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

Infertilitet är en utbredd problematik som drabbar många par världen över. Olika behandlingsformer finns att tillgå för att hjälpa infertila par att uppnå en graviditet. IVF-behandling är en vanlig behandlingsform. Många studier visar att IVF-behandling påverkar kvinnan mer än mannen psykiskt. Syftet med denna studie var att beskriva hur kvinnor upplever processen att genomgå IVF-behandling. Metod: En litteraturstudie gjordes på tio kvalitativa artiklar för att besvara syftet. En innehållsanalys gjordes på dessa artiklar, där meningsbärande enheter identifierades i artiklarnas resultat. Dessa enheter kondenserades, grupperades och formade sub-kategorier och teman. Livsvärldsperspektivet användes som en teoretisk referensram. Resultatet av denna litteraturstudie blev sju teman; Tro på behandlingen; Okunskap; Påverkan på vardagen; Bemötande i relationer; Negativ hantering; Rädsla, samt Väntan. Slutsatser som drogs av denna litteraturstudie var att kvinnornas upplevelser var övervägande negativt präglade, men en tro på behandlingen var ändå en central upplevelse. Det är viktigt för berörda kvinnors omgivning att inneha ett livsvärldsperspektiv inför kvinnornas upplevelser av behandlingen. En ökad förståelse från kvinnornas omgivning skulle kunna ge kvinnorna bättre upplevelser av att genomgå behandlingen. / Infertility is a widespread problem that affects many couples worldwide. Various treatments are available to help infertile couples achieve conception, and IVF is a common form of treatment. The psychological impact of the IVF-treatment affects the woman more than the man. The aim of this study was to describe how women experience the process of going through IVF treatment. Method: A literature review was done on ten qualitative articles to answer the aim of the study. A content analysis was done on these articles, and meaningful units were identified in the articles‟ results. These units were condensed, grouped and then formed into sub-categories and themes. A Life-World perspective was used as a theoretical framework. The result of this study was seven themes: Faith in the treatment; Ignorance: The impact on daily life; Responding in relationships, Negative handling, Fear, and Wait. Conclusions of this study were that women's experiences were predominantly negatively characterized, but a faith in the treatment was still a central experience. It is important for the women‟s surroundings to hold a Life-World perspective to women's experiences of the treatment. A greater understanding from the women's surroundings may improve the women‟s experiences of going through the treatment.

Reproduction in mammals is regulated by neurons that synthesize and secrete gonadotropin-releasing hormone (GnRH) and across the species these neurons are present in few numbers scattered in the hypothalamus. Due to limited neurogenesis of these neuronal cell types outside the brain in the olfactory placode, these neurons are subjected to tight regulation during embryonic development to reach their final targets in the hypothalamus, from birth until puberty for minimal secretion of hormone and during adults to achieve pulsatile secretion of the hormone. Deregulation in any of these mechanisms may lead to deleterious effects on adult reproduction and clinical pathologies like absence of puberty, hypogonadism, sterility, amenorrhea, etc. Kallmann syndrome (KS), one of these severe reproductive pathologies is an inherited disorder and patients affected with this syndrome display anosmia (inability to smell) and hypogonadotropic hypogonadism (HH). Genetic screening of molecules in these patients lead to identification of genes like KAL1, FGFR1, FGF8, PROK2, PROKR2, WDR11 and CHD7 encoding proteins that play an important role in migration and targeting of olfactory system during embryonic development however these genes account only for 30% of KS cases emphasizing the need for further characterization and identification of other genes. While these proteins are involved in ontogenesis olfactory and GnRH system, genetic screening of molecules in patients suffering from normosmic idiopathic HH lead to identification of genes encoding for Kisspeptin receptor-GPR54, TAC-TACR3, LEP-LEPR, PCSK-1, GnRH receptor-GnRHR and GnRH-1 itself that play a crucial role in occurrence of puberty or adult reproduction. Here, for my PhD thesis, we focused on studying the role of guidance molecule Semaphorin3A (Sema3A)-Neuropilin1 (Nrp1) interactions in ontogenesis of GnRH neurons during embryonic development while in adults we first addressed the question if hypothalamic Kisspeptin neurons interact with neurons containing neuronal nitric oxide synthase (nNOS), the mutation of which causes HH in mice, and physiological significance of this interactions in regulation of GnRH neurons and neuroendocrine control of female reproduction. Finally our results demonstrate that Sema3A-Nrp1 interactions are implicated in ontogenesis of olfactory and GnRH neurons during embryonic development and nNOS neurons are important mediators of peripheral estrogens-kisspeptin signaling onto GnRH neurons and adult reproduction and propose to further study the implication of nNOS neurons in reproductive pathologies.

