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Sperm DNA fragmentation : implications in assisted reproductive technologiesHoogendijk, Christiaan F. (Christiaan Frederik) 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Male fertility has for many years been defined in vitro as the ability of sperm to fertilize
oocytes and to obtain early cleavage-stage embryos. Spermatozoa comprise of an
extraordinary high percentage of polyunsaturated fatty acids in their plasma membrane.
Due to an extremely low content of cytoplasm, sperm cells have a particularly low
potential to scavenge reactive oxygen species (ROS), and are therefore highly sensitive
to oxidative processes, which lead to sperm nucleus DNA damage/fragmentation.
Normally, DNA fragmentation occurs in every ejaculate and can be induced by an
excessive ROS production of active leukocytes or the spermatozoa themselves. Under
distressed conditions, DNA fragmentation may also occur in the testis as a result of
oxidative processes in the apoptotic cascade. These DNA fragmentations can be
regarded as late signs of programmed cell death (apoptosis).
Clinically, DNA fragmentation in spermatozoa results in significantly decreased
implantation and pregnancy rates especially in patients with oligo- and/or
teratozoospermia. The p-pattern normal sperm morphology has been shown to give
poorer fertilization rates in vitro than the g- and n-patterns. In this study there is
reported on the significant correlation found between the p-pattern normal sperm
morphology and sperm DNA fragmentation as measured with the terminal
deoxynucleotidyl transferase-mediated dUDP-biotin end labeling (TUNEL) assay. This
finding further explains the lower fertility potential of patients presenting with p-pattern
normal sperm morphology.
In addition, this study explores the intricate relations between ROS in the semen, DNA
fragmentation of the spermatozoa, as measured with the TUNEL assay and the sperm
chromatin structure assay (SCSA ), spermatozoa apoptotic status and sperm
parameters as measured with a standard semen analysis. Positive correlations were
found between ROS and the apoptotic status of the sperm, as well as between sperm
with non-fragmented DNA and sperm concentration and percentage motility. The
results emphasize the importance of sperm selection especially when the treatment of
choice is intracytoplasmic sperm injection (ICSI). An early sign of programmed cell death, also known as apoptosis, is the externalization
of phosphatidylserine (PS) from the inner membrane leaflet to the outer leaflet. PS
shows a high affinity to Annexin V. Apoptotic spermatozoa are able to fertilize oocytes,
but embryo senescence may occur at the time when the paternal genes are activated.
In this study there is reported on a novel method whereby spermatozoa can be
separated on the basis of their apoptotic status through flow cytometry. Results showed
that the normal sperm morphology, according to strict criteria, of the resultant nonapoptotic
sperm fraction is significantly higher than that of the apoptotic counterpart.
With refinement of this technique, it will be possible in future to use these separated
non-apoptotic sperm cells during ICSI for fertilization.
From the above it is apparent that the spermatozoon has to play a vital role in the
development of the embryo from fertilization to implantation and pregnancy. It is,
however, important to note that besides the gametes, there are other critical factors
which contribute to a successful in vitro fertilization (IVF) cycle, among these are the in
vitro culture conditions. In this regard, this study compared two sequential embryo
culture systems. It was found that the more complex medium resulted in better day
three embryo quality and a better blastocyst formation rate and pregnancy rate.
These findings highlight the importance of a holistic perspective towards the complexity
of the factors involved in affecting embryo quality and pregnancy outcome. / AFRIKAANSE OPSOMMING: Manlike fertiliteit is vir baie jare gedefinieer as die in vitro vermoë van ‘n spermsel om ‘n
eiersel te bevrug om sodoende embrios te verkry. Die spermsel se plasmamembraan
bestaan uit ‘n hoë persentasie poli-onversadigde vetsure. As gevolg van die klein
hoeveelhede sitoplasma van die spermsel het dit ‘n beperkte weerstand teen reaktiewe
suurstof spesies (ROS) en is gevolglik baie sensitief vir oksidasie. Oksidasie lei tot
DNS skade/fragmentasie. DNS fragmentasie kom in spermselle van alle ejakulate voor
en is gewoonlik die gevolg van ROS produksie deur die leukosiete in die semen of
vanaf die spermselle self. Onder sekere omstandighede kan DNS fragmentasie ook
voorkom in die testis waar dit deel vorm van apoptose. Hierdie tipe DNS skade word
gesien as laat tekens van geprogrammeerde seldood (apoptose).
In oligo- en/of teratozoospermiese mans lei DNS fragmentasie tot verlaagde
implantasie- en swangerskapssyfers. Die p-patroon normale sperm morfologie groep
gee laer in vitro bevrugting en swangerskapsyfers as die g- en n-patrone. In hierdie
studie doen ons verslag oor die statisties betekenisvolle korrelasie wat gevind is tussen
die p-patroon normale sperm morfologie en DNS fragmentasie soos gemeet met die
‘terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling’ of te wel
TUNEL toets. Hierdie bevinding is ‘n verdere verklaring vir die laer fertiliteits potensiaal
van pasiënte wat voordoen met p-patroon sperm morfologie.
‘n Verdere doel van die studie was om die moontlike verband tussen ROS in die semen,
spermatozoa DNS fragmentasie, apoptotiese status van die sperms en die motiliteits
parameters van die spermatozoa te bepaal. ‘n Positiewe korrelasie is gevind tussen
ROS en sperm apoptotiese status. Sperms met ongeframenteerde DNS is ook positief
gekorreleer met sperm konsentrasie en motiliteit. Die resultate beklemtoon die
belangrikheid van spermseleksie veral in pasiënte waar die keuse van behandeling
intrasitoplasmiese sperm inspuiting (ICSI) is.
‘n Vroeë teken van apoptose is die eksternalisering van ‘phosphatidylserine’ (PS) vanaf
die interne oppervlakte van die plasmamembraan na die eksterne oppervlak. PS het ‘n
hoë affiniteit vir Annexin V. Apoptotiese sperms het die vermoë om ‘n oösiet te bevrug, maar kan lei tot die staking van embrio deling wanneer die vaderlike gene ‘n rol begin
speel in embrio ontwikkeling. In hierdie studie het ons ‘n nuwe metode ontwikkel
waarvolgens die spermatozoa in die ejakulaat op grond van hul apoptotiese status
geskei kan word in apoptotiese en nie-apoptotiese fraksies. Die normale sperm
morfologie van die nie-apoptotiese fraksie is betekenisvol beter as dié van die
apoptotiese fraksie. Verdere verfyning van die tegniek kan daartoe lei dat dit in die
toekoms toegepas kan word om vir nie-apoptotiese sperms te selekteer veral voor die
uitvoering van ICSI.
Uit die bogenoemde is dit duidelik dat die spermsel ‘n baie belangrike rol in die
ontwikkeling van ‘n embrio, vanaf bevrugting tot implantasie en swangerskap, speel. Dit
is egter ook belangrik om in gedagte te hou dat daar ander bydraende faktore tot ‘n
suksesvolle in vitro swangerskap is, soos laboratorium toestande en embrio
kultuursisteem. Om hierdie rede is daar ook twee kultuurmedia in hierdie studie
vergelyk. Daar is bevind dat die meer komplekse medium beter kwaliteit embrios op
dag drie lewer, asook meer blastosiste en ‘n hoër swangerskapsyfer.
Dit is dus duidelik dat dit uiters belangrik is om ‘n holistiese perspektief te hê op die
komplekse faktore wat ‘n invloed mag hê op bevrugting, embrio kwaliteit asook die
swangerskapsyfer.
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Evaluation of gamete dysfunction as a cause of failed human in vitro fertilizationEsterhuizen, Aletta Dorothea 12 1900 (has links)
Thesis (D.Phil.)--Stellenbosch University, 2000. / 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. / 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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The role of sperm morphology in assisted reproduction (ART)Kruger, Theunis Frans 12 1900 (has links)
Thesis (DSc)--Stellenbosch University, 2012. / Please refer to full text for abstract.
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The evaluation of different embryo markers and their subsequent effect on embryo development, implantation and pregnancy outcome in an in-vitro fertilization programKotze, Dirk Jacobus 12 1900 (has links)
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.
