| 1. |
- Helmestam, Malin
(author)
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Effects of Endocrine Disrupting Chemicals on Human Endometrial Endothelial Cells In Vitro
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Evidence from an abundant number of studies suggests that human female reproductive functions have become impaired over the past half century and that there might be a relationship between endocrine disrupting chemicals (EDCs) and reduced fertility. It is, however, not known by what mechanisms EDCs affect different reproductive functions such as endometrial receptivity, embryo implantation and placentation.The endometrium is continuously changing its morphological and functional properties, responding to cyclic changes of oestrogen and progesterone levels during the menstrual cycle. These changes include monthly preparation for embryo implantation through changed endometrial angiogenic activity and consequent changes in endometrial vasculature.Use of primary human endometrial endothelial cells (HEECs) in this work was evaluated as a possible screening tool for effects caused by EDCs on human endometrial vasculature and subsequently on various endometrial functions.In this study HEEC and endometrial stromal cells were isolated. HEECs were grown in monocultures, and together with stromal cells in co-cultures, and exposed to endocrine active substances. These were cadmium, which has oestrogenic properties, tamoxifen, with anti-oestrogenic effects, mifepristone, which is an anti-progestin, and bisphenol A, with oestrogenic properties. The effects were evaluated by using proliferation and viability assays, migration and tube formation assays, quantitative PCR (qPCR), immunohistochemistry and western blot.Cadmium affected the expression of angiogenesis-related genes, and caused different effects in HEECs cultured alone vs. HEECs co-cultured with stromal cells. Tamoxifen altered the expression of angiogenesis-related genes and reduced HEEC migration, thