingskoerse is nie beduidend verskillend tussen Groep A [52/353 (15%)] en Groep B [73/417 (18%)] (p<0.05) nie. Die aantal swangerskappe wat tot niet gegaan het tydens die eerste trimester was bykans 12 keer hoër in Groep A [25/52 (48%)]. Die slotsom was dat die miskraamsyfer beduidend laer in Groep B as in Groep A is en die swangerskap syfer betekenisvol beter was wanneer die selektering van embrios op GES plus sHLA-G gebaseer is. HOOFSTUK 4 Verskeie studies het „n assosiasie getoon tussen die teenwoordigheid van oplosbare menslike leukosiet antigeen-G (sHLA-G) in menslike embrio kultuur en swangerskaps uitkoms in vitro. „n Retrospektiewe studie is op 2040 ICSI pasiënte by 6 verskillende sentra gedoen om die effek van s-HLAG verder te bestudeer. Alle embrios is individueel gekweek om die teenwoordigheid van sHLA-G in „n kultuurmedium rondom die embrios te identifiseer. In alle sentra is „n positiewe sHLA-G uitslag met „n toename in die waarskynlikheid van veelvuldige inplantings geassosieer (OR: 1.48, 95% CI: 1.07 tot 2.05, p-waarde: 0.0170), asook „n toename in waarskynlikheid van meervoudige swangerskappe wat voortduur (OR: 1.66, 95% CI: 1.10, 2.51, p-waarde: 0.0170). Data uit die multisentriese studie beklemtoon dat sHLA-G uitdrukking „n waardevolle nie-indringende embriomerker is om by te dra tot die verbetering van swangerskapsuitkoms, asook die teoretiese potensiaal om meervoudige swangerskappe te verminder. „n Kombinasie van sHLA-G uitdrukking en verlengde embrio kultuur tot die blastosist stadium mag moontlik „n toekomstige hulpmiddel wees waardeur enkele embrios vir terugplasing geselekteer kan word. Daardeur kan die risiko van meervoudige swangerskappe beperk word sonder om die swangerskapkoerse in gevaar te stel. HOOFSTUK 5 In dié hoofstuk word die ploïdie status van die eerste en tweede poolliggaampies en Dag 3 blastomere, embrio morfologie en biochemiese (sHLA-G) eienskappe gekorrelleer met blastosist ontwikkelling en uiteindelike swangerskapsuitkoms. Alle oösiete/sigote en embrios is individueel tot die blastosist stadium gevolg. PB-I, PB-II en blastomere het “volledige kariotipering ondergaan deur gebruik te maak van die toets “comparative genome hybridization (CGH)”. Elke embrio se kultuurmedium supernatant is versamel en ontleed vir sHLA-G uitdrukking op Dag 2. Die volgende uitslae is gerapporteer: Sewe-en-vyftig mature (MII) donor oösiete is verkry; 33/57 (57.9%) is aneuploïd, 21/57 (36.8%) is euploïd en 3/57 (5%) is onbeslis. Geen verwantskap is gevind tussen CGH status van PB-I, PB-II en die GES-telling. Geen verwantskap is gevind tussen CGH status van sHLA-G. Verder was daar geen verwantskap gevind tussen PB-I CGH uitslae en blastosist morfologie graad nie. Daar was „n beduidende korrelasie tussen PB-I CGH en blastomeer CGH uitslae. Euploïde en aneuploïde PB-I het onderskeidelik in 58% en 67% blastosiste ontwikkel. Daar is „n positiewe verwantskap tussen die ploïdie van PB-I, PB-II en die blastomere aangetoon [Kappa (>0.7)]. Dit is afgelei dat na ICSI en sekwensiële genetiese kariotipering van die oösiet/sigoot en daaropvolgende blastomere, die meerderheid oösiete bevrug is en die daaropvolgende sigote ontwikkel het tot blastosiste, ongeag hul ploïdie status. Ons afleiding is dus dat blastosist ontwikkelling nie aan ploïdie verwant is nie. HOOFSTUK 6 In hierdie hoofstuk bespreek ons waarnemings wat betref seleksie kriteria om die beste embrios te kies vir terugplasing wat uiteindelik tot „n suksesvolle swangeskap sal lei. Morfologiese, biochemiese en genetiese faktore is ondersoek. „n Onderskeiding is gemaak tussen nie-indringende (mikroskopiese en biochemiese) en indringende (embrio biopsie, aCGH) tegnieke. 'n Kombinasie van nie-indringende observasies, wat insluit pronukliere mofologie, vroee sigoot verdeling en vroeë embrio morfologie/kwalitieit op Dag-3 het beperkte inligting verskaf wat betref swangerskapkans. Verlengde embrio kweking tot die blastosist stadium (Dag-5) plus „n positiewe sHLA-G resultaat gee egter veel meer voordelige inligting aan die embrioloog met die embrio seleksie proses, voor embrio terugplasing of bevriesing. Laasgenoemde inligting sal die swangerskap syfer bevoordeel en die meervoudige swangerskap kans verlaag. Wat die indringende tegniek (aCGH) betref, word veel meer data benodig rakend die potensiele voor- en nadele wat betref swangerskap uitkoms, voordat „n sinvolle gevolgtrekking gemaak kan word. HOOFSTUK 7 „n Volledige literatuur oorsig dui daarop dat alle beskikbare riglyne om embrios te kies vir terugplasing, ingespan moet word. In die studie is daar gekyk na „n kombinasie van hierdie voorstelle. Daar is begin met die morfologie van die pronukliere stadium, gevolg deur vroeë sigoot-verdeling, asook beoordeling van embrios se morfologie/kwaliteit op Dag-3 van ontwikkeling. Daar word voorgestel dat die keuse van „n blastosist op Dag 5, gekombineerd met „n positiewe oplosbare menslike leukosiet antigeen G (shla-G) die embrioloog van hulp kan wees om die beste embrio te kies vir terugplasing of bevriesing. Hierdie nie-indringende riglyn behoort swangerskap-uitkoms te verbeter asook meervoudige swangerskappe te verminder. Indringende tegnieke soos ACGH benodig verdere in diepte navorsing en data verkryging om die waarde van hierdie toets te kan beoordeel.

