Dissertations / Theses on the topic 'Fertilization in vitro, Human'

Thesis (M.A.)--Central Connecticut State University, 2000.<br>Thesis advisor: Kathy Martin. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 23-27).

For Muslims all over the world, whether in North America where they form minorities or in all-Muslim societies, their religion permeates every aspect of their lives and ethical decision-making. It is no wonder that when deliberating the treatment of infertility or the introduction of cloning to the world, Muslims look to their Islamic scholars and await their decision on such matters. They are the ones with the most knowledge of the Quran, Sunnah, and other sources used in Islam. This thesis will explore the ethics of assisted reproduction technologies and human reproductive cloning from an Islamic ethical perspective. I will investigate the principles and regulations that are used today in the Islamic analysis of both practices.

Thesis (D.Phil.)--Stellenbosch University, 2000.<br>ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme.<br>AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.

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Orientador: José Gonçalves Franco Júnior<br>Coorientador: Edson Borges Júnior<br>Banca: João Batista Alcântara Oliveira<br>Banca: Joelcio Francisco Abade<br>Banca: Mario Cavagna<br>Banca: Júlio Elito Junior<br>Resumo: Não disponível<br>Abstract: Not available<br>Doutor

Thesis (PhD)--Stellenbosch University, 2012.<br>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.<br>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.

<p>Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB.</p><p>We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.</p>

The cellular and molecular environment of the uterus during the pre - and peri - implantation period of early pregnancy is critical for implantation success and optimal fetal and placental development. Perturbations to this environment not only have consequences for the success of pregnancy and neonatal health and viability, but can also drive adverse health outcomes in the offspring after birth, particularly the development of metabolic disorders such as obesity, hypertension and insulin resistance. The influence of seminal plasma on the cytokine and immune uterine environment has been previously well characterised in mice, however the effects of disruption in uterine seminal plasma exposure for pregnancy outcome have not been investigated. The studies described in this thesis employed the use of surgical seminal vesicle ablation in males and embryo transfer experiments to investigate the physiological significance of uterine seminal plasma exposure on programming fetal and neonatal outcomes, and growth and metabolic status in adult offspring. We demonstrate that in the absence of seminal plasma, oocyte fertilisation and embryo implantation are reduced, showing that seminal plasma acts primarily to facilitate fertilisation, possibly by promoting sperm transport and survival in the reproductive tract. In addition we show that pregnancies initiated in the absence of seminal plasma give rise to offspring which display accelerated growth after birth and increased adiposity in adulthood, compared to those developed in a tract exposed to seminal plasma at the time of conception. Offspring conceived in the absence of seminal plasma also displayed alterations in serum leptin and adiponectin content, similar to those known to be associated with obesity in the mouse. Using embryo transfer experiments, we showed that some, but not all aspects of the perturbed postnatal development are recapitulated when embryos fertilised in the presence of what semen are transferred to a recipient tract which has not been exposed to seminal plasma. More severe perturbations were seen in 2 - cell transfer than in blastocyst transfer experiment. Additionally, there was a significant effect of the embryo transfer procedure, irrespective of seminal plasma exposure, on fetal and postnatal development that confounded interpretation of these experiments. In addition, we investigated the potential mechanisms by which the influence of seminal plasma is exerted. Mediators of pre - implantation embryo development, implantation and the modulation of the maternal immune response to pregnancy were all assessed for regulation by seminal plasma using QRT - PCR. It was demonstrated that seminal plasma exposure induces the up - regulation of key embryotrophic factors, LIF, GM - CSF and IL - 6, in the oviduct following insemination. Factors important in tissue remodelling required for implantation and angiogenesis, MMP - 2, MMP - 3 and VEGF - C, were also shown to be increased at the time of implantation after seminal plasma exposure. Additionally the generation of T - regulatory cells in uterine tissues, demonstrated by the up - regulation of the transcription factor FOXp3 was shown to be dependent on semen exposure. The influence of seminal plasma on embryonic development, implantation and modulation of the maternal immune response to pregnancy may therefore be mechanisms which contribute to the adverse outcomes seen in pregnancies initiated in the absence of seminal plasma. Together these experiments show a role for seminal plasma signalling at the time of insemination in influencing the pre - implantation embryo to program later fetal and neonatal development, thereby impacting on the metabolic health of offspring. We conclude that seminal plasma is not simply a transport medium for sperm, but acts also as a key regulator of a female tract environment providing optimal support for the developing embryo.<br>Thesis (Ph.D.)--School of Paediatrics and Reproductive Health, 2006.

<p>The chromatin integrity in sperm cells is vital for successful pregnancy. In this</p><p>study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization</p><p>(IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were</p><p>purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test</p><p>Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation</p><p>Index (DFI) of 30 % and the results correlated with fertilization, cleavage and</p><p>pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower</p><p>cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro</p><p>may be independent of the degree of DNA-damage, the embryonic development could be</p><p>seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a</p><p>DFI threshold of 30 %.</p>

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.

Dissertation (PhD)--University of Stellenbosch, 2007.<br>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.<br>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.