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Human endometrial gene expression profiling and receptivity in patients undergoing in vitro fertilization (IVF) treatmentLiu, Yunao., 劉蘊奡. January 2009 (has links)
published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy
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HÁBITOS ALIMENTARES EM MULHERES SUBMETIDAS AO TRATAMENTO DE FERTILIZAÇÃO IN VITRO E A PROMOÇÃO DA SAÚDE NA AGENDA 21 DE GOIÂNIAOliveira, Lídia Regina Zanatta de 28 February 2008 (has links)
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Previous issue date: 2008-02-28 / This study had as objective to analyze the alimentary habits, the body mass index
(BMI) and some varied of the women's treatment submitted to in vitro fertilization
by the technique of ICSI and to stand out the presence of actions of promotion of
alimentary habits in Agenda 21 of Goiânia. The present idea is reinforced in the
Agenda 21 that the health promotion and the nutritional surveillance are of
extreme importance for the quality of life of the population in Goiânia. The
research was accomplished in a clinic specialized in human reproduction located
in Goiânia in the period of September of 2006 to April of 2007. The alimentary
ingestion was evaluated by the reminding of 24 hours through which was possible
to observe the macro and micronutrients ingestion, as well as the presence of the
fat acid, glutamic acid and caffeine. BMI was calculated through the measures of
height and weight. The program DietPRO was used to convert homemade
amounts in measures in ingestion levels. The variables of the treatment were
obtained from the medical handbook after the procedures. For the statistical
analysis of the data the program was used MATLAB version 6.0, the statistical
method used to correlate the nutritional variable with the one of the treatment the
coefficient of Pearson s correlation. The correlations were classified as: positive or
negative light, acceptable, moderate, substantial and almost perfect. The results
showed that BMI, proteins, caffeine, copper, vitamin E, zinc and carbohydrates
had a moderate correlation with the gonadotropins levels used in the induction of
the ovulation. BMI, copper, vitamin E and zinc had moderate correlation with the
days of induction of the ovulation. The number of follicles, observed in the last
analysis had a moderate correlation with the potassium and with the folic acid. The
folic acid still presented a correlation moderated with the oocyte number, identified
after the collection. The correlation among the nutrients and the oocyte number
classified as immature (Vg and M1) it was mainly negative except for the sodium,
manganese and of the proteins, however these correlations just presented
acceptable values. The mature oocyte (M2) had a moderate correlation with the
calcium and folic acid. As the results found in this research are not conclusive, is
suggested some study to confirm the relationship between human reproduction
and alimentary habits. / Este estudo teve como objetivo analisar os hábitos alimentares, o índice de
massa corporal (IMC) e algumas variáveis do tratamento de mulheres submetidas
à fertilização in vitro pela técnica de ICSI e ressaltar a presença de ações de
promoção de hábitos alimentares na agenda 21 de Goiânia. Reforça-se a idéia
presente na agenda 21 de que a promoção da saúde e a vigilância nutricional é
de extrema importância para a qualidade de vida da população de Goiânia. A
pesquisa foi realizada em uma clínica especializada em reprodução humana
assistida localizada em Goiânia no período de Setembro de 2006 a Abril de 2007.
Avaliou-se a ingestão alimentar através de um recordatório de 24 horas onde se
pode observar a ingestão de macro e micronutrientes, bem como a presença dos
ácidos graxos, ácido glutâmico e cafeína. O IMC pode ser calculado através da
aferição das medidas antropométricas (altura e peso). O programa DietPRO foi
utilizado para converter quantidades caseiras em medidas em níveis de ingestão.
As variáveis do tratamento foram obtidas diretamente do prontuário das pacientes
no final do tratamento. Para a análise estatística dos dados foi utilizado o
programa MATLAB versão 6.0, sendo o método estatístico empregado para
correlacionar as variáveis nutricionais com as do tratamento o coeficiente de
correlação de Pearson com nível de significância de 5%. As correlações foram
classificadas como positivas ou negativas leve, aceitável, moderada, substancial e
quase perfeita. Os resultados mostraram que o IMC, proteínas, cafeína, cobre,
vitamina E, zinco e carboidratos tem uma correlação moderada com os níveis de
gonadotrofinas utilizados na indução da ovulação. O IMC, cobre, vitamina E e
zinco apresentaram correlação moderada com os dias de indução da ovulação. O
número de folículos, observados na última análise ecográfica, apresentou uma
correlação moderada com o potássio e com o ácido fólico. O ácido fólico ainda
apresentou uma correlação moderada com o número de oócitos identificados
após a coleta. A correlação entre os nutrientes e o número de oócitos
classificados como imaturos (Vg e M1) foi predominantemente negativa com
exceção do sódio, manganês e das proteínas, no entanto estas correlações
apresentaram apenas valores aceitáveis. Os oócitos maduros (M2) apresentaram
uma correlação moderada com o cálcio e com o ácido fólico. Este trabalho abre
caminho para novas pesquisas na área da reprodução humana assistida que
aprofundam a importância dos hábitos alimentares de pacientes que irão se
submeter ao tratamento de fertilização in vitro.
