Academic literature on the topic 'In vitro fertilization-embryo transfer'

This essay hopes to convey the problematic framework of outcomes for assisted reproductive technologies (ARTs) as they are published today, reframe the logic to focus on human embryo mortality, and quantify a population of human beings generally not accounted for in publicly available in-vitro fertilization data. This analysis, conducted using the CDC’s 2015 National Summary data in conjunction with a retrospective study conducted by one of the nation’s largest ART delivery systems, is one of the first attempts to estimate pre-transfer embryo mortality in IVF. This analysis focuses on fresh non-donor cycles only, that is, rounds of IVF treatment that include immediate transfer of at least one embryo and in which the embryos transferred were engendered using the recipient’s own eggs.

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.

Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional potential from cells used therapeutically. One of the reasons for this gap is because the fluidic environment of mammalian cells in vivo is microscale and dynamic whereas typical in vitro cultures are macroscopic and static. This presentation will give an overview of efforts in our laboratory to develop programmable microfluidic systems that enable spatio-temporal control of both the chemical and fluid mechanical environment of cells. The technologies and methods close the physiology gap to provide biological information otherwise unobtainable and to enhance cellular performance in therapeutic applications. Specific biomedical topics that will be discussed include subcellular signalling in normal and cancer cells, in vitro fertilization on a chip, studies of the effect of physiological and pathological fluid mechanical stresses on endothelial and epithelial cells, and microfluidic stem cell engineering. In the nanoscale regime, tunable nanochannels that can manipulate single DNA molecules will be discussed.

Background: Cardiovascular diseases (CVDs) are considered the major cause of death worldwide. Therapeutic delivery to the cardiovascular system may play an important role in the successful treatment of a variety of CVDs, including atherosclerosis, ischemic-reperfusion injury, and microvascular diseases. Despite their clinical benefits, current therapeutic drugs are hindered by their short half-life and systemic side effects. This limitation could be overcome using controlled drug release with the potential for targeted drug delivery using a nanomedicine approach. In the current study, we have assessed the use of a highly porous nano-sized preparation of iron-based Metal-organic Framework (MOF) commonly referred to as MIL-89 as potential drug carriers in the cardiovascular system. Aims: To assess the effect of MOFs on the viability and cytotoxicity of human vascular cells and the cellular uptake in vitro, and the organ-system toxicity of MOF in vivo using the Zebrafish model. Methods: Human pulmonary endothelial cells (HPAECs) and pulmonary smooth muscle cells (HPASMCs) were treated with variable concentrations of MOFs. The viability, cytotoxicity and anti-inflammatory effects were measured using AlamarBlue, LDH assay and ELISA. The cellular uptake of MOFs were assessed using light, confocal, and transmission electron microscopes and EDS analysis. Moreover, Zebrafish embryos were cultured and treated with MOFs-nanoparticles at 0 hours post fertilization (hpf) followed by different organ-specific assays at 24, 48, and 72 hpf. Results: Although MOFs affect the viability at high concentrations, it does not cause any significant cytotoxicity on HPAECs and HPASMCs. Interestingly, MOFs were shown to have an anti-inflammatory effect. Microscopic images showed an increased (concentration-dependent) cellular uptake of MOFs and transfer to daughter cells in both cell types. Moreover, the in vivo study showed that high concentrations of MOFs delay zebrafish embryos hatching and cause heart deformation, which is currently investigated using cardiotoxicity markers. Conclusion: MOFs is a promising nanoparticle prototypes for drug delivery in the cardiovascular system with high cellular uptake and anti-inflammatory effects. Further investigations of MOFs, including diseased models and drug- loaded formulation is required.

The overall objective of the current proposal is to develop procedures to improve the pregnancy rate achieved following transfer of fresh or cryopreserved embryos produced in the laboratory into heat-stress recipients. The overall hypothesis is that pregnancy rate in heat-stressed lactating cows can be improved by use of embryo transfer and that additional gains in pregnancy rate can be achieved through development of procedures to cryopreserve embryos, select embryos most likely to establish and maintain pregnancy after transfer, and to enhance embryo competence for post-transfer survival through manipulation of culture conditions. The original specific objectives were to 1) optimize procedures for cryopreservation (Israel/US), 2) develop procedures for identifying embryos with the greatest potential for development and survival using the remote monitoring system called EmbryoGuard (Israel), 3) perform field trials to test the efficacy of cryopreservation and the EmbryoGuard selection system for improving pregnancy rates in heat-stressed, lactating cows (US/Israel), 4) test whether selection of fresh or frozen-thawed blastocysts based on measurement of group II caspase activity is an effective means of increasing survival after cryopreservation and post-transfer pregnancy rate (US), and 5) identify genes in blastocysts induced by insulin-like growth factor-1 (IGF-1) (US). In addition to these objectives, additional work was carried out to determine additional cellular determinants of embryonic resistance to heat shock. There were several major achievements. Results of one experiment indicated that survival of embryos to freezing could be improved by treating embryos with cytochalasin B to disrupt the cytoskeleton. An additional improvement in the efficacy of embryo transfer for achieving pregnancy in heat-stressed cows follows from the finding that IGF-1 can improve post-transfer survival of in vitro produced embryos in the summer but not winter. Expression of several genes in the blastocyst was regulated by IGF-1 including IGF binding protein-3, desmocollin II, Na/K ATPase, Bax, heat shock protein 70 and IGF-1 receptor. These genes are likely candidates 1) for developing assays for selection of embryos for transfer and 2) as marker genes for improving culture conditions for embryo production. The fact that IGF-1 improved survival of embryos in heat-stressed recipients only is consistent with the hypothesis that IGF-1 confers cellular thermotolerance to bovine embryos. Other experiments confirmed this action of IGF-1. One action of IGF-1, the ability to block heat-shock induced apoptosis, was shown to be mediated through activation of the phosphatidylinositol 3-kinase pathway. Other cellular determinants of resistance of embryos to elevated temperature were identified including redox status of the embryo and the ceramide signaling pathway. Developmental changes in embryonic apoptosis responses in response to heat shock were described and found to include alterations in the capacity of the embryo to undergo caspase-9 and caspase-3 activation as well as events downstream from caspase-3 activation. With the exception of IGF-1, other possible treatments to improve pregnancy rate to embryo transfer were not effective including selection of embryos for caspase activity, treatment of recipients with GnRH.and bilateral transfer of twin embryos. In conclusion, accomplishments achieved during the grant period have resulted in methods for improving post-transfer survival of in vitro produced embryos transferred into heat-stressed cows and have lead to additional avenues for research to increase embryo resistance to elevated temperature and improve survival to cryopreservation. In addition, embryo transfer of vitrified IVF embryos increased significantly the pregnancy rate in repeated breeder cows.