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 (PhD)--Stellenbosch Uni versity, 2000.<br>ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI) introduced a revolutionary way of treatment for male factor infertility. With the exception of some cases of non-obstructive azoospermia, all other male factor infertility cases have the potential to be successfully treated with ICS!. The only prerequisite seems to be the presence of a motile or viable immotile sperm cell for each oocyte. In this study we report on our own experience with the development and implementation of the ICSI method in the Reproductive Biology Unit at Tygerberg Hospita!. An analysis of 5 years of ICSI experience showed that semen parameters, sperm morphology, motility and concentration did not influence fertilization and pregnancy rates adversely. In most cases, patients who could not be treated with in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), due to poor semen parameters or fertilization failure, were treated successfully with ICS!. Even a case of globozoospermia was treated successfully with ICS!. Testicular spermatozoa, fresh or frozen-thawed, also resulted in excellent fertilization and pregnancy rates. Cryopreservation of testicular samples facilitated the management of the infertile couple, aiding the coordination of the recovery of vital gametes from both partners and also limiting the repetition of testicular biopsies. Incubation (maturation) of testicular spermatozoa also induced an enhancement in pregnancy rates. It can be concluded that ICSI proved to be a treatment method with success similar to that of in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), in spite of a severe male factor. The study also indicated transfer route and embryo quality (viability) to be very important factors in the success of ICS!. The tubal transfer route was shown to be a significant contributor to the pregnancy success (compared to uterine transfer) as was the transfer of embryos that showed early division to the 2-cell stage, 26 hours post injection. The transfer of early dividing embryos into the fallopian tube resulted in a pregnancy rate of almost 40%, a result similar to that of GIFT with a mild male factor. The role of the oocyte in fertilization and pregnancy success was also revealed indirectly by the introduction of ICS!. Visual observation of denuded oocytes was possible and many morphological features, normal and abnormal, can be observed. Immature oocytes can also be identified and it was shown that they could be successfully matured in vitro before injection. In this study transmission electron microscopy (TEM) was used to study abnormalities in oocyte morphology. The standard method was adapted and modified for single cell TEM. The abnormalities observed included lysosomal and non-lysosomal degeneration (yellowish or darkly coloured oocytes), degeneration and vacuole formation (vacuolated oocytes), large secondary lysosomes filled with multiple small lipid droplets - lipofuscin body (refractile body) and a fragmented oocyte. It was also possible to study at ultrastructural level, possible reasons for fertilization failure in ICS!. Different stages of oocyte activation failure, cytoplasmic immaturity, sperm cell extrusion, abnormal sperm cell decondensation, female spindle abnormalities and technique related factors were observed. TEM was also successfully implemented to elucidate the reason for infertility in a patient with a longstanding, unexplained history of infertility. TEM evaluation of two of the patient's unfertilized oocytes revealed a spindle abnormality with contributing cytoskeletal anomalies at ultrastructural level. The modified TEM technique offers a valuable tool to study this small, but important group of patients with unexplained infertility. ThisTEM study opened up a new, valuable and interesting avenue of research with both diagnostic and prognostic value for patients with unexplained infertility. ICSI is therefore a valuable method in the treatment of especially male factor infertility. It is the most advanced fertilization technique developed in the last decade in this field. Not only can almost all male factor patients be treated, but unexplained female infertility can also be exposed, studied and hopefully in future also be treated with micromanipulation methods.<br>AFRIKAANSE OPSOMMING: Die ontwikkeling van die mikromanipulasie tegniek "Intracytoplasmic sperm injection" (ICSI) het die behandeling van die manlike faktor in infertiliteit, revolusionêr verander. Met die uitsondering van sommige gevalle van nie-obstruktiewe asoospermia, kan potensieel alle ander manlike infertiliteits faktore suksesvol met ICSI behandel word. Die enigste voorvereiste blyk "n bewegende of "n nie-bewegende, maar bewese lewende spermsel te wees. In hierdie studie word verslag gedoen oor die ontwikkeling en toepassing van die ICSI metode in die Eenhed vir Reproduktiewe Biologie by Tygerberg Hospitaal. 'n Analise van 5 jaar se resultate na die implementering van die ICSI metode het gewys dat die semen parameters, sperm morfologie, motiliteit en konsentrasie, nie "n effek op bevrugting- en swangerskapsyfers gehad het nie. Pasiënte wat, as gevolg van ontoereikende semen parameters, nie met die klassieke metodes, in vitro bevrugting (IVB) of gameet intrafallopiusbuis terugplasing (GIFT) behandel kon word nie, kon suksesvol met ICSI behandel word. Daar was selfs "n geval van manlike infertiliteit as gevolg van globosoospermie, wat suksesvol met ICS behandel is. Die ICSI metode het dit ook moontlik gemaak om uitstekende bevrugting- en swangerskap resultate met testikulêre spermatosoa .(vars en gevries) te bereik. Die bevriesing van testisweefsel het ook bygedra tot beter hantering van sulke pasiënte. Herhaalde testisbiopsies word uitgeskakel en die koórdinasie van die verkryging van die manlike en vroulike gamete, word ook vergemaklik wanneer testisweefsel in gevriesde vorm beskikbaar is. Die studie het verder getoon dat wanneer testikulêre weefsel geïnkubeer word (om spermatosoa te laat matureer), die swangerskapsyfers verhoog was. Dit is dus duidelik dat die ICSI metode net so suksesvol soos die IVB en GIFT metodes toegepas kan word, selfs en veral in gevalle van erge manlike faktor infertiliteit. Die studie het ook verder getoon dat die plek waar embrios teruggeplaas word, asook die embriokwalitiet van teruggeplaasde embrios, belangrike bydraende faktore in die ICSI swangerskapsukses was. Embrioterugplasing in die buis van fallopius en terugplasing van embrios wat vroeë 2-sel deling, 26 uur na ICSI getoon het, is uitgewys as faktore wat ICSI swangerskap betekenisvol verbeter het. Dit was moontlik om "n swangerskapsyfer van ongeveer 40%, sootgelyk aan die van GIFT sonder "n erge manlike faktor, te bereik met die terugplasing van ten minste een vroeë deler embrio in die fallopiese buis. Die ICSI tegniek het ook indirek bygedra tot nuwe insigte met betrekking tot die rol wat die vroulike eiersel (oësief in ICSI bevrugting speel. Oósiete word gestroop van hulomringende selle vir die ICSI proses en kan dan maklik vir hul normale en abnormale morfologiese eienskappe evalueer word. Oësiete wat immatuur is kan ook so geïdentifiseer word en dit is moontlik om hulle suksesvol te matureer voor mikro-inspuiting. Transmissie-elektronmikroskopie (TEM) is in die studie gebruik om die ultrastruktuur van onbevrugde en abnormale oësiete te bestudeer. Hiervoor is "n bestaande tegniek gemodifiseer vir die hantering van "n enkele sel, in hierdie geval die oosiet. Lisasomale en nie-lisosomale degenerasie (oósiete wat geelof donker van kleur voorkom), degeneratiewe tekens en vakuole (oësiete met vakuole), groot sekondêre lisosome gevul met klein lipieddruppels ('refractile body') en 'n gefragmenteerde oosiet was van die morfologies abnormale eienskappe wat ultrastruktureel geïdentifiseer is. Moontlike faktore wat 'n rol kan speel in nie-bevrugting na ICSI kon ook op ultrastrukturele vlak met die tegniek geïdentifiseer word. Hierdie faktore het die volgende ingesluit: die onvermoë van verskillende stadiums van oosiet aktivering, sitoplasmatiese immaturiteit, uitwerping van die spermsel na die periviteliene spasie, abnormale spermsel dekondensasie, vroulike spoelvormings abnormaliteite en tegniekgekoppelde faktore. Die TEM tegniek is ook suksesvol aangewend om die infertiliteitsprobleem van 'n pasiënt wat vir etlike jare aan onverklaarbare infertiliteit gely het, te identifiseer. TEM het op die ultrastrukturele vlak gewys dat daar 'n spoel abnormaliteit in twee van haar onbevrugde oëslete was. TEM kan dus baie vrugbaar gebruik word in hierdie groep pasiënte om onverklaarbare infertiliteit, wat andersins ongeïdentifiseerd sou bly, te verklaar. Die ICSI metode is die mees revolusionêre tegniek wat die afgelope dekade vir die behandeling van veral manlike infertiliteit ontwikkel en baie suksesvol toegepas is. Die metode ook kan 'n bydraende rol speel in die hantering van onverklaarbare infertiliteit veral ten opsigte van die vroulike gameet. In die toekoms is dit moontlik dat selfs hierdie probleem met nuwe mikromanipulasietegnieke opgelos sal kan word.