Fertilization and cleavage of bovine embryos depend not only on maternal involvement, but also on the paternal contributions that involve more than just providing the haploid male genome. Therefore, the overall objective of this project was to determine the impact of morphologically abnormal spermatozoa on fertilization, subsequent embryonic development, and embryo quality at the cellular level. Four experiments used morphologically abnormal semen samples collected and cryopreserved from four Holstein bulls before (Pre) and after a scrotal insulation (PI) period of 48 h. Zygotes were cultured for 8 d when a developmental score was assigned to each embryo; subpopulations were subjected to either the TUNEL or caspase assays to determine apoptosis. In the final experiment pronuclear decondensation for presumptive zygotes was evaluated by differential interference contrast microscopy at 3 h time intervals from 6 to 18 h post in vitro insemination (hpi). Morphological evaluation of semen samples revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the PI samples in comparison with the Pre samples for Bulls I and Bull III (74 to 22.3% and 67.7 to 0.5 %, respectively) and the scrotal insulation effects persisted from the time of cleavage through blastocyst formation for Bulls I and III and corresponded with a similar decrease in blastocyst development for PI samples in experiment 1 regardless of which semen separation method was used. Likewise, the overall pronuclear decondensation rate for the PI zygotes of Bull I and III showed no increase over time and remained predominantly at PN1 stage (1.5 ± 0.17; 1.8 ± 0.22, respectively). In contrast, the development for Bull II and Bull IV were unaffected. The embryo quality assessment revealed that the caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the PI embryo groups compared to those of Bull II (98 ± 115) and Bull IV (90 ± 111). In conclusion, the tested separation methods used seemed inadequate in their ability to provide potentially competent sperm for IVF. The decrease in embryonic development appears to be multifaceted and related to the changes in head shape morphology and we suggest the failure in normal pronuclear formation is associated with an absence of normal decondensation of the penetrating spermatozoon. The inability to consistently measure apoptosis in early stage embryos complicates the assessment of differences in embryo quality. These observations support the hypothesis of uncompensable seminal traits in IVF with abnormal spermatozoa and provide compelling evidence that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus is manifested during the early stages of fertilization.
Ph. D.

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

Introdução. A fertilização in vitro (FIV) é uma opção de tratamento para pacientes inférteis com endometriose (EDT), e a escolha dos embriões a serem transferidos é um passo fundamental para o sucesso do processo. A endometriose é capaz de interferir em todas as etapas do processo de FIV, a qual pode estar associada à foliculogênese anormal, a defeitos de implantação e à qualidade oocitária inferior, bem como a alterações na receptividade endometrial. Em estágios mais severos da doença, pode-se observar um importante substrato anatômico associado. O Escore de Graduação Embrionário (GES) é uma classificação baseada numa combinação da morfologia pronuclear, clivagem precoce e morfologia no terceiro dia após a fertilização que permite avaliar quais embriões terão maiores chances de implantação/gestação. Ainda não há, na literatura, relatos de comparação do GES com pacientes portadoras de endometriose. Objetivo. Determinar o escore de graduação embrionário médio entre as pacientes inférteis com endometriose submetidas à fertilização in vitro e comparar com pacientes inférteis sem endometriose. Métodos. Foram comparados, através de uma coorte prospectiva, 706 embriões (162 pacientes), sendo 472 embriões pertencentes às pacientes do grupo controle sem endometriose (n=109; infertilidade tubária ou por fator masculino) e 234 embriões de pacientes com endometriose (n=53). Todas as pacientes foram submetidas à fertilização in vitro, utilizando o protocolo de indução da ovulação com antagonista do GnRH. Todos os embriões foram transferidos no dia 3 após fertilização. O escore de graduação embrionário (GES) foi realizado através de avaliação de todos os embriões por 3 vezes, nos tempos: 16 – 18 horas, 25 – 27 horas e 64 – 67 horas, sempre pelo mesmo embriologista. Para pontuação do GES, avaliaram-se: citoplasma, morfologia pronuclear, fragmentação, alinhamento nucleolar, posição do corpo polar, número de blastômeros/morfologia e simetria. Consideramos escore médio a soma dos escores embrionários dividida pelo número de embriões obtidos; e o escore embrionário médio transferido como a soma dos GES dividido pelo número de embriões transferidos. Foi considerado estatisticamente significativo quando P < 0,05 utilizando-se os testes t Student e qui-quadrado. Resultados. Os grupos foram comparados quanto à idade, índice de massa corporal, perfil obstétrico (infertilidade primária, secundária, paridade, abortamentos) e perfil hormonal, não sendo encontrada diferença estatisticamente significativa. Apesar de o número de embriões transferidos ser maior em pacientes com endometriose quando comparadas ao grupo controle (2,38 ± 0,66 versus 2,15 ± 0,54, respectivamente; P=0,001), não se observou diferença no escore de graduação embrionário médio entre os grupos de estudo e controle (71 ± 19,8 versus 71,9 ± 23,5, respectivamente; P=0,881). Quanto às taxas de fertilização, houve semelhança entre os grupos, correspondendo a 61% nas pacientes com endometriose e 59% naquelas do grupo controle (P=0,511). Também não houve diferença estatisticamente significativa quando os grupos foram comparados quanto às taxas de implantação (21% versus 22%, respectivamente; P=0,989) e gestação (26,4% versus 28,4%, respectivamente; P=0,989). Para estes parâmetros de avaliação, os diferentes graus de severidade da endometriose não interferiram significativamente nos resultados. Conclusão. A presença de endometriose não impactou significativamente na qualidade embrionária. Da mesma forma, não foram observadas diferenças nos desfechos reprodutivos avaliados entre pacientes inférteis com e sem endometriose.