The ability to introduce novel genetic material into the genome of commercial poultry has been impeded by a lack of kowledge regarding the origin in the early embryo of the target cell of interest, namely, the germ cell. Hence, this project investigated the emergence of primordial germ cells (PGCs) during the early development of the avian embryo to aid in efforts to produce transgenic poultry on a routine basis. The strategy was to introduce foreign DNA into the area of the unincubated embryo that is destined to give rise to the germ line. The objectives of this project were: 1) to identify and localize a subpopulation of cells in the early embryo which will give rise to PGCs, 2) to determine the best location and stage of development to transfer donor cells for efficient germline chimerism, and 3) to transfect donor cells to produce transgenic/germline chimeric embryos. We show that by using the monoclonal antibody SSEA-1 and by various cell culture techniques that germ cells appear to segregate from the somatic lineages at St. X., a process that is gradual and continues through St. XIV. Using microsurgical transplantation between quail and chick embryos, we demonstrated that the inner 1/3 of the area pellucida between states X-XII gives rise to about 2/3 of the germ cell population at the time of their residence in the germinal crescent. Because of the non-localized emergence of PGCs, attempts to introduce foreign DNA into clonal precursors of germ cells through liposome-mediated transfection yielded unacceptable levels of efficiency. However, through our investigation of germ cell origins, an in vitro model of germ cell differentiation was developed that could offer a means of determining the factors required for the long term culture of avian PGCs thereby providing a convenient means of manipulating the avian genome.

The long-term motivation for this research is to transfer useful traits from a broad based gene pool of wild species into the narrow base of a cultivated crop in Cucumis. Our primary focus was to use polyploid prior to fertilization as a tool to overcome fertility barriers in the cross between C. melo and C. metuliferus. In conducting this research, we explored all combinations of tetraploid and diploid parents, in reciprocal combinations. Pollinations were made in both the field and greenhouse, using emasculated flowers, moneocious females, and open pollination by insect vectors, with morphological selection criteria. After observations of thousands of ovaries, we still have no definitive proof that this hybridization yielded viable embryos. The most promising results came from using tetraploid C. metuliferus, as the maternal parent in the interspecific hybridization, that set fruit were seeds contained small embryos that did not germinate. To obtain fruit set, it was important to rear plants in a cooler sunny greenhouse, as would be found in late winter/early spring. A second interspecific hybrid between wild and cultivated Cucumis, C. hystrix x C. sativus, yielded fertile progeny for the first time, while concomitantly working toward our primary goal. Two distinct treatments were necessary; 1) special plant husbandry was necessary to have the wild species produce fruit in cultivation, and 2) embryo rescue followed by chromosome doubling in vitro was required for fertility restoration. Backcrosses to crop species and resistance to nematodes are compelling areas for further work.

The BARD program includes two main parts. In the first, experiments were conducted to complete our understanding of the mechanisms responsible for the impairment of reproductive functions under heat stress. Experiments focused on follicular development and function, since results obtained in our previous BARD project indicate that the preovulatory follicle is susceptible to heat stress. The theca cells, sensitive to thermal stress, produced less androgen during the summer, as well as during the autumn. Similarly, luteinized theca cells obtained from cows in summer produced much less progesterone than in winter. Granulosa cells and luteinized granulosa cells were less susceptible to heat stress. A delayed effect of heat stress on follicular development, on suppression of dominance and on steroid production by theca and granulosa cells was noted. This may be related to the low fertility of cows during the cool months of autumn. In the second part, experiments were conducted aiming to improve fertility in summer. The timed AI program was developed using two injections of GnRH coupled with PGF2a. It was found effective in improving reproductive performance in lactating cows. Limitations induced by heat stress on estrus detection were eliminated with the timed AI management program. Replacing the second injection of GnRH with hCG instead of GnRH agonist increased plasma progesterone levels post ovulation but did not improve fertility. Use of the timed AI program in summer, shortened days open and increased the net revenue per cow, however, it did not protect the embryo fiom temperature-induced embryonic mortality. Incorporation of a GnRH-agonist implant into the timed AJ program was examined. The implant increased plasma progesterone and LH concentrations and altered follicular dynamics. The use of a GnRH-implant enhanced pregnancy rate in cows with low body conditions. In a timed embryo transfer experiment, the use of fresh or frozen in vitro produced embryos was compared in the summer to improve fertility. The use of flesh embryos (but not frozen ones) improved pregnancy rate, however, substantial embryonic death occurred between 21 and 45 days. The timed AI program, which is now being used commercially, shortened days open, and increased pregnancy rate during summer. Other approaches which were found to improve fertility in small-scale studies, need to be tested again in large-scale field trials.

Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.