La interacción entre gametos es crucial para la fecundación. La zona pelúcida (ZP) se considera responsable de bloquear la polispermia, pero in vitro estas funciones no son totalmente eficientes. La polispermia es frecuente en fecundación in vitro (FIV) en porcino y bovino, mientras que la interacción heteróloga espermatozoide-ovocito ha sido demostrada. Los objetivos fueron estudiar el bloqueo de la polispermia para mejorar los resultados de FIV e investigar las interacciones heterólogas entre espermatozoide humano y ovocito porcino. Los resultados demuestran que se produce endurecimiento de la ZP de ovocitos bovinos y porcinos de forma previa a la fecundación, utilizando DTSP o fluido oviductal bovino. Cuando se utilizan estos ovocitos en FIV aumenta la monospermia y el rendimiento final. En las interacciones heterólogas los espermatozoides humanos pueden unirse a ZP porcina y sufren la reacción acrosómica, pero no penetran los ovocitos sin ZP. En ICSI activan el ovocito y forman pronúcleos.<br>The interaction between gametes is crucial to fertilization. The zona pellucida (ZP) is responsible to block of polyspermy, but in vitro these functions are not efficient. The polyspermy is frequently in bovine and porcine in vitro fecundation. Besides the heterologous interaction between spermatozoa-oocyte had been described. The aims were study the block of polyspermy to improve the output of IVF and research the heterologous interactions between human spermatozoa and porcine oocyte.The results show that there is hardening of bovine and porcine ZP previously at fertilization, in vivo and using DTSP or bovine oviductal fluid. When these oocytes are used in IVF improve the monospermy and the output. In heterologus interactions the human spermatozoa could bind to porcine ZP and it triggers the acrosome reaction, but not penetration in ZP-free oocyte was observed. In ICSI the oocyte activation and

Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>Nesta dissertação de Mestrado em Direito Civil procura-se demonstrar que as inovações biotecnológicas, principalmente da área da medicina reprodutiva, têm trazido consequências impactantes no direito de família e das sucessões, criando, assim, a necessidade de (re)adaptar o sistema jurídico brasileiro à nova (e dinâmica) realidade social, à luz dos princípios e normas constitucionais. Nesse sentido, por exemplo, repensar e reestruturar o modelo tradicional de família parece ser um passo necessário para esta (re)adaptação, uma vez que novas tecnologias, como as técnicas de reprodução assistida, criam a possibilidade de interferência externa em processos naturais de procriação, de maneira não antes prevista pela sociedade e pelo Direito. Assim, verifica-se a possibilidade de procriação de um novo descendente anos após a morte do genitor. Este trabalho busca entender e examinar as implicações de tais inovações biotecnológicas para o sistema jurídico brasileiro. Mais especificamente, busca-se entender e examinar as consequências jurídicas da reprodução medicamente assistida post mortem no âmbito sucessório, demonstrando diversas dificuldades de ordem prática, a exemplo da necessidade do respeito à igualdade entre filhos, por um lado; e a necessidade de definição da questão sucessória, por outro. Busca-se também demonstrar o modo de garantir os direitos hereditários do filho póstumo, tanto na sucessão legítima, como na testamentária. Ademais, pretende-se provocar reflexões secundárias acerca da necessidade de autorização expressa do de cujus para implantação de seu material genético no útero materno, bem como a necessidade de existência de um prazo para essa utilização, evitando que haja insegurança jurídica.<br>This Master in Civil Law dissertation aims at demonstrating that biotechnological innovations, most especially in the field of reproductive medicine, have fundamental implications for family law and inheritance law, thus creating the need for (re)adapting the legal system to the new (and dynamic) social reality, in light of the Brazilian constitutional principles and norms. In this regard, for instance, rethinking and restructuring the traditional family model seems to be a necessary step within this legal (re)adaptation, since new technologies, such as techniques of assisted human reproduction, create the possibility of interfering in natural processes of procreation in ways not foreseen by society and the law. In this regard, it is now possible to imagine the birth of a child years after the death of the parent. This research seeks to understand and examine the implications of such biotechnological innovations for the Brazilian legal system. Most specifically, it aims at understanding and examining the implications of post mortem assisted reproduction for the law of succession, demonstrating and commenting on various practical difficulties, such as the need for respecting the equality of children, on the one hand, and the necessity of defining the succession problem, on the other. This work also seeks to demonstrate how to ensure the inheritance rights of the posthumous son, both in the legitimate succession and in the testament succession. Furthermore, it aims at suggesting and provoking secondary reflections on the need for previous and explicit authorization from the deceased parent for implanting his genetic material into the womb of the widow, and the need for clarifying and determining the period in which this use of genetic material may be authorized, avoiding legal uncertainty.

Abstract The aim of the present study was to evaluate the efficacy of the first trimester screening for Down syndrome (DS) in an unselected low-risk Finnish population. The study involved 4,617 women who attended screening between the 8th and 14th weeks of pregnancy in 1998-2000. They gave a blood sample for the measurement of pregnancy associated plasma protein A (PAPP-A) and free beta human chorionic gonadotrophin (β-hCG). Of these women, 3,178 also had an ultrasound examination for the measurement of fetal nuchal translucency (NT). The risk figure for every screened woman was calculated using a computerized risk figure program. The risk 1 in 250 was used as a cut-off. The subgroup of screen positives comprised 5.8% of the study group. There were 16 DS cases. The combined method (maternal age, NT and the biochemical markers) detected 77% of the affected pregnancies. NT combined with maternal age gave a detection rate of 69%. Serum markers without NT combined with maternal age found 75% of the Down's. In 49 consecutive singleton in-vitro-fertilization pregnancies, the β-hCG value was more often elevated compared to spontaneous pregnancies, increasing the false positive rate. In 67 twin pregnancies, the serum marker levels were approximately double those in singletons. Smoking reduced PAPP-A by 20% making the smokers more likely to get a positive screening result. To determine the impact of the screening on the live born incidence of DS, two historical populations were compared. The first group was screened by second trimester serum samples (β-hCG and AFP) and the second group by first trimester ultrasound examination. When detection rates were at the same level, the second trimester screening reduced the number of live born Down's children more effectively. In conclusion, the first trimester combined method (maternal age, NT, β-hCG and PAPP-A) for Down syndrome screening is efficient in an unselected low risk population. The biochemical screening is not recommended in IVF-pregnancies.

O avanço da ciência, em especial na área da reprodução humana, permitiu a ocorrência de concepção humana de forma extracorpórea através do processo de fecundação in vitro a ser feita em laboratório especializado em reprodução humana assistida. Os embriões não implantados em útero materno são criopreservados. Este trabalho busca trazer os questionamentos da bioética, do biodireito, a cerca deste tema e das questões nele envolvidas. Também examina o enquadramento destes embriões sob a ótica da proteção Constitucional do patrimônio genético, da inviolabilidade da vida e da dignidade da pessoa humana. Trata também do Meio Ambiente quando aborda a Constituição Federal Brasileira de 1988, que em seu artigo 225 § 1º, inciso I, legisla sobre a preservação e restauração dos processos ecológicos e, no inciso II, determina que incumbe ao poder público preservar a diversidade e a integridade do patrimônio genético do País ficando demonstrado que tratar de embriões é tratar de patrimônio genético e de Meio Ambiente.Trata ainda da Nova Lei de Biossegurança/2005, a qual veio permitir pesquisas com célulastronco de embriões criopreservados, bem como a regulamentação desta Lei, tratando ainda sobre a Ação Direta de Inconstitucionalidade (ADIN) 3.510-0 que fora proposta junto ao Supremo Tribunal Federal (STF), questionando a constitucionalidade da permissão legal destas pesquisas. Dessa forma, é feita uma análise sobre a legislação existente com vistas a obter uma visão sobre eventuais direitos e proteção dos embriões criopreservados.<br>Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-04T19:54:25Z No. of bitstreams: 1 Dissertacao Denise Kempf.pdf: 1443498 bytes, checksum: 1586cffbe1630fc158628d51f0cd295a (MD5)<br>Made available in DSpace on 2014-06-04T19:54:25Z (GMT). No. of bitstreams: 1 Dissertacao Denise Kempf.pdf: 1443498 bytes, checksum: 1586cffbe1630fc158628d51f0cd295a (MD5)<br>The advancement of science, particularly in the area of human reproduction, allowed the occurrence of human conception in a bypass through the process of in vitro fertilization to be made in a laboratory specializing in assisted human reproduction. The embryos not implanted in the uterus are cryopreserved. This work seeks to bring the questions of bioethics, the biolaw, about this issue and the issues involved. It also examines the framework of these embryos from the perspective of Constitutional protection of genetic heritage, the sanctity of life and human dignity. It also deals with the Environment when addresses the Brazilian Constitution of 1988, which in article 225 § 1, section I, legislation on the preservation and restoration of ecological processes and, in section II, provides that "it is for the government to preserve the diversity and integrity of the genetic heritage being shown that treating embryos is to address genetic heritage and also the Middle Ambiente.Trata Biossegurança/2005 New Law, which has allowed research on stem cells from cryopreserved embryos, as well as regulation this Act, still dealing with on the direct action of unconstitutionality (ADIN) 3510-0 which was proposed to the Supreme Court (STF), questioning the constitutionality of the legal permission of this research. Thus, an analysis is made on the existing legislation in order to get an insight into possible rights and protection of cryopreserved embryos.