Bakgrund: Infertilitet är när en man och kvinna under minst tolv månader av regelbundna oskyddade samlag inte lyckas åstadkomma en planerad graviditet. Det är ett vanligt problem som drabbar mellan tio till femton procent av alla par.  Syfte: Syftet var att beskriva män och kvinnors upplevelser av ofrivillig barnlöshet vid infertilitet. Samt att beskriva de valda artiklarnas undersökningsgrupp. Metod: En litteraturstudie. Datainsamling har skett från databaserna CINAHL, PubMED och PsycINFO. 14 kvalitativa vetenskapliga artiklar ligger till grund för denna litteraturstudie. Resultat: Infertila män och kvinnor upplevde många olika känslor av att leva med ofrivillig barnlöshet, både som par och individuellt. De upplevde ofta en förändring i relationen, både i kommunikationen och samlivet med sin partner. Infertila män och kvinnor hade ett ökat behov av stöd från sin partner, människor i sin omgivning samt sjukvården. För att de infertila personerna skulle kunna hantera sin situation och gå vidare i livet, var det viktigt för dem att använda sig av copingstrategier som t.ex. att finna andra saker i livet som var meningsfulla. Slutsats:Ofrivillig barnlöshet vid infertilitet påverkade hela människan, både individuellt och som par. Det var vanligt att personerna drabbades av en livskris i samband med infertiliteten, vilket upplevdes på många olika sätt. Utifrån den föreliggande litteraturstudien framkom det att sjuksköterskan hade ett ökat behov av att få bättre förståelse och kunskap om infertila personers upplevelser av ofrivillig barnlöshet, för att bättre kunna bemöta, stödja och informera dem på bästa sätt. / Background: Infertility is when a man and a woman for at least twelve months of regular unprotected intercourse fails to conceive a planned pregnancy. Infertility is a common issue affecting ten to fifteen percent of all couples. Aim: The aim was to describe men and women's experiences of involuntary childlessness caused by infertility, and to describe the selected articles study groups. Method: A literature study. Data was collected from the databases CINAHL, PubMed and PsycINFO. 14 qualitative scientific articles form the basis of this study. Result: Infertile men and women experienced many different emotions caused by their involuntary childlessness. They often experienced a change in their relationship, both in communication and intimacy with their partner. Infertile men and women often had a greater need for support from their partner, people in their surroundings and healthcare professionals. To be able to handle their situation and move on in life, it was important for infertile people to use coping strategies and find other meaningful things in life. Conclusion: Involuntary childlessness caused by infertility affected the men and women both individually as well as a couple. It was common to experience a life crisis associated with infertility, which was expressed in many ways. Based on the present literature study, nurses need to gain understanding and knowledge of the experiences infertile men and women can witness because of their involuntarily childlessness, to be able to better cherish and support these people in the best possible way.

Infertility is a frequently occurring chronic health condition, which often persists throughout the reproductive years. Heightened anxiety symptoms often are comorbid with infertility diagnoses. Women experiencing infertility, and particularly those with anxiety symptoms, characterize an emerging population that deserves special attention. However, women experiencing infertility have identified barriers to seeking psychotherapy (e.g., fears of being dismissed from fertility treatment and/or stigmatized). Consequently, women diagnosed with infertility need a psychotherapy that not only can reduce these symptoms, but can also be private and convenient. The current study translated an empirically tested in-person mind/body protocol into an internet-based intervention to suit the needs of this population. Seventy-one women were randomly assigned to the intervention or a wait-list control. At the close of the study, only three participants had completed the intervention. At mid-assessment, relative to the wait-list group, the intervention group had a lower level of depressive symptoms and, for those with elevated anxiety symptoms at baseline, a lower level of anxiety symptoms. The findings suggest that even a partially completed internet-based intervention can reduce the anxiety and depressive symptoms of women with a diagnosis of infertility.

This master thesis deals with infertility and its treatment. Both in the theoretical and empirical part, it focuses on the female aspects of the issue. The theoretical part summarizes an overview of key information important to understand the presented problems. We define the terms, describe the psychological and physiological causes of infertility, the process of treatment of infertility and aspects thereof. We focus on the depression rate related to infertility and its treatment, furthermore, we deal with type locus of control related to the experienced rate of depression. Our aim in the empirical part is to describe the depression rate, type of locus of control within the two samples of women acquired by us and capture the possible connections. The sample is made of 58 women who are currently undergoing a treatment for infertility and of the control group, which is made of 58 healthy women. To collect the data, we used the Beck Depression Inventory BDI-II, the Rotter's Internal- External Scale and the anamnestic questionnaire. Based on acquired data, we found out some specifity in depression rate and type of locus of control within our samples of women.

Le gène Spatial est exprimé par des cellules hautement polarisées : les cellules épithéliales du thymus, neuronales du système nerveux central et germinales du testicule. La différenciation de ces cellules est accompagnée d'une polarisation de la distribution de Spatial, dans des structures microtubulaires hautement organisées telles que la manchette, le flagelle et la dendrite.Mon projet a porté sur l’identification de la fonction du gène Spatial au niveau du thymus et du cerveau murins. De plus, j’ai été impliquée dans la caractérisation du gène Spatial chez l’homme et l’évaluation de son impact sur la spermatogenèse et l’infertilité humaine.Nos résultats montrent qu’au niveau du thymus, Spatial est détecté tout au long de l’organogenèse thymique jusqu’aux stades adultes. Son profil d’expression correspond à un « promiscuous gene » impliqué dans l’acquisition de la tolérance des lymphocytes T. Au niveau du système nerveux central, Spatial présente une distribution somatodendritique dans les cultures de neurones hippocampiques et son expression est fortement détectée lors de la poussée dendritique. Nous avons montré que le transport de Spatial du corps cellulaire vers les dendrites est dépendant de la kinésine Kif17. De plus, Spatial semble être impliqué dans la formation des dendrites via la voie de signalisation stimulée par le Nerve Growth Factor.Chez l’homme, le gène H-Spatial est fortement exprimé au niveau du testicule et son expression est spécifique de la spermiogenèse, étape de différenciation des spermatides rondes en spermatozoïdes. Chez les patients infertiles asthéno- et/ou tératozoospermiques, présentant des anomalies de mobilité et de forme des spermatozoïdes, le niveau d’ARNm d’H-Spatial est fortement réduit. Ces résultats suggèrent qu’H-Spatial est un marqueur potentiel de l’infertilité masculine.J’ai également participé à la validation des dendrimères PAMAM (poly-amidoamine) comme vecteurs efficaces pour le transfert de siRNA et d’ADN in vitro sur différents lignées cellulaires et in vivo sur des thymus murins. Ce système pourrait être un moyen thérapeutique pour traiter des immunodéficiences liées aux lymphocytes T. / Spatial gene is expressed in highly polarized cells such as, thymic epithelial cells, testicular germ cells and neuronal cells of the central nervous system. The differentiation of these cells is accompanied by the polarized distribution of Spatial in highly organized microtubule structures such as the manchette, the flagellum and dendrites. This project aims to identify the function of Spatial gene in the thymus and in the brain. Moreover, we characterize Spatial gene in humans and evaluate its impact on spermatogenesis and human infertility.Our results showed that, in the thymus, Spatial is detected throughout the thymic development, until adulthood. Its expression profile corresponds to a ‘promiscuous gene’, implicated in the acquisition of T lymphocyte tolerance.At the level of the central nervous system, Spatial showed a somatodendritic distribution in hippocampal neuron cultures. Moreover, its expression was highly detected during dendritic growth. We have also shown that the transport of Spatial from the cell body to the dendrites is dependent on the kinesin Kif17. In addition, our results suggest that Spatial seems to be implemented in dendrite formation by the Nerve Growth Factor mediated signaling pathway.In the humans, H-Spatial is highly expressed in the testis and its expression is specific to spermiogenesis: the phase of differentiation of round spermatids to spermatozoids. In infertile astheno and/or teratozoospermic patients with sperm shape and mobility anomalies, H-Spatial levels were drastically reduced. These results propose H-Spatial as a potential marker for human male infertility.Finally, we have equally participated in the validation of PAMAM (poly-amidoamine) dendrimers as efficient vectors for the transfer of DNA and siRNA in vitro, in different cell lines; and in vivo, in murine thymi. This system could serve as a new therapeutic model for treating diseases linked to T lymphocytes.

