This study was conducted to investigate the requirement for sperm processing in microsurgical subzonal placement of sperm in rabbit oocytes. Fertilization rates with standard in vitro fertilization and microsurgical subzonal sperm placement were found to be similar (56 and 55%) when sperm treated with high-ionic strength Brackett's defined inedium to initiate capacitation were used. Statistically significant reductions in fertilization rates for both standard in vitro fertilization and subzonal placement were noted when twice-washed spermatozoa were used. Initiation of capacitation of spermatozoa results in higher fertilization results even when the zona pellucida is bypassed during fertilization.

Thesis (MMed)--Stellenbosch University, 2105. / ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI), as well as other micromanipulation assisted reproductive technology methods, such as physiologic ICSI (PICSI) and intracytoplasmic morphologically selected sperm injection (IMSI), are routinely used in many fertility laboratories around the world. An integral part of these methods is the manipulation of spermatozoa in preparation of the injection into the oocyte. It is common practice to place prepared spermatozoa in a viscous holding medium to facilitate the handling, manipulation and slowdown of spermatozoon movement during the immobilization and injection processes of ICSI. The possible effect of these holding mediums on basic semen parameters, as well as the sperm deoxyribonucleic acid (DNA) and structural integrity of spermatozoa, is of importance. Hamilton Thorne IVOS® developed an automated software solution for live sperm morphology evaluation under high magnification, called IMSI StrictTM. It combines Tygerberg Strict Criteria morphological classification of human spermatozoa with motile sperm organelle morphology examination (MSOME) and provides software-based categorization. The IMSI StrictTM software was developed to aid in the IMSI spermatozoon selection process that enables objective classification of spermatozoa to remove inter-technician variation. For good optics and spermatozoon evaluation in IMSI StrictTM, spermatozoa need to be moving very slowly or be immotile, but still viable. This can be achieved by placing spermatozoa in a viscous holding medium, either polyvinylpyrrolidone (PVP) or SpermSlowTM, sometimes for a substantial time period. Before marketing the clinical use of IMSI StrictTM, the possible toxicity or deleterious effect of PVP and SpermSlowTM on spermatozoa needs to be excluded. The primary objective of this study was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa after different exposure times using a viability stain, CASA motility and kinetic parameters, chromatin packaging analysis (CMA3 staining analysis) and DNA fragmentation analysis (TUNEL analysis). The secondary objective was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa‟s ultrastructure with Transmission Electron Microscopy. This prospective analytical study was conducted at Drs Aevitas Fertility Clinic (Vincent Pallotti Hospital, Cape Town, South Africa) as well as the Fertility Unit at Tygerberg Hospital (Cape Town, South Africa) between July 2013 and October 2014. A total of 90 separate (no duplication) semen samples were analysed for the quantitative analysis (primary objective) and 1 sample for the descriptive analysis (secondary objective). Results showed that although PVP and SpermSlowTM treated sperm outcomes often differed significantly after typical statistical analysis, clinically these two mediums were shown to be equivalent (using a specific statistical test for equivalence) for the tested outcomes. PVP and SpermSlowTM had no detrimental effect clinically on sperm viability, motility parameters, chromatin packaging and DNA fragmentation rate. The secondary investigation indicated that SpermSlowTM might exert a disintegrating effect on various sperm membranes, and as a secondary consequence of the eventual necrotic process, alteration of chromatin and