Les études sur les couples traités par fécondation in vitro (FIV) ont jusqu'à présent porté essentiellement sur l'évaluation du succès en FIV. Très peu de données sont disponibles sur le devenir à long terme de couples traités par FIV. L'objectif de ce travail était d'estimer la fréquence de réalisation du projet parental à long terme, et d'étudier les facteurs associés aux interruptions précoces des traitements et aux naissances naturelles.L'enquête DAIFI-2009 a inclus 6 507 couples ayant débuté un programme de FIV en 2000-2002 dans l'un des 8 centres de FIV participant à l'étude. Les données médicales des couples et leur parcours dans le centre ont été obtenus à partir des dossiers médicaux des centres de FIV pour tous les couples. L'information sur le devenir des couples après le départ du centre a été obtenue par questionnaire postal auprès des couples en 2008-2009 (38% de participation 7 à 9 ans après l'initiation des FIV). L'étude des facteurs associés à la participation à l'enquête postale suggérait que la fréquence de réalisation du projet parental estimée sur les répondants seulement pourrait être biaisée. Les différentes méthodes mises en œuvre pour corriger la non réponse (pondération, imputation multiple) n'ont pas modifié l'estimation de la fréquence de réalisation du projet parental. Au total, 7 à 9 ans après l'initiation des FIV, 60% des couples ont réalisé leur projet parental de façon biologique, suite à un traitement ou suite à une conception naturelle. Lorsque les adoptions sont aussi prises en compte, 71% des couples ont réalisé leur projet parental. Après l'échec d'une première tentative de FIV, un couple sur 4 (26%) a interrompu les FIV dans le centre d'inclusion. Globalement, les couples avec de mauvais facteurs pronostiques ont un plus grand risque d'interrompre les FIV. Cependant, la proportion plus importante d'interruption parmi les couples avec une origine inexpliquée de l'infécondité pourrait s'expliquer par la survenue plus fréquente de naissance naturelle dans ce sous-groupe de couples. Parmi les couples n'ayant pas eu d'enfant suite aux traitements, 24% ont ensuite conçu naturellement en médiane 28 mois après l'initiation des FIV. Parmi les couples ayant eu un enfant suite aux traitements, 17% ont ensuite conçu naturellement en médiane 33 mois après la naissance de l'enfant conçu par AMP. Les facteurs associés aux naissances naturelles sont des indicateurs d'un meilleur pronostic de fertilité, particulièrement chez les couples sans enfant AMP.L'enquête DAIFI-2009 a permis d'apporter des informations sur le parcours à long terme des couples traités par FIV qui n'avait jusqu'à présent été que peu étudié, souvent sur de faibles effectifs et avec un suivi plus court. Ces résultats doivent apporter de l'espoir aux couples inféconds, puisque la majorité d'entre eux ont finalement réalisé leur projet parental, même si cela peut prendre de nombreuses années.