Objective. To determine embryo quality (Mean Graduated Embryo Score, MGES) in infertile patients with endometriosis submitted to in vitro fertilization (IVF-ET). Design. Prospective cohort. Setting. Private Human Reproduction Center. Patients. We compared 706 embryos (162 patients) divided in two groups: 472 embryos derived from patients without endometriosis (n=109, infertile patients with tubal or male infertility) and 234 embryos from patients in study group (n=53, infertile patients with endometriosis). Interventions. All patients were submitted to IVF using an estradiol-antagonist-recombinant FSH protocol for ovarian stimulation. MGES was performed evaluating all embryos three times: 16 – 18 hours, 25 – 27 hours and 64 – 67 hours, by the same embryologist. Embryo evaluation was performed according to the following parameters: fragmentation, nucleolar alignment, polar body apposition, blastomere number/morphology and symmetry. Main Outcome Measures. MGES scores, fertilization, implantation and pregnancy rates. Results. Groups were compared for age, body mass index (BMI), past ob/gyn characteristics (primary or secondary infertility, parity, abortions) and hormonal profile. Although the number of embryos transferred was greater in patients with endometriosis than in the control group (2.38 ± 0.66 versus 2.15 ± 0.54, respectively; P = 0.001), the MGES was similar in both groups (71 ± 19.8 versus 71.9 ± 23.5, respectively; P = 0.881). Likewise, fertilization rate was similar in groups, 61% in patients with endometriosis and 59% in control group (P=0,511). Moreover, no significant differences were found in implantation rates (21% versus 22%, respectively [P=0,989]) and in pregnancy rates (26,4% versus 28,4%, respectively [P=0,989]). The severity of endometriosis did not influence the results. Conclusions. Embryo quality measured by MGES was not influenced by endometriosis. Likewise, the reproductive outcomes evaluated were similar between infertile patients with and without endometriosis.

Statistical modelling of data from the embryo transfer process of In-Vitro Fertilization (IVF) treatments is motivated by the need to perform statistical inference for potential factors and to develop predictive models for these treatments. The biggest issue arising when modelling these treatments is that a number of embryos are transferred but unless all of the embryos get implanted or fail to implant then it is not possible to identify which of the embryos implanted. Little work has been done to address this partial observability of the outcome as it arises in this context. We adopt an Embryo-Uterus (EU) framework where a patient response has distinct uterine and embryo components. This framework is used to construct statistical models, expand them to allow for clustering effects and develop a package that will enable the fitting and prediction of these models in STATA. The capabilities of this package are demonstrated in two real datasets, aimed in investigating the effect of a new embryo prognostic variable and the effect of patient clustering in these treatments. In a simulation study EU models are shown to be capable of identifying a patient covariate either as a predictor of uterine receptivity or embryo viability. However a simulation case study shows that a considerable amount of information about the embryo covariate is lost due to the partial observability of the outcome. Further simulation work evaluating the performance of a number of proposed alternatives to the EU model shows that these alternatives are either biased or conservative. The partially observed cycles are finally considered as a missing data problem and two novel modelling approaches are developed which are able to handle the structure of these treatments. These novel models, based on multiple imputation and probability weighting, are compared to the EU model using simulation in terms of predictive accuracy and are found to have similar predictive accuracy to the EU model.

Os objectivos desta tese foram implementar a técnica de produção in vitro de embriões de suíno e investigar os efeitos da modulação lipídica durante a maturação in vitro (MIV) usando o isómero trans-10,cis-12 do ácido linoleico conjugado (t10,c12 CLA) e a forscolina. A modulação lipídica durante a MIV induziu alterações na morfologia do oócito e perfil em ácidos gordos (AG)/ plasmalogénios dos complexos cumulus-oócito. Após 44 h de suplementação com t10,c12 CLA ocorreu aclaramento citoplásmico mas a taxa de blastocistos diminuiu. A suplementação em períodos longos com forscolina poderá reduzir o crescimento do oócito, a progressão nuclear e a capacidade fertilizante, ou apenas interferir na morfologia e composição das gotas lipídicas (2 h de suplementação). Os dois suplementos em simultâneo melhoraram a taxa de maturação. Apesar das alterações na composição em AG/ plasmalogénios dos oócitos tratados, não se detetaram efeitos sobre a crio-resistência dos blastocistos; ABSTRACT: The purpose of this thesis was to implement the in vitro technique for porcine embryo production and to investigate the effects of lipid content modulation during oocyte in vitro maturation (IVM) using trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) and forskolin. Alterations in oocyte morphology and fatty acids (FA)/ plasmalogens profile were induced by chemical modulation, during IVM. After 44 h of t10,c12 CLA supplementation, a lighter cytoplasmic colour tone was achieved but the blastocyst rate was reduced. Long supplementation with forskolin reduced oocyte growth, nuclear progression, fertilizing ability and interfered with lipid droplet morphology. These disturbances disappeared by reducing supplementation with forskolin to only 2 h. When added simultaneously both supplements improved oocyte maturation rate. Despite modifications in FA/ plasmalogens in treated oocytes, no effects on blastocyst cryo-resistance could be detected.