cytoskeletal components. PVP medium on the other hand did not show these disintegrating effects. This finding needs to be further investigated since only one semen sample was evaluated. Based on this study‟s results, either PVP or SpermSlowTM can be used for IMSI StrictTM purposes. However, the study did not include the technical aspects of the usage of PVP and SpermSlowTM. / AFRIKAANSE OPSOMMING: Intrasitoplasmiese sperm inspuiting (ICSI), sowel as ander mikro-manipulasie voortplantings tegnieke, soos fisiologiese ICSI (PICSI) en intrasitoplasmiese morfologies geselekteerde sperm inspuiting (IMSI), word in baie fertiliteitsklinieke regoor die wêreld gebruik. 'n Integrale deel van hierdie metodes is die manipulasie van spermatosoa ter voorbereiding van die inspuitproses. Dit is algemeen om voorbereide spermatosoa in 'n viskose medium te plaas om die hantering, manipulasie en vertraging van spermatosoön beweging tydens die immobilisasie en inspuitproses van ICSI te fasiliteer. Die effek van hierdie mediums op basiese semenparameters, sowel as die sperm deoksiribonukleïensuur (DNS) en strukturele integriteit van spermatosoa, is van belang. Hamilton Thorne IVOS® het 'n sagteware oplossing, IMSI StrictTM, vir lewende sperm morfologie evaluering onder hoë vergroting ontwikkel. Hierdie sagteware bied sagteware-gebaseerde morfologiese klassifikasie deur die Tygerberg streng kriteria morfologiese klassifikasie met beweeglike spermorganel morfologie ondersoek (MSOME) te kombineer. Die IMSI StrictTM sagteware is ontwikkel om die objektiewe klassifikasie van spermatosoa vir IMSI spermatosoön seleksie moontlik te maak. Spermatosoa moet baie stadig beweeg of immotiel, maar steeds lewensvatbaar wees om goeie optika en spermatosoön evaluering vir IMSI StrictTM te verseker. Dit sal bereik kan word deur spermatosoa in 'n viskose medium, hetsy PVP (“polyvinylpyrrolidone”) of SpermSlowTM, vir 'n aansienlike tydperk te inkubeer. Voordat IMSI StrictTM vir kliniese gebruik bemark kan word moet die moontlike toksisiteit of nadelige effek van PVP en SpermSlowTM op spermatosoa uitgesluit word. Die primêre doel van hierdie studie was om die effek van PVP en SpermSlowTM op menslike spermatosoa na verskillende inkubasie tye te evalueer deur ʼn lewensvatbaarheid kleuring toets, twee sperm DNS toetse (CMA3 en TUNEL) en rekenaar geëvalueerde sperm beweeglikheid toetse te gebruik. Die sekondêre doel was om die effek van PVP en SpermSlowTM op menslike spermatosoa se ultrastruktuur deur middel van Transmissie Elektronmikroskopie te evalueer. Hierdie studie is by Drs Aevitas Fertiliteitskliniek (Vincent Pallotti Hospitaal, Kaapstad, Suid-Afrika) sowel as die Fertiliteitseenheid by Tygerberg Hospitaal (Kaapstad, Suid-Afrika) tussen Julie 2013 en Oktober 2014 uitgevoer. 'n Totaal van 90 semenmonsters vir die kwantitatiewe analise (primêre doel) en een vir die beskrywende analise (sekondêre doel) is ontleed. Resultate het getoon dat alhoewel PVP en SpermSlowTM geïnkubeerde spermuitkomste dikwels na ʼn tipiese statistiese analise betekenisvol verskil, hierdie twee mediums vir die geëvalueerde uitkomste klinies ekwivalent (bepaal deur middel van spesifieke statistiese toetse vir ekwivalensie) is. Die mediums het ook nie klinies 'n nadelige effek op sperm lewensvatbaarheid, beweeglikheid parameters, chromatien verpakking en DNS fragmentasie koers getoon nie. Die sekondêre ondersoek het getoon dat SpermSlowTM hoofsaaklik 'n effek van disintegrasie op verskeie spermmembrane getoon het. Hierdie nekrotiese proses kan lei tot verandering van chromatien en sitoskelet komponente. PVP medium het egter nie dieselfde disintegrerende effek getoon nie. Hierdie bevinding moet egter verder ondersoek word, aangesien slegs een semenmonster geëvalueer is. Alhoewel hierdie studie nie die tegniese aspekte van die gebruik van PVP en SpermSlowTM geëvalueer het nie, kan aanbeveel word dat óf PVP óf SpermSlowTM op grond van geëvalueerde uitkomste tydens die IMSI StrictTM sperm seleksie proses gebruik word.

No description available.