Background: Obese and overweight women are frequent users of in-vitro fertilization (IVF) owing to the association between increased body mass index (BMI) and infertility. Policies that impose BMI restrictions for access to IVF exist in some countries, based on limited evidence suggesting that obese women experience reduced IVF success rates. However, there is a paucity of data on the frequency and risk of preeclampsia in the obese IVF population. More data would help inform such policies. Objectives: (i) To evaluate the respective effects of an elevated BMI (> 25 kg/m2) and the use of IVF on the risk of preeclampsia; (ii) to assess whether the risk of preeclampsia associated with an elevated BMI is different between twins and singletons; and (iii) to assess whether the effect of elevated BMI is modified by IVF treatment. Methods: The study population originated from a cohort of births delivered at the Royal Victoria Hospital in Montreal, Canada recorded between 2001 and 2008 in the McGill Obstetric and Neonatal Database (MOND). Associations were investigated with descriptive statistics, univariate and multivariate logistic regressions. We assessed effect modification by comparing observed to expected combined effects of a raised BMI and IVF treatment on preeclampsia. Results: Preeclampsia complicated 4.4% of pregnancies. There was an increased risk of preeclampsia among overweight and obese mothers (adjusted OR 3.1, 95% CI 2.5, 3.7). IVF treatment did not confer a heightened risk of preeclampsia in multivariate analyses (adjusted OR 1.0, 95% CI 0.7, 1.5). There was heterogeneity of the effect of BMI in singletons vs. multiples. There was evidence for synergism between IVF and obesity among singletons. Conclusions: Our study confirms the strong association between high BMI and preeclampsia. Despite a null effect of IVF on its own, obese women with singleton pregnancies who use IVF have a higher relative and absolute risk of preeclampsia than spontaneous singleton pregnancies. Given that preeclampsia is a serious complication of pregnancy, its risk should be considered in policy decisions about access to IVF treatment in the overweight and obese population.<br>Contexte: Les femmes en surpoids sont des utilisateurs fréquents de fécondation in-vitro (FIV) en raison de l'association entre l'augmentation de l'indice de masse corporelle (IMC) et l'infertilité. Les politiques qui imposent des restrictions IMC pour l'accès à la FIV existent dans certains pays, basé sur des données limitées suggérant que les femmes obèses ont des taux de succès de FIV réduits. Cependant, il y a un manque de données sur la fréquence et le risque de prééclampsie chez la population de FIV obèses. Plus de données aideraient à informer ces politiques. Objectifs: (i) dévaluer les effets respectifs d'un IMC élevé (> 25 kg/m2) et l'utilisation de la FIV sur le risque de pré-éclampsie, (ii) évaluer si le risque de pré-éclampsie associé à un IMC élevé est différent entre jumeaux et singletons, et (iii) évaluer si l'effet de l'IMC élevé est modifié par un traitement de FIV. Méthodes: Notre cohorte comprend les naissances de l'Hôpital Royal Victoria, à Montréal, Canada enregistrées entre 2001 et 2008 dans la base de données obstétriques McGill et néonatale (MOND). Les associations ont été étudiées avec les statistiques descriptives, univariées et des régressions logistiques multivariées. Nous avons évalué la modification de l'effet observé en comparant les effets combinés attendus d'un IMC élevé et un traitement de FIV sur la pré-éclampsie. Résultats: La prééclampsie complique 4,4% des grossesses. Il y avait un risque accru de pré-éclampsie chez les mères en surpoids et obèses (OR ajusté 3,1, IC 95% 2.5, 3.7). FIV ne conférait pas un risque accru de pré-éclampsie dans les analyses multivariées (OR 1,0, IC 95% de 0,7, 1,5). Il y avait hétérogénéité de l'effet de l'IMC dans les grossesses de singletons vs multiples. Il y avait en évidence d'une synergie entre FIV et un IMC élevé chez les singletons. Conclusions: Notre étude confirme la forte association entre un IMC élevé et la pré-éclampsie. Les femmes en surpoids avec des grossesses uniques qui utilisent la FIV ont un risque plus élevé relative et absolue de la pré-éclampsie que pendant les grossesses spontanées. Étant donné que la pré-éclampsie est une complication grave de la grossesse, le risque doit être pris en compte dans les décisions politiques concernant l'accès à un traitement par FIV dans la population en surpoids et obèses.

In 1984, The National Institute of Child Health and Human Development awarded a grant to The Eastern Virginia Medical School and The Children's Hospital of The King's Daughters to study the IVF children conceived a The Jones Institute for Reproductive Medicine in Norfolk, Virginia. The purpose of the study was to assess the children comprehensively using a multi-disciplinary team to determine whether the IVF process resulted in higher than average physical and/or behavioral deficits. The children were psychologically tested on the Bayley Scales of Infant Development; they also received pediatric, neurological, cardiac, and ultrasound examinations of their internal organs. 83 of 105 eligible IVF children were examined. These children were matched on the following criteria: maternal age, child's age, race, gender, births/pregnancy, and socioeconomic status. The controls were obtained from a 100 mile radius of Norfolk, Virginia. Ninety-three children served as controls. All were between 12 and 30 months of age.;The results indicate that these families are different from the general population in several respects: they are older, better educated, more affluent, almost all white, and have a higher rate of multiple births. The groups did not differ in their rate of congenital defects. While prematurity was common, the children demonstrated no adverse effects from their prematurity.;The psychological results indicated that both groups were above the national norms for the Bayley Scales on both their MDIs and PDIs; they did not differ significantly, but the IVF group was higher on both scores. Two IVF children with physical handicaps were cognitively normal. Behaviorally the groups did not differ at the p =.01 level on any of the Behavioral Record variables.;The NICHHD study concludes that the risk of the IVF process is acceptable from a medical viewpoint. The children who are born do not demonstrate a higher rate of physical or psychological abnormalities based on current information.

This study focused on factors that influenced clinician embryo disposition decision-making in in vitro fertilization (IVF). Evidence-based medicine (EBM) should be built on the premise of shared decision-making. This paradigm is often skewed, resulting in clinician stress, a higher probability of errors, reduced productivity, or ineffective decisions. Guided by the theory of planned behavior and the self-perception theory, this study assessed the independent variables' religiosity, ethnicity, level of burnout, gender, age, years of experience, and clinical role in relationship to the dependent variable "decision-making" as measured by the Lyerly Frozen Embryo Survey, Maslach Burnout Inventory, and the Areas of Worklife Survey. IVF clinicians throughout the United States and Europe (n = 151) completed an online survey via a nonrandom, cross-sectional methodology. Study results indicated the factors were not significant. A vast majority of participants identified as: White, (84%), and female (75%), and that religion was very important (73%). The bulk of participants had a moderate level of burnout (85%), which showed that the multitude of participants were not experiencing overly high levels of emotional exhaustion, were not emotionally detached from their patients' needs, and felt a high degree of personal accomplishment. Recommendations included using a larger sample size, different variables, or developing a new survey as the decision making process may have been more multifaceted than anticipated. There are more areas to be studied around factors and decision making to fully understand these concepts. The positive social change implications include an increased awareness of factors that have the potential for impacting clinician decision-making as a reminder of the importance to be cognizant and sensitive of the needs of patients.