High concentrations of intracellular glutathione enhance the in vitro production of porcine embryos. Six experiments were conducted to study the effects of varying concentrations of different supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and in vitro culture (IVC), and evaluate subsequent embryo viability. In Exp. 1, 2, 3, and 4, porcine oocytes were matured in either 3.3 mM cysteine, 150 μM cysteamine, 3.3 mM cysteine and 150 μM cystemaine; 1.0 mM glycine, 2.5 mM glycine, 5.0 mM glycine; 1.0 mM L-glutamate, 2.5 mM L-glutamate, 5.0 mM L-glutamate; and 3.3 mM L-α-aminobutyrate, 25 μM β-mercaptoethanol, 3.3 mM cysteine and 25 μM β-mercaptoethanol, or 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol. After IVM (44 h), concentrations of intracellular glutathione (GSH) were determined using a colorimetric assay based on absorbency. The supplements that elicited significantly (P < 0.05) the greatest increase in GSH concentrations were 3.3 mM cysteine, 1.0 mM L-glutamate, 3.3 mM L-α-aminobutyrate, and 3.3 mM L-α-aminobutyrate with 25 25 μM β-mercaptoethanol. In Exp. 5, oocytes matured with 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol had a significantly less (P < 0.05) occurrence of polyspermy and greater occurrence of MPN formation during IVF compared to the other treatment groups and a significantly greater percentage (P < 0.05) of embryos reaching the 2 cell developmental stage by 48 h post-IVF and blastocyst stage of development by 144 h post-IVF compared to the other treatment groups. In Exp. 6, treatment had no effect on the time of cell death. The times at which embryo mortality was significantly the greatest (P < 0.05) were located within the middle of IVC. The approximate time of the onset of cell death occurred between 24 to 42 h post-IVF with the greatest occurrence around 36 h. These results suggest that supplementing 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol into the IVM medium 1) increases intracellular GSH concentrations by the end of IVM, 2) decreases the occurrence of polyspermy during IVF, 3) increases the MPN formation during IVF, and 3) increases embryo development parameters during IVC. Supplementation to the maturation media does not have an effect on cell death during embryo development. The onset of cell death appears to occur between 24 to 42 h post-IVF with the greatest occurrence around 36 h post-IVF. In order to increase the success of in vitro derived porcine embryos and offspring, the basic fundamentals of the system need to be fully understood.
Master of Science

La calidad de los oocitos es sinónimo de competencia oocitaria y se define como la capacidad de un oocito para reanudar la meiosis, dividirse después de la fertilización, desarrollarse hasta la etapa de blastocisto, inducir la preñez y conseguir el nacimiento de un animal sano. La selección de los oocitos más competentes es un punto clave en los programas de producción in vitro de embriones (PIVE). El test de Azul de Cresol Brillante (BCB) es un método no invasivo que separa los oocitos que han finalizado su crecimiento (BCB+: citoplasma azul) de los oocitos con la enzima G6PDH activa o oocitos en proceso de crecimiento (BCB-: citoplasma incoloro). En esta tesis, hemos llevado a cabo 3 estudios para probar la capacidad del BCB en seleccionar los oocitos más competentes para la producción in vitro de embriones utilizando corderas como donantes de oocitos. Además se estudiará la competencia oocitaria en relación con el citoplasma y factores moleculares, sus respuestas a las diferentes técnicas de fecundación y nuevos medios de maduración para mejorar la PIVE. En el Experimento 1, se analizaron diferentes concentraciones del BCB (13, 26, 39 y 52 µM) y los oocitos fueron MIV-FIV y CIV. Se evaluó: diámetro de los oocitos, actividad mitocondrial, actividad del factor promotor de la maduración (MPF), la expresión de genes relacionados con el metabolismo (ATP1A1 y COX1) y la función constitutiva de la célula (CPEB y S100A10) y el desarrollo hasta el estadio de blastocisto. Los resultados mostraron que 13 µM de BCB es una concentración adecuada que diferencia los oocitos más grandes (BCB+: 123,66 µm), competentes para desarrollar hasta blastocisto (21%) y con más células (69,71±6,19) que los BCB- (106,82 µm, 9% y 45,91±3,35, respectivamente). La actividad mitocondrial fue mayor en los BCB+ que en los BCB- (3369 y 1565 unidades arbitrarias, respectivamente) y también la actividad de MPF (1,479±0,09 y 1,184±0,05 densidad óptica, respectivamente). La expresión de genes no mostró correlación con la calidad de los oocitos. El objetivo del experimento 2 fue mejorar la producción in vitro de los oocitos BCB- mediante la adición de insulina transferrina selenio y ácido ascórbico durante 12 y 24 h de la MIV. El MPF y el contenido de ATP se midieron antes y después de la MIV. Los resultados no mostraron diferencias en la producción de blastocistos entre los oocitos BCB-. El MPF y el ATP aumentaron en todos los grupos (P <0,001) después de la MIV. En el Experimento 3 se estudiaron técnicas de fecundación como la FIV, ICSI y la activación partenogénetica (AP) en los oocitos BCB+ y BCB-. Se analizó el contenido del ATP intracelular. Los oocitos BCB+ tuvieron un desarrollo hasta blastocito significativamente mayor tras la FIV y la AP (31,7% y 20,5%, respectivamente) que los BCB- (6,7% y 8,8%, respectivamente). Sin embargo los oocitos inyectados (ICSI) no mostraron diferencias en los blastocistos producidos entre los BCB+ y BCB- (14,3% vs 11,8%, respectivamente). Los oocitos BCB+ mostraron tener mayor concentración de ATP que los BCB-. Finalmente podemos concluir que el test del BCB es una metodología fácil, rápida y adecuada para seleccionar los oocitos de corderas más competentes. Los oocitos BCB+ mostraron una mayor producción de blastocistos, actividad mitocondrial y del MPF y mayor contenido de ATP que los BCB-. El test de BCB es un método útil para ser incorporado en los protocolos de PIVE cuando se trabaja con un gran y heterogéneo número de oocitos. Sin embargo este test es menos interesante cuando se trabaja con un pequeño número de oocitos, como en laparoscópica o ICSI.