No description available.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2013-09-29 23:45:35.395

One in six couples wishing to start a family are infertile. The many causes of infertility include genetic defects that can be single gene, multifactorial or chromosomal (including Y deletions, karyotype abnormalities and gamete aneuploidy). This thesis is concerned with the association between infertility and increased sperm aneuploidy. Specific questions are: should males be screened for sperm aneuploidy before intracytoplasmic sperm injection (ICSI)? Is there a relationship between individual semen parameters and sperm aneuploidy for specific chromosome pairs? What is the role of genome organisation in male gametes and its association with infertility? Whether use of alternative therapy (in this case, traditional Chinese Medicine (TCM)) can be used to improve sperm disomy levels. Statistical analysis of questionnaire data revealed that infertility specialists believed there to be merit in screening sperm aneuploidy levels before ICSI. Evidence is presented for possible chromosome-specific and semen parameter specific mechanisms for sperm aneuploidy as is evidence of genome organisation that may be perturbed in infertile males. Finally, in six males studied, sperm aneuploidy levels improved significantly coincident with TCM. Closer investigation of the biological activity of individual therapeutic herbs and treatment cocktails revealed strong anti-oestrogenic and anti-oxidant properties. This suggests a possible mechanism of action of the herbs and provides the basis from which future placebo controlled clinical trials might continue. Possible criticisms of the work presented here include the unavailability of blood samples from many of the patients (thus preventing karyotype analysis) and the absence of a second control group in our studies on semen parameters. Nevertheless significant steps have been made towards establishing the need for, and the implementation of, a pre-ICSI screening test. Moreover progress has been made towards further understanding the aetiology of sperm aneuploidy and towards the implementation of a new treatment that may, ultimately, augment, or even replace ICSI.

Aujourd’hui, au moins 5 millions d’enfants à travers le monde, sont nés suite au recours de leurs parents à l’AMP. Les traitements de l’infertilité ont significativement évolué, le plus souvent cela a eu lieu en dehors des protocoles expérimentaux classiques. L’exemple le plus marquant a été l’introduction de la FIV avec micro-injection intracytoplasmique d’un spermatozoïde (ICSI). Le manque d’évaluation de la santé des enfants nés de ces techniques reste la faiblesse de cette spécialité. Nous avons mis en place un suivi longitudinal d’une cohorte mono-centrique au sein de l’hôpital Saint-Joseph (Marseille). Le recueil a été fait par la collecte des photocopies des pages du carnet de santé des enfants et de questionnaires remplis par les parents. Notre étude est une des rares études françaises présentant un suivi à long terme, pouvant aller jusqu’à 5 ans, sur une cohorte à grande échelle. L’étude de l’IMC jusqu’à l’âge de 5 ans, n’a pas révélé d’effet de la FIV, comme cela a pu être pressenti dans la littérature. D’autres investigations méritent d’être conduites. Il est important de construire un système d’information cohérent autour de la santé des enfants nés après FIV à cause de l’apparition constante des nouvelles techniques dans cette spécialité, toutes étant potentiellement responsables de risques sur la santé future de l’enfant. La faisabilité de la collecte de données couvrant à la fois l’environnement maternel, conceptionnel et les indicateurs de santé de l’enfant doit être pensée à l’échelle nationale. A cette fin le développement des méthodes de liaison entre les différents registres existants en France serait une des solutions les plus opportunes. / Today, at least 5 million children worldwide were born following the enrollment of their parents in ART program. Infertility treatments have changed significantly; most often these changes took place outside traditional experimental protocols. The most striking event was when IVF with intracytoplasmic sperm injection (ICSI) was introduced in ART practices in 1995. The lack assessment of the health of children born after this technique remains the major weak in this discipline. We established a longitudinal monocentric follow-up study in Saint-Joseph Hospital (Marseille). The data were collected by asking parents to send copies of child health records and questionnaires filled out by them. This investigation is one of the few French studies involving a long-term follow- up to 5 years, in a large scale cohort. The study of BMI up to age 5 years didn’t show the suspected epigenetic influence of IVF reported in literature. Further investigations need to be conducted. It is important to build a coherent information system around the health of children born after IVF. The feasibility of collecting a series of data covering both maternal and conceptional environment, and child health indicators should be considered at the national level through the development of connection methods between different registers developed in France.