Master of Science / Department of Clinical Sciences / Maria S. Ferrer / Presence of anti-sperm antibodies (ASA) is associated with infertility in many species, including bulls but there is no standardized direct technique that allows detection of ASA bound to the sperm surface. The overall objective was to standardize a flow cytometric technique for detection of IgG and IgA directly attached to bovine sperm. The effects of fixation using phosphate buffer solution (PBS) or diluted formalin buffer solution (dFBS), exclusion of dead cells from the analysis, and aliquot variability were assessed using healthy bulls classified as Satisfactory Potential Breeders (SPB, n=9) and bulls with experimentally induced ASA (n=4) (Experiment1). The effect of freezing on the percentage of IgG- and IgA- bound sperm was assessed in samples from immunized bulls (n=4) (Experiment 2). Anti-sperm antibodies on the sperm surface were induced in yearling bulls by intramuscular injection of autologous semen and an adjuvant. Fixation of sperm cells did not affect the percentage of IgG- or IgA-bound sperm in any group of bulls. Exclusion of dead cell from the analysis did not affect the percentage of IgG-bound sperm (p= 0.0922 and p= 0.1525 for immunized and reproductively normal bulls, respectively). The exclusion of dead cells significantly increased the percentage of IgA-bound sperm in semen samples from immunized bulls (p= 0.0152) and significantly decreased the percentage of IgA- bound sperm in semen samples from reproductively normal bulls (p= 0.0012). Variability was < 10% in samples from immunized and reproductively normal bulls for percentage of IgG- and IgA-bound sperm. Freezing did not affect the percentage of IgG- (p=0.1287) or IgA-bound sperm (p=0.4175). Based on these results, fixation is neither necessary nor detrimental for analysis, and the percentage of antibody-bound cell should be calculated gated on the population of live cells only, especially when evaluating IgA binding. The percentage of ASA-bound sperm can be assessed on frozen-thawed samples. The development of this technique allows for further studies on ASA-bound sperm in populations of normal and abnormal bulls.

Cette recherche de thèse porte sur le travail réalisé avec des sujets demandant une prise en charge en médecine procréative. Nous envisageons la question du travail collaboratif entre psychanalyse et médecine dans le champ de l’assistance médicale à la procréation, sur la base de deux approches. La première, historique, porte sur l’évolution de la représentation culturelle de l’infertilité, et la seconde, clinique, d’inspiration psychanalytique, a été construite sur la base de notre expérience au sein d'un centre d’assistance médicale à la procréation et dans un cabinet privé. Nos hypothèses visent à montrer comment les pré-conceptions médicales et psychologiques barrent le dialogue entre le sujet demandant une prise en charge et celui qui l’accueille. À cet effet, nous envisagerons le processus d’objectivation subi par le concept de désir d’enfant, de plus en plus assimilé à celui de projet d’enfant, et nous abordons aussi les difficultés que l’infertilité pose aux hommes dans une époque où elle commence à être conçue comme une maladie du couple et non pas comme une maladie des femmes. À partir d’une mise à jour de la notion d’infertilité, telle qu’elle est comprise par la médecine et par la loi, et de la reprise des théorisations freudiennes autour du symptôme et de la pratique clinique, nous chercherons à montrer la valeur d’une perspective qui n’assume pas d’emblée que toute demande de prise en charge en médecine procréative est une demande d’enfant à la médecine. / This research focuses on work with subjects requiring care in medically assisted procreation. Based on two approaches: an historical one, on the evolution of the cultural representation of infertility. The other clinical, from a psychoanalytic perspective, developed working in a center of assisted reproduction and in private practice. Our work addresses the question of collaborative work between psychoanalysis and medicine in the field of reproductive medicine. Our hypotheses are intended to show how medical and psychological preconceptions difficult the dialogue between the subject requiring care and those who receive him. To this end we will consider the process of objectification suffered in the concept of desire for a child, now assimilated to that of the project of a child. We also address the challenges that infertility imposes on men when it begins to be seen as a couple’s disease and not only as women’s disease. From an update of the notion of infertility, as it is comprised now by medicine and law, and the recovering of Freudian theories about the symptom and clinical practice, we intend to show the value of a perspective that does not automatically assume the demand for care in reproductive medicine as a demand of a child to medicine.

L'Organisation Mondiale de la Santé définit l'infertilité comme une maladie et un échec de l'appareil reproducteur à parvenir à une grossesse après 12 mois ou plus de rapports sexuels réguliers non protégés. De nos jours, l'infertilité est devenue un phénomène commun affectant 1 couple en âge de procréer sur 5. Une origine idiopathique est le plus souvent associée à un système immunitaire actif qui pourrait produire des niveaux élevés d'anticorps anti-liquide séminal ou anti-sperme. L'auto-immunisation, aussi bien que l'iso-immunisation, joue un rôle significatif dans jusqu'à 30% des cas signalés. Le liquide séminal, qui est défini comme un fluide complexe contenant le sperme, les vésicules cellulaires et autres cellules et composantes, pourraient immuniser l'appareil génital féminin. Cette thèse est liée à l'infertilité féminine immune, en particulier à l'iso-immunisation féminine. Une meilleure compréhension de cette manifestation physiopathologique consiste en (1) la détermination des isotypes d'anticorps jouant un rôle significatif dans cette maladie, puis en (2) la caractérisation et l'identification des antigènes de liquide séminal ou de sperme reconnus par ces anticorps, (3) la proposition de marqueurs diagnostiques potentiels afin d'adapter des thérapies spécifiques, et, en outre, la conception d'un outil de diagnostic miniaturisé basé sur les marqueurs sélectionnés, (4) la suggestion d'une éventuelle immuno-intervention. En se fondant sur la distribution des isotypes d'anticorps spécifiques au liquide séminal/sperme, nous suggérons que les immunoglobulines E, M, A1,2, et G3 ne sont pas impliquées dans la sensibilisation physiopathologique chez les femmes. Les IgG4, semblent constituer la sous classe majeure interagissant avec les protéines de sperme. A l’inverse, les IgG1 semblent être principalement impliquées dans la réactivité vis-à-vis des protéines séminales. Nous avons également élargi le groupe déjà existant d’IgGs liés aux protéines de sperme à d’autres protéines, parmi lesquelles la protéine de choc thermique 70 1A/1B, la protéine apparentée aux protéines de choc thermique 71kDa et l’alpha-énolase ont été reconnues, pour la première fois, être liés à l’iso-immunisation féminine. Nous avons mis en évidence le rôle des protéines séminales dans l’iso-immunisation et pas seulement dans l’hypersensibilité au sperme par l’intermédiaire d’IgE. En particulier, l’antigène spécifique de la prostate, la phosphatase acide prostatique et la protéine à doigt de zinc 778 ont été décrites comme immuno-dominants parmi les protéines séminales reconnues par des IgGs liés aux protéines de sperme. La détermination des sous classes d’IgG de sérum féminin, spécifiques au liquide séminal/sperme, pourrait rendre le diagnostic des patients plus complet. Les IgG1 et IgG4 anti-liquide séminal/sperme pourraient présenter un intérêt pour l’immunothérapie. Par ailleurs, les protéines décrites, dans notre étude, pourraient se révéler être des bio marqueurs utiles pour de telles pathologies. Le dispositif miniaturisé pourrait être de type LFIA (Lateral Flow Immuno Assay), se basant sur la détection immuno-chimique d’anticorps spécifiques. L’immuno-intervention envisagée pourrait reposer sur l’effet d’immunoglobulines intraveineuses. / The World health Organization reports infertility as a disease and a failure of reproductive tract to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse. Nowadays, infertility has become a common life phenomenon affecting 1 out of 5 couples at reproductive age. Idiopathic cause is mostly associated with active immune system which may produce high levels of anti-seminal and/or anti-sperm antibodies. Auto-immunization as well as iso-immunization has a significant role in up to 30% of reported cases of infertility. Semen that is defined as a complex fluid containing sperm, cellular vesicles and other cells and components, could immunize the female genital tract. This thesis is related to female immune infertility, in particular to female iso-immunization. The better understanding of this pathophysiological event consists of (1) the determination of antibody isotype playing a significant role in this disease, then (2) the characterization and identification of semen antibody-binding proteins, seminal and/or sperm, (3) the proposal of potential diagnostic markers to adapt specific therapy and, in addition, the design of miniaturized diagnostic tool based on the selected markers, (4) the suggestion of potential immuno-intervention. Based on the distribution of seminal/sperm-specific antibody isotypes, we suggest that immunoglobulins E, M, A1,2, G3 are not involved in the primary pathophysiological female sensitization. IgG4 appears to be the major subclass interacting with sperm proteins. On a contrary, IgG1 seems to be the one mainly involved in the reactivity towards seminal proteins. We have also extended the existing group of IgG-binding sperm proteins, among which heat shock protein 70 1A/1B, heat shock cognate protein 71 kDa and alpha-enolase have been shown, for the first time, to be related to female iso-immunization. We have put the emphasis on the role of seminal proteins in iso-immunization and not only in the IgE-mediated semen hypersensitivity as known so far. In particular, prostate-specific antigen, prostatic acid phosphatase and zinc finger protein 778 have been determined as immunodominant among IgG-binding seminal proteins. The determination of female serum seminal/sperm-specific IgG subclasses could make the patient diagnoses more comprehensive. Anti-seminal/sperm IgG1,4 might be of interest for immunotherapy. Furthermore, the herein described proteins could be useful biomarkers of such pathology. The miniaturized chip could be a lateral flow immunoassay-based device acting on the immunochemical detection of specific antibodies. The intended immuno-intervention could consist of the effect of intravenous immunoglobulins.