The oocyte quality is used as synonymous of oocyte competence defined as the ability of an oocyte to resume meiosis, cleave following fertilization, develop to the blastocyst stage, induce a pregnancy and bring the offspring to term in good health. The selection of more competent oocytes is an important point in in vitro embryo production programs. The Brilliant Cresyl Blue (BCB) test has been successfully used as a non invasive methodology to classify oocytes according to their cytoplasm coloration as grown (BCB+: blue cytoplasm) or growing (BCB-: colorless cytoplasm) oocytes. To our knowledge there are no previous reports using this test to select competent oocytes in sheep. We have carried out 3 studies to test the ability of the BCB to select the most competent prepubertal sheep oocytes for in vitro blastocyst embryo production. Furthermore, we pretend to improve the knowledge about oocyte competence related to their cytoplasmic and molecular performances, their responses to different techniques of fertilization and to test new maturation media to improve the blastocyst production. In Experiment 1, different concentrations of the BCB test (13, 26, 39 and 52 µM) were analyzed. After BCB culture all of oocytes were IVM-IVF and IVC. The parameters assessed in this study were: oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity, mRNA relative expression (RE) of genes related to metabolism (ATP1A1 and COX1) and constitutive function of the cell (CPEB and S100A10) and the blastocyst development. The results showed that 13 µM BCB during 60 min could be a suitable concentration to differentiate largest (BCB+, 123.66 µm) and most competent oocytes to develop to the blastocyst stage (21%) and with a higher number of cells (69.71±6.19 S.E.M.) compared with non-stained BCB- oocytes (106.82 µm, 9% and 45.91±3.35 S.E.M., respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565 Arbitrary Units, respectively) and the MPF activity, assessed by CDC2 kinase activity assay, showed significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479±0.09 and 1.184±0.05 optical density, respectively). The mRNA RE of the different genes analyzed in this work did not show a correlation with the oocyte quality. In Experiment 2, the objective was to improve blastocyst production of small (BCB-) oocytes by addition of Insulin Transferrin Selenium and Ascorbic Acid during 12 and 24 h of IVM. MPF and ATP content were measure before and after IVM. Results showed no differences in blastocyst production between BCB oocytes. The MPF and ATP increased in all groups (P<0.001) after the IVM. In Experiment 3, the aim was to test the blastocyst production after IVF, ICSI and PA of BCB+ and BCB- oocytes. ATP content was analyzed. BCB+ oocytes developed significantly higher up to the blastocyst stage after IVF and PA (31.7% and 20.5%, respectively) than BCB- (6.7% and 8.8%, respectively) oocytes. However, ICSI treated oocytes did not show these differences between BCB+ and BCB- oocytes (14.3% vs 11.8%, respectively). BCB+ oocytes had significantly more ATP content than BCB- oocytes. Finally we can conclude that the BCB test is an easy, fast and suitable methodology to select the more competent sheep oocytes. Good quality oocytes (BCB+) showed higher blastocyst production, mitochondrial and MPF activity and ATP content than BCB- oocytes. The BCB test is a useful method to be incorporated in the embryo production protocol where a large and heterogeneous number of oocytes are use to be in vitro fertilized. However, BCB test is less interesting when working with a small number of oocytes such as in Laparoscopic Ovum Pick Up (LOPU) or ICSI.

Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Male fertility has for many years been defined in vitro as the ability of sperm to fertilize oocytes and to obtain early cleavage-stage embryos. Spermatozoa comprise of an extraordinary high percentage of polyunsaturated fatty acids in their plasma membrane. Due to an extremely low content of cytoplasm, sperm cells have a particularly low potential to scavenge reactive oxygen species (ROS), and are therefore highly sensitive to oxidative processes, which lead to sperm nucleus DNA damage/fragmentation. Normally, DNA fragmentation occurs in every ejaculate and can be induced by an excessive ROS production of active leukocytes or the spermatozoa themselves. Under distressed conditions, DNA fragmentation may also occur in the testis as a result of oxidative processes in the apoptotic cascade. These DNA fragmentations can be regarded as late signs of programmed cell death (apoptosis). Clinically, DNA fragmentation in spermatozoa results in significantly decreased implantation and pregnancy rates especially in patients with oligo- and/or teratozoospermia. The p-pattern normal sperm morphology has been shown to give poorer fertilization rates in vitro than the g- and n-patterns. In this study there is reported on the significant correlation found between the p-pattern normal sperm morphology and sperm DNA fragmentation as measured with the terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling (TUNEL) assay. This finding further explains the lower fertility potential of patients presenting with p-pattern normal sperm morphology. In addition, this study explores the intricate relations between ROS in the semen, DNA fragmentation of the spermatozoa, as measured with the TUNEL assay and the sperm chromatin structure assay (SCSA ), spermatozoa apoptotic status and sperm parameters as measured with a standard semen analysis. Positive correlations were found between ROS and the apoptotic status of the sperm, as well as between sperm with non-fragmented DNA and sperm concentration and percentage motility. The results emphasize the importance of sperm selection especially when the treatment of choice is intracytoplasmic sperm injection (ICSI). An early sign of programmed cell death, also known as apoptosis, is the externalization of phosphatidylserine (PS) from the inner membrane leaflet to the outer leaflet. PS shows a high affinity to Annexin V. Apoptotic spermatozoa are able to fertilize oocytes, but embryo senescence may occur at the time when the paternal genes are activated. In this study there is reported on a novel method whereby spermatozoa can be separated on the basis of their apoptotic status through flow cytometry. Results showed that the normal sperm morphology, according to strict criteria, of the resultant nonapoptotic sperm fraction is significantly higher than that of the apoptotic counterpart. With refinement of this technique, it will be possible in future to use these separated non-apoptotic sperm cells during ICSI for fertilization. From the above it is apparent that the spermatozoon has to play a vital role in the development of the embryo from fertilization to implantation and pregnancy. It is, however, important to note that besides the gametes, there are other critical factors which contribute to a successful in vitro fertilization (IVF) cycle, among these are the in vitro culture conditions. In this regard, this study compared two sequential embryo culture systems. It was found that the more complex medium resulted in better day three embryo quality and a better blastocyst formation rate and pregnancy rate. These findings highlight the importance of a holistic perspective towards the complexity of the factors involved in affecting embryo quality and pregnancy outcome.