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

Male infertility may be due to oligozoospermia, asthenozoospermia and teratozoospermia. Intracytoplasmic sperm injection is used to address male infertility. However, the percentage of viable embryos obtained by this technique is very low. Pronucleus formation has been identified as one of the key events in fertilisation and gamete decondensation is vital for this process to take place. Decondensation can be initiated by chemicals such as DTT that reduce the disulphide groups between the protamine proteins that keep the DNA of the gamete condensed. An increase in decondensation should translate into a higher fertilization rate and a higher yield of embryos. The research from this thesis has compared the decondensation ability via DTT in human spermatozoa and bovine spermatozoa, to study pronucleus formation in bovine zygotes and bovine embryo formation in the presence of DTT; and lastly the cytotoxic effect of DTT using somatic cells in culture has been investigated. In this study 12 semen samples for either fertile or subfertile subjects were collected, isolated and exposed to 25 mM DTT for 0, 5, 7, and 10 minutes, washed and the morphological changes associated with decondensation was evaluated by phase contrast microscopy. After 5 and 7 minutes 11 of the 12 samples underwent decondensation while after 10 minutes several samples showed a lower rate of decondensation and this was associated with and unusual hypercondensed state, CMA3 staining revealed all spermatozoa samples evaluated were mature. However, after treatment with DTT for 5, 7 and 10 minutes an increase in fluorescence was observed indicating increased protamine thiol group reduction and subsequently increased CMA3 accessibility. For some samples reduced fluorescence was observed possible due to the supercoiling of the DNA. DTT successfully induces decondensation of human spermatozoa, however does this lead to the formation of viable embryos? Due to ethical issues associated with working with human embryos all further studies were done using bovine embryos. Spermatozoa used were derived from Friesian bulls and the samples were pooled to prevent sample bias and interindividual variation. Spermatozoa were exposed to 25 mM of DTT at 5, 7, and 10 minutes as used for human spermatozoa. No decondensation was observed using the same conditions as for human spermatozoa, therefore the ‘swim up’ medium containing heparin and regularly used in IVF procedures for bovines was used, and this resulted in successful decondensation of bovine spermatozoa after 30 minutes. The effects of DTT on pronucleus formation and embryo development were evaluated in three bovine specimens. In the first group, DTT had no significant effect on the parameters measured, namely the number of oocytes that were in metaphase II, with one pronucleus, with two pronuclei, with degeneration of the nucleus and polyspermia. In the second group the percentage cleavage and embryo formation was determined on Day 1 (group 2) and 7 (group 3) respectively and statistical differences were obtained between the control and the DTT group. DTT had no significant effect on all the early parameters measured however later in development DTT had a significant adverse effect on cleavage and eventual embryo development. <p)Cleavage and embryo formation is a process of multiple mitotic divisions resulting in an increase in the number of cells that become smaller with each cell division, while somatic cells also undergo mitotic division although the cell size remains constant. Therefore the L929 cell line, a standardized system used to test toxicity, can be used to investigate the toxic effects of DTT on a dividing cell population. In this study L929 cells were expose to 25mM DTT for 30 minutes, and lysosomal membrane integrity, cell viability and number was determined immediately following exposure and after 48 hours growth. In another experiment the L929 cell line was exposed to all concentrations used in this and other studies for 5, 10 and 20 minutes. At all concentrations and exposure times DTT was found to be cytotoxic to the L929 cell line. How exactly DTT mediates this toxic effect is unknown, however due to its high solubility DTT can cross the cell membranes. The tertiary structure of proteins, enzymes and DNA is vulnerable to the reducing effects of DTT. In conclusion, although DTT induces decondensation in human and bovine spermatozoa, in the bovine model it does not lead to viable embryo formation and this has been confirmed in cell culture where DTT at all concentrations used was found to be cytotoxic. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted