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

Infertility affects 15% of reproductive-aged couples. The milieu surrounding the growing embryo is of outmost importance, and should be optimised during in vitro fertilisation (IVF). Many biological processes, such as angiogenesis, coagulation, and immune processes need to be well regulated for a pregnancy to occur and progress normally. Histidine-rich glycoprotein (HRG) is a plasma protein that regulates components of these systems by building complexes with various ligands. A single nucleotide polymorphism (SNP) in HRG, denoted HRG C633T, seem to be of importance for IVF treatment outcomes. The aim of this thesis was to further investigate the proposed human fertility effects of the HRG C633T SNP. According to the findings of this thesis, the HRG C633T genotype is associated with primary recurrent miscarriage. Male HRG C633T genotype is associated with semen characteristics in infertile men, and pregnancy rates following IVF. However, the distribution of the HRG C633T SNP does not differ between infertile and fertile couples. We further examined the role of the region surrounding the HRG C633T SNP for regulation of endometrial angiogenesis and human embryo development. The region affects primary endometrial endothelial cell migration, proliferation and tube-formation in vitro but does not appear to affect human embryo development. No effect of the HRG peptide was noted on the secretome of human embryos. However, early embryos secrete proteins into the surrounding culture media and the level of secretion of VEGF-A, IL-6, EMMPRIN and PlGF is greater in embryos of higher developmental stages. In conclusion, the HRG C633T genotype appears to play a role only if infertility is established. The region surrounding HRG C633T SNP is of relevance in vitro for regulation of human endometrial endothelial cell angiogenesis. To predict which embryos to transfer in IVF, we have highlighted a number of proteins of interest for further investigation.