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The in vitro produced cow embryo : factors affecting development and metabolismSteeves, Tracey Elizabeth, 1968- January 2000 (has links)
Abstract not available
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Factors affecting the developmental competence of pig oocytes matured in vitro.Bagg, Melanie Anna January 2007 (has links)
Pre-pubertal pig oocytes possess lower developmental competence than those from adult pigs following in vitro maturation (IVM). Previous studies have demonstrated that exposure of pre-pubertal oocytes to 1 mM dibutyryl cAMP (dbcAMP), a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, for the first 20 h of IVM improves the rate of blastocyst development. Developmental competence of in vitro matured pig oocytes has been reported to increase with increasing follicle size. In this thesis, experiments were carried out using pre-pubertal and adult pig oocytes to investigate the relationship between donor age, intra-oocyte cAMP level and follicle size in terms of oocyte maturation and developmental competence. These experiments demonstrated that, while ovarian, follicular and oocyte morphology are immediately altered with the onset of puberty, pre-pubertal oocytes must be exposed to more than the first oestrous cycle to achieve improved developmental competence in vitro. Later experiments demonstrated that pre-pubertal oocytes accumulate less cAMP during IVM, undergo more rapid meiotic progression and display reduced rates of blastocyst development compared to in vitro matured adult oocytes. Treatment with dbcAMP for 22 h IVM increased the cAMP content of pre-pubertal oocytes, slowed meiotic progression during IVM and improved the rate of blastocyst formation. While the cAMP concentration of pre-pubertal oocytes was increased to levels similar to that of adult oocytes, rates of blastocyst formation remained lower, suggesting that additional factor(s) are required for oocyte maturation. This thesis also examined the follicle size cohorts that make up the 3-8 mm aspiration range on pig ovaries. The surface of pre-pubertal ovaries contained around double the number of 3 mm follicles compared with adult ovaries. Blastocyst development of pre-pubertal oocytes increased with increasing follicle size and was highest using oocytes from 5-8 mm follicles, while adult oocytes from all follicle size cohorts displayed similar high rates of blastocyst formation. The interaction between follicle size and cAMP content in pre-pubertal oocytes was examined next. Cumulus-oocyte complexes (COCs) from 3 mm follicles accumulated less intra-oocyte and inter-COC cAMP and displayed reduced cumulus expansion compared with COCs from 5-8 mm follicles. While dbcAMP treatment increased the cAMP content of oocytes from 3 mm follicles, it had no effect on the cAMP content of the whole COC. These findings suggest that inadequate levels of intra-oocyte cAMP during IVM contribute to the low developmental competence of pre-pubertal oocytes from 3 mm follicles, suggesting that cAMP transfer, production or degradation processes are incomplete. Analysis of steroid content from different follicle size cohorts revealed that the progesterone content of prepubertal follicular fluid (FF) increased with increasing follicle size, yet overall was lower than that of adults. This suggests that differences may exist in the gonadotropinstimulated steroidogenic activity of granulosa cells of pre-pubertal COCs from different follicle sizes. Since progesterone secretion did not differ between pre-pubertal and adult COCs, it appears that the downstream pathway from the granulosa cell response rather than the actual quantity of progesterone is important for subsequent maturation processes. These studies then examined gap junction communication (GJC) within the pre-pubertal COC during IVM to examine whether the positive effects of increasing follicle size and dbcAMP on intra-oocyte cAMP levels relates to improved cAMP transfer between the cumulus cell layer and oocyte. Cumulus cell-oocyte GJC during IVM was maintained for a longer period in pre-pubertal COCs from 3 mm follicles than in those from 5-8 mm follicles. Treatment with dbcAMP had minimal effect on GJC in either COC type, thus the dbcAMP-induced increase in intra-oocyte cAMP levels appears independent of GJC. Differences in GJC during IVM together with the COCs ability to increase intraoocyte cAMP levels during IVM, suggests that differences may exist in the quantity of gonadotropin receptors, which are responsible for cAMP production, within the cumulus layer of COCs from 3 mm compared with 5-8 mm follicles. In conclusion, this thesis has demonstrated that an increase in intra-oocyte cAMP is necessary during maturation for completion and synchronisation of maturation and high developmental competence of the pig oocyte. Comparison of 3, 4 and 5-8 mm follicle sizes in the pre-pubertal pig, as described here, provides an excellent model for further investigation into the role of cAMP and the other factors required for co-ordination of oocyte nuclear and cytoplasmic maturation and subsequent embryo production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297309 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007
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Screening soybean genotypes for high temperature tolerance by in vitro pollen germination, pollen tube length and physiological techniquesSalem, Mohammed A., January 2005 (has links)
Thesis (Ph.D.) -- Mississippi State University. Department of Plant and Soil Sciences. / Title from title screen. Includes bibliographical references.
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Gene expression in preimplantation embryos of the mouse, pig and cow /Larson, Melissa Anne, January 2000 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.
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