With increasing incidences of early onset of cancer and the delay of reproductive age, the demand for fertility preservation in women have increased. Even though most results of oocyte cryopreservation have been obtained from ovarian stimulation protocols for in vitro fertilization (IVF), ovarian stimulation with gonadotropins may not be suitable for many women, especially those who are with hormone-dependent cancers, or those require immediate chemotherapy. However, it is agreed that the outcome of in vitro matured (IVM) oocytes are not as good as in vivo matured ones. In this thesis, we tested the interfering of oocytes during IVM, by the addition of antioxidant and the blocking of mitochondrial function, and observed their survival and the outcome of embryonic development after vitrification and IVF. For the first part, our study showed that the presence of the antioxidant, cysteamine during IVM significantly (P<0.05) reduced the fragmentation rate in cumulus-intact group from 61.9% to 40.8% following vitrification-thawing and IVF. However, the same trend wasn't shown in denuded groups. Nevertheless, there were no significant differences in terms of maturation and cleavage rate with the supplementation of cysteamine in either COC or denuded groups. We hypothesized that cysteamine facilitated effect during IVM might have occurred by affecting mtDNA accumulation during IVM. Fluorescent real-time quantitative analysis of mtDNA showed that the mtDNA copy number were similar in cysteamine treated and untreated group with a ratio close to 1, which implied that the effect of cysteamine during IVM may not be directly related to the mtDNA replication. In the second part, a mitochondria blocker, rotenone, was added to the IVM medium in order to test the vitrification outcome and the subsequent embryonic development. The testing of rotenone concentrations from 100nM to 50µM, showed a lethal dose of around 50µM where all the oocytes were lysed during IVM. With the increase of rotenone concentration from 2µM to 50µM, the oocyte maturation rate dropped significantly in a dose dependent manner and eventually reaching 0 when the concentration was at 50µM. Within the concentration window of 250nM to 2µM, where no difference has been found in terms of the oocyte death and maturation rates compared to the control, there is a significant drop of cryo-survival rate at a concentration of 2µM, from 93.3% (in the control) to 55.6%. The percentage of embryos that developed to 4-8 cell stage were significant lower in the 2µM group (17.5%), compared to the group with a lower concentration of 250nM (50%). This study confirmed the strong positive dependency of mitochondrial functionality during oocyte maturation on its later development as well as the cryopreservation outcomes. Our work suggested that as two individual ART applications, the understanding and improvement of IVM could improve the outcome of vitrification since the impact of IVM on the oocytes could significantly determine the oocytes ability to survival and to develop following vitrification and IVF.<br>Suite a làugmentation de l'incidence des pathologies cancéreuses chez les jeunes et au recul de l'âge parental, les demandes de préservation de la fertilité sont de plus en plus fréquentes. Bien que la majorité des résultats sur la cryopréservation des ovocytes aient été obtenu suite a un protocole de stimulation ovarienne dans le cadre d'un traitement par fécondation in vitro (FIV), làdministration de gonadotrophines est inappropriées pour un certain nombre de patientes, en particulier celle atteintes de cancer hormono-dépendant, ou celle nécessitant une chimiothérapie sans délai possible. D'autre part, il est bien établi que le taux de fécondation des ovocytes maturés in vitro (IVM) n'est pas aussi élevé que lorsque les ovocytes sont maturés in vivo. Dans cette thèse, nous avons testé l'effet de la présence d'antioxydants et d'inhibiteurs de la fonction mitochondriale dans le milieu de maturation in vitro sur le taux de maturation ovocytaire (IVM) ainsi que sur le taux de survie et de développement embryonnaire après vitrification. Dans un premier temps, nous avons démontré que la présence d'antioxydants, la cysteamine, pendant l'IVM réduit significativement le taux de fragmentation après vitrification des ovocytes maturés avec leur cellules du cumulus (complexes ovocyte-cumulus, COC), de 61.9% dans le groupe non traité à 40.8% dans le groupe traité (p<0.05). Cette différence n'est pas observée dans le groupe des ovocytes maturés en l'absence de leur cellules du cumulus (ovocytes dénudés). La présence de cysteamine n'a pas d'effet significatif sur le taux de maturation et de clivage embryonnaire que les ovocytes soient dénudés ou non. Nous avons ensuite vérifié si l'effet bénéfique de la cysteamine durant l'IVM était lié à l'accumulation de DNA mitochondrial durant la maturation ovocytaire. Le nombre de copies d'aDN mitochondrial dans les ovocytes maturés en présence ou non de cysteamine a donc été évalué par PCR quantitative et le résultat ne montre pas de différence significative entre les deux groupes (ratio ~1). Ces données ne sont donc pas en faveur d'un effet de la cysteamine sur la réplication de l'aDN mitochondrial lors de la maturation ovocytaire in vitro. Dans un deuxième temps, un inhibiteur de la fonction mitochondriale, la rotenone, a été ajoutée au milieu de maturation in vitro afin de tester son effet sur la vitrification et le développement embryonnaire ultérieur. Les tests d'échelonnement de concentrations, de 100nM a 50µM, ont permis de définir une dose l'etale de 50µM, pour laquelle la totalité les ovocytes sont lysés. Entre 2µM et 50µM, le taux de maturation diminue de manière dose dépendante, atteignant 0 à la dose maximale. Dans une échelle de concentration allant de 250nM à 2µM, aucune différence significative n'est observée en terme de taux de survie et de maturation entre le groupe traité et le groupe contrôle. Par contre le taux de survie après vitrification diminue en présence de 2µM de roterone dans le milieu IVM, passant de 93.3% dans le groupe contrôle à 55.6% dans le groupe traité. Le taux d'embryons atteignant 4-8cellules après fécondation est également significativement plus bas dans le groupe traité à une concentration de 2µM comparé à un groupe traité à une dose plus faible de 250nM (respectivement 17.5% et 50%). Cette étude confirme le rôle essentiel des mitochondries durant la maturation ovocytaire, le développement ultérieur et la survie après cryopréservation. Notre travail montre l`interaction entre deux techniques utilisées en Procréation Médicalement Assistée : la maturation in vitro d'une part et la vitrification d'autre part. La compréhension et le développement des milieux de maturation ovocytaire in vitro ont en effet un impact majeur sur la survie des ovocytes après vitrification et sur leur développement embryonnaire ultérieur.