AFRIKAANSE OPSOMMING: Manlike fertiliteit is vir baie jare gedefinieer as die in vitro vermoë van ‘n spermsel om ‘n eiersel te bevrug om sodoende embrios te verkry. Die spermsel se plasmamembraan bestaan uit ‘n hoë persentasie poli-onversadigde vetsure. As gevolg van die klein hoeveelhede sitoplasma van die spermsel het dit ‘n beperkte weerstand teen reaktiewe suurstof spesies (ROS) en is gevolglik baie sensitief vir oksidasie. Oksidasie lei tot DNS skade/fragmentasie. DNS fragmentasie kom in spermselle van alle ejakulate voor en is gewoonlik die gevolg van ROS produksie deur die leukosiete in die semen of vanaf die spermselle self. Onder sekere omstandighede kan DNS fragmentasie ook voorkom in die testis waar dit deel vorm van apoptose. Hierdie tipe DNS skade word gesien as laat tekens van geprogrammeerde seldood (apoptose). In oligo- en/of teratozoospermiese mans lei DNS fragmentasie tot verlaagde implantasie- en swangerskapssyfers. Die p-patroon normale sperm morfologie groep gee laer in vitro bevrugting en swangerskapsyfers as die g- en n-patrone. In hierdie studie doen ons verslag oor die statisties betekenisvolle korrelasie wat gevind is tussen die p-patroon normale sperm morfologie en DNS fragmentasie soos gemeet met die ‘terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling’ of te wel TUNEL toets. Hierdie bevinding is ‘n verdere verklaring vir die laer fertiliteits potensiaal van pasiënte wat voordoen met p-patroon sperm morfologie. ‘n Verdere doel van die studie was om die moontlike verband tussen ROS in die semen, spermatozoa DNS fragmentasie, apoptotiese status van die sperms en die motiliteits parameters van die spermatozoa te bepaal. ‘n Positiewe korrelasie is gevind tussen ROS en sperm apoptotiese status. Sperms met ongeframenteerde DNS is ook positief gekorreleer met sperm konsentrasie en motiliteit. Die resultate beklemtoon die belangrikheid van spermseleksie veral in pasiënte waar die keuse van behandeling intrasitoplasmiese sperm inspuiting (ICSI) is. ‘n Vroeë teken van apoptose is die eksternalisering van ‘phosphatidylserine’ (PS) vanaf die interne oppervlakte van die plasmamembraan na die eksterne oppervlak. PS het ‘n hoë affiniteit vir Annexin V. Apoptotiese sperms het die vermoë om ‘n oösiet te bevrug, maar kan lei tot die staking van embrio deling wanneer die vaderlike gene ‘n rol begin speel in embrio ontwikkeling. In hierdie studie het ons ‘n nuwe metode ontwikkel waarvolgens die spermatozoa in die ejakulaat op grond van hul apoptotiese status geskei kan word in apoptotiese en nie-apoptotiese fraksies. Die normale sperm morfologie van die nie-apoptotiese fraksie is betekenisvol beter as dié van die apoptotiese fraksie. Verdere verfyning van die tegniek kan daartoe lei dat dit in die toekoms toegepas kan word om vir nie-apoptotiese sperms te selekteer veral voor die uitvoering van ICSI. Uit die bogenoemde is dit duidelik dat die spermsel ‘n baie belangrike rol in die ontwikkeling van ‘n embrio, vanaf bevrugting tot implantasie en swangerskap, speel. Dit is egter ook belangrik om in gedagte te hou dat daar ander bydraende faktore tot ‘n suksesvolle in vitro swangerskap is, soos laboratorium toestande en embrio kultuursisteem. Om hierdie rede is daar ook twee kultuurmedia in hierdie studie vergelyk. Daar is bevind dat die meer komplekse medium beter kwaliteit embrios op dag drie lewer, asook meer blastosiste en ‘n hoër swangerskapsyfer. Dit is dus duidelik dat dit uiters belangrik is om ‘n holistiese perspektief te hê op die komplekse faktore wat ‘n invloed mag hê op bevrugting, embrio kwaliteit asook die swangerskapsyfer.

Master of Science
Department of Animal Sciences and Industry
Karol E. Fike
This thesis involves two separate studies that evaluate the effects of different beef cattle management practices on reproduction. The objective of the first study was to determine if feedlot heifers administered melengestrol acetate (MGA) and growth promoting implants could serve as viable oocyte donors for in vitro embryo production. Ovaries from heifers administered MGA and growth promotants (MGA-Implant) and ovaries from heifers not administered either substance (Control) were collected from heifers post-slaughter. Oocytes were harvested and in vitro maturation, in vitro fertilization (IVF), and in vitro culture were completed. Treatment and time interacted to affect the number of oocytes aspirated per ovary (P = 0.07) and the number of zygotes per ovary (P = 0.07). Fertilization (P = 0.90) and cleavage rates (P = 0.80) did not differ between treatments. Blastocyst rates (P = 0.30) and the number of embryos per ovary (P = 0.50) did not differ between treatments. We concluded that beef feedlot heifers fed MGA and implanted with growth promotants seem to be a viable source of oocytes for in vitro embryo production. In the second study, we hypothesized that continuous fenceline exposure of prepubertal beef bulls to cycling beef females would hasten the onset of puberty as well as increase the percentage of bulls passing their initial breeding soundness examination (BSE). Bulls were either exposed to estrous females (exposed) or were not exposed (control). Monthly scrotal circumference (SC) measurements, blood samples, semen evaluations, and bull behavior assessments were conducted. Age at puberty (P = 0.40), SC at puberty (P = 0.50), and weight at puberty (P = 0.30) did not differ between treatments. A similar (P = 0.50) percentage of bulls passed their initial BSE at 363 ± 21.5 d of age (exposed: 87.8%; control: 74.2%). Treatment, month, and stage of the estrous cycle of cows interacted to affect the number of mount attempts (P = 0.05) and the number of flehmen responses (P < 0.001). In conclusion, bulls given continuous fenceline exposure to cycling beef females were neither younger at puberty nor did a greater percentage pass their initial BSE.

Alterações morfogênicas do aparelho cardiorespiratório de bovinos provenientes de fecundação in vitro e transferência nuclear são um dos principais fatores responsáveis pela alta incidência de mortalidade embrionária, fetal e pós-natal. Utilizamos técnicas empregadas em microscopia de luz para estudar o desenvolvimento do coração e pulmão nestes animais. Verificamos que em embriões provenientes de fecundação in vitro, aos 28 dias de gestação, aparece o tubo laringotraqueal e sua septação através da prega traqueoesofágica. Neste mesmo período os embriões apresentaram cavidade pericárdica, átrio dividido em direito e esquerdo, cone cardíaco, seio venoso, camada de miocárdio e epicárdio. O brônquio traqueal foi observado em embriões com idade gestacional de 36 dias a partir de um brotamento na porção lateral direita da traquéia, cranial a sua bifurcação. Aos 44 dias de gestação os brotos pulmonares dos embriões apresentaram brônquios principais originando brotamentos de brônquios segmentares. O mesênquima de sustentação em diferenciação continha vasos sangüíneos dispersos, diferentemente de embriões provenientes de TN, que com 68 dias de gestação apresentou pulmão em fase pseudoglandular contendo brotos de bronquíolos e poucos vasos sangüíneos nos cortes obtidos e analisados. Com 70 dias, o coração do feto apresentava ventrículo significativamente grande, pequeno átrio e pulmão não desenvolvido. A partir dos nossos resultados, concluímos que alterações genéticas, incompletas informações e comunicações celulares e modificação no metabolismo celular são os prováveis responsáveis pelas anomalias presentes nas técnicas de manipulação de embrião, causando um desenvolvimento mais lento e falho quando comparados com embriões in vivo, explicando o alto índice de perda gestacional
Morphogenetic changes of cardio-respiratory system of bovine from in vitro fertilization and nuclear transfer is a major factor responsible for high incidence of both periods embryonic, fetal and postnatal mortality. We used techniques of light microscopy to study the development of heart and lung in these animals. We found that in embryos derived from in vitro fertilization, at 28 days of gestation, appears the laryngotracheal tube and its fold through tracheoesophageal septation. In the same period the embryos showed pericardial cavity, atrium divided into left and right, cardiac cone, venous sinus, layer of myocardium and epicardium. In embryos with 36 days of gestational age was observed the tracheal bronchus from a bud in the right lateral portion of the trachea, cranial to its bifurcation. At 44 days of gestation, the lung buds of the embryos showed main bronchi giving rise to budding of segmental bronchi. The sustentation mesenchyme in differentiation contained scattered blood vessels, unlike embryos from TN, which with 68 days of gestation showed lung in pseudoglandular stage, containing bronchioles buds and few blood vessels in histological sections obtained and analyzed. With 70 days, the fetal heart ventricle had significantly large, small atrium and lungs not developed. From our results we conclude that genetic alterations, incomplete information in cellular communications and change in cellular metabolism are likely responsible for the anomalies in the techniques of embryo manipulation, causing a slower and flawed development when compared to embryos in vivo, explaining the high rate of pregnancy loss

Šiame magistro baigiamajame darbe lyginamuoju metodu yra analizuojamas preimplantacinės diagnostikos reguliavimas įvairiose pasaulio valstybėse, tuo pačiu atskleidžiant pagrindines iš to kylančias problemas. Tyrimas atliktas siekiant palyginti skirtingų valstybių teisės aktus ir patirtį šioje biomedicinos srityje. Atlikta analizė rodo, kad preimplantacinė diagnostika vis dar yra pakankamai nauja ir atsargiai vertinama procedūra, sukelianti daug etinių ir teisinių diskusijų, o teisinis reguliavimas priklauso nuo valstybės teisinių, religinių, kultūrinių, socialinių tradicijų.
These master theses analyze the regulation of preimplantation genetic diagnosis in comparative aspect in different countries, simultaneously revealing main problems arising. The aim of this research is to compare legal acts and experience of different countries in this biomedical field. The analysis shows, that preimplantation genetic diagnosis is still innovative and well considered procedure, which give a rise to a lot of ethical and legal discussions, and legal regulation of this procedure depends on legal, religious, cultural and social traditions of the country.

Background: Since the initiation of assisted reproduction techniques, several studies has been performed to improve treatment results by development of culture conditions like embryo oil and culture media used. In this study, two embryonic oils from different companies, Nidoil and Ovoil were examined.Method: In this study, 47 human embryos were used. All embryos were donated for research purposes by couples who had been treated at the clinic in Uppsala University Hospital. The embryos were divided into two groups, one group was cultured with Ovoil and the other with Nidoil.Results: There was no difference between the two oils, the embryo quality was the same in both groups.CONCLUSION: The result was expected because both oils had the same composition and purity.

Thesis (M.A.)--Indiana University, 2008.
Department of Philosophy, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Peggy Zeglin Brand, Jason T. Eberl, Michael B. Burke. Includes vitae. Includes bibliographical references (leaves 79-84).