Scientific discoveries, developments in medicine and health issues are the constant focus of media attention and the principles surrounding the creation of so called ‘saviour siblings’ are of no exception. The development in the field of reproductive techniques has provided the ability to genetically analyse embryos created in the laboratory to enable parents to implant selected embryos to create a tissue-matched child who may be able to cure an existing sick child. The research undertaken in this thesis examines the regulatory frameworks overseeing the delivery of assisted reproductive technologies (ART) in Australia and the United Kingdom and considers how those frameworks impact on the accessibility of in vitro fertilisation (IVF) procedures for the creation of ‘saviour siblings’. In some jurisdictions, the accessibility of such techniques is limited by statutory requirements. The limitations and restrictions imposed by the state in relation to the technology are analysed in order to establish whether such restrictions are justified. The analysis is conducted on the basis of a harm framework. The framework seeks to establish whether those affected by the use of the technology (including the child who will be created) are harmed. In order to undertake such evaluation, the concept of harm is considered under the scope of John Stuart Mill’s liberal theory and the Harm Principle is used as a normative tool to judge whether the level of harm that may result, justifies state intervention or restriction with the reproductive decision-making of parents in this context. The harm analysis conducted in this thesis seeks to determine an appropriate regulatory response in relation to the use of pre-implantation tissue-typing for the creation of ‘saviour siblings’. The proposals outlined in the last part of this thesis seek to address the concern that harm may result from the practice of pre-implantation tissue-typing. The current regulatory frameworks in place are also analysed on the basis of the harm framework established in this thesis. The material referred to in this thesis reflects the law and policy in place in Australia and the UK at the time the thesis was submitted for examination (December 2009).

Mestrado em Biologia Molecular e Celular<br>Mundialmente é estimado que aproximadamente 70 milhões de casais tenham problemas de infertilidade, o que corresponde a um em cada sete casais em idade reprodutiva. O rápido aumento de problemas reprodutivos nas últimas décadas sugere uma maior probabilidade deste aumento ser devido a factores do estilo de vida e/ou ambientais, do que resultado de uma variação genética. Em Portugal 2,2% dos bébes nascidos resultam de técnicas de reprodução medicamente assistida. A transferência de vários embriões realizada, por vezes, no decurso destas técnicas pode resultar em gravidezes múltiplas, o que advém em complicações bem conhecidas para as mães e os bébes. São necessárias novas ferramentas de diagnóstico para que se possa transferir menos embriões com resultados similares ou melhores. Neste estudo foi investigado o impacto de vários factores do estilo de vida no potencial reprodutivo de 47 casais que recorreram a técnicas de reprodução medicamente assistida. Para além disso, foi realizada também, a correlação entre os níveis de expressão de um marcador da apoptose (caspase-3 clivada) nas células do cumulus do ovócito e a obtenção de uma gravidez (n=30). Uma concentração significativamente (p<0.01)) maior de caspase-3 clivada foi observada nas células do cumulus dos casais que não obtiveram uma gravidez. Dada a dificuldade de obter respostas reais nos questionários dos voluntários e a pequena dimensão da amostra para avaliar parâmetros com tanta variação inter-individual, o estudo não conseguiu obter resultados estatísticamente significativos na correlação do impacto de factores do estilo de vida no potencial reprodutivo. O estudo permitiu concluir que o nível de caspase-3 clivada nas células do cumulus parece ser um bom marcador da qualidade ovocitária e um bom predictor do resultado de gravidez.<br>Worldwide it is estimated that approximately 70 million couples suffer from infertility, which corresponds to one in each seven couples at fertility age. The rapid increase in reproductive problems in recent decades suggests that they are more likely to be caused by lifestyle and/or environmental factors than as a result of genetic variations. In Portugal 2.2% of the babies born result from an assisted reproduction technology (ART) technique. The multiple embryo transfer performed sometimes in ART techniques may result in multiple pregnancies, which have well known complications for mothers and babies. New diagnostic tools are needed to improve embryo selection, in order to transfer less embryos to achieve similar or better results. In this study the impact of lifestyle factors on the reproductive potential of 47 couples who resort to ART was investigated. Also, the correlation between the expression levels of an apoptotic marker (cleaved caspase-3) in oocytes cumulus cells and the achievement of pregnancy was performed (n=30). Significant (p<0.01) higher concentration of cleaved caspase-3 was observed in cumulus cells of couples who did not achieve pregnancy. Given the difficulty in obtain reliable answers from the volunteers in the questionnaires and the small sample size to evaluate parameters with such a wide inter-subject variability it failed to give conclusive statistical significant data in the lifestyle impact into reproductive potential. The present study allowed concluding that the level of cleaved caspase 3 in cumulus cells appears to be a good marker of oocytes quality and predictor of pregnancy outcome.