O imprinting genômico é um processo epigenético essencial para o desenvolvimento normal de mamíferos com placenta e refere-se à expressão gênica alelo-específica, de acordo com a origem parental. A expressão dos genes marcados por imprinting é controlada por regiões diferencialmente metiladas (DMRs), situadas em regiões controladoras de imprinting (ICRs). O cromossomo 29 de Bos taurus possui dois domínios cromossômicos semelhantes à região 11p15.5 de humanos, que são denominados KvDMR1 (na ICR2) e H19DMR (na ICR1). Essas ICRs controlam um cluster de genes importantes para o crescimento e desenvolvimento, sendo a KvDMR1 metilada no alelo materno e a e H19DMR metilada no alelo paterno. No presente trabalho, foi verificado o padrão de metilação da KvDMR1 e da H19DMR em oócitos não maturados (Vg) e maturados in vitro (MII) e nos blastocistos inicial (Bi) e expandido (Bx) bovinos e em placentas bovinas e humanas de primeiro trimestre. Foram coletados oócitos e embriões pré-implantação no estágio de blastocisto produzidos pela técnica de Fertilização in vitro. Também foram coletados o tecido placentário e de um feto bovino de 49 dias e de uma placenta humana, com idade gestacional de 12 semanas. O DNA genômico foi extraído e modificado com bissulfito de sódio. O padrão de metilação das regiões KvDMR1 e H19DMR foi verificado por meio de clonagem e seqüenciamento do DNA modificado com bissulfito de sódio. Para as análises de expressão gênica nos oócitos e blastocistos, foi realizada a extração do RNA e em seguida o cDNA foi produzido para a quantificação relativa da expressão gênica por meio da técnica de PCR em tempo real. Os resultados de metilação para a amostra controle de espermatozóide apresentaram um perfil hipometilado para a KvDMR1 e hipermetilado para a H19DMR. Os oócitos Vg e MII mostraram um perfil hipermetilado para a KvDMR1 e nos Bi e Bx foi observado um perfil hipermetilado e hipometilado, respectivamente. Para a H19DMR, foi observado um perfil hipermetilado para as amostras Vg e MII, sendo que para os Bi foi observado um perfil hipometilado e, para os Bx, um perfil monoalélico de expressão. A expressão dos genes LIT1 e IGF2 foi relativamente baixa nas amostras analisadas, sendo que o gene LIT1 foi expresso nos mesmos níveis para todas as amostras e o IGF2 não foi expresso nos Bi e Bx. Os oócitos MII apresentaram altos níveis de expressão do IGF2 quando comparados com os oócitos Vg. Nas placentas precoces de bovinos, a porcentagem de metilação para a KvDMR1 variou entre os cotilédones de 39,6%. e 88,9%. A porcentagem de metilação para a H19DMR nos cotilédones variou entre 35,0% e 57,0% , sendo que apenas uma amostra apresentava-se completamente demetilada para esta ICR. Nas análises das vilosidades humanas, foi observado um perfil hipermetilado em todas as amostras analisadas, em que as porcentagens de metilação para a KvDMR1 e H19DMR variaram entre 84,4% e 97,9%. Os resultados mostram um perfil alterado de metilação nos oóctios MII para as duas regiões analisadas, e uma alteração nas amostras de oócitos Vg para a H19DMR. Para os blastocistos, o esperado seria um perfil monoalélico para as duas regiões, no entanto, esse resultado só foi encontrado para os Bx na H19DMR. Em bovinos, as DMRs apresentaram um funcionamento antagônico, enquanto a KvDMR1 tende a uma hipermetilação a H19DMR tende a uma hipometilação. O resultado das análises comparativas das placentas bovina e humana não sugerem uma relação do padrão de metilação dessas regiões entre essas duas espécies, no entanto, servem de base para o conhecimento do imprinting na placenta. Os estudos nos oócitos e blastocistos realizados representam um passo inicial na investigação da influencia das tecnologias de reprodução assistida no desenvolvimento embrionário, sendo o primeiro relato do funcionamento dessas DMRs nas amostras de oócitos e embriões pré-implantação bovinos.
Genomic imprinting is an epigenetic process that plays an essential role in the development of placental mammals with a parent-of-origin-specific manner of gene expression, in which only one allele is expressed. The imprinted gene expression is controlled by differentially methylated regions (DMRs), located in imprinting control regions (ICRs). In Bos Taurus, chromosome 29 presents two imprinted domains similar to human 11p15.5 region which are named KDMR1 (in the ICR2) and H19DMR (in the ICR1). Several genes that play an essential role in growth and development are under the control of the ICRs, in which the KvDMR1 is methylated on the maternal allele and the H19DMR is methylated on the paternal allele. In this study, the DNA methylation status of the KvDMR1 and H19DMR was verified in bovine non-matured germinative vesicle (GV) in vitro matured (MII) oocytes, as well in early (EA) and expanded (EX) blastocysts, and in bovine and human early placenta. The oocytes and blastocysts were collected after in vitro fertilization (IVF) techniques. Tissues from bovine placenta and fetus with 49 days of gestational age and human placenta with 12 weeks of gestational age were also collected. The DNA was extracted and modified by sodium bisulfite. The methylation pattern of KvDMR1 e H19DMR was verified by cloning and bisulfite sequencing. RNA extraction and cDNA synthesis for the relative quantification of gene expression by real time PCR were performed for oocytes and blastocysts. The methylation profile for the control sample of sperm was hypomethylated for KvDMR1 and hypermethylated for H19DMR. The GV and MII oocytes showed a hypermethylated pattern for KvDMR1 and in the EA and EX was hypermethylated and hypomethylated, respectively. The H19DMR displayed a hypermethylated pattern for GV and MII oocytes. For EA was observed a hypomethylated profile and EX presented a monoallelic expression. The LIT1 and IGF2 gene expression were low for all samples, however the LIT1 had the same level of expression in all samples while the IGF2 was not expressed in EA and EX. The MII oocytes showed high levels of IGF2 gene expression when compared with GV oocytes. The methylation levels for KvDMR1 in bovine early placenta varied between the cotyledons (39,6% and 88,9%). The percentage of methylation for H19DMR in cotyledons varied between 57.0% to 35.0%. Only one sample was not methylated for this ICR1. In human villous, a hypermethylated profile was observed for all samples, and the percentage of methylation for KvDMR1 and H19DMR varied between 84.4% e 97.9%. The results show an altered methylation profile in MII oocytes for two analysed regions and an alteration of H19DMR in GV oocytes. The expected for blastocysts was a monoallelic profile for KvDMR1 and H19DMR, however these results were observed only in EX for H19DMR. In bovine, the methylation levels of KvDMR1 and H19DMR seems to work antagonistically. While the KvDMR1 tended to hypermethylation, the ICR1 tended to a hypomethylation. The comparative analysis of bovine and human early placentas does not suggest a relationship between the methylation patterns of these regions in these two species. However, these studies provide the basis for the understanding of imprinting in the placenta. The study in oocytes and blastocysts represent an initial investigation in understanding how the assisted reproductive technologies affect the embryo growth and development, and this is the first report on the methylation patterns of KvDMR1 and H19DMR in bovine oocytes and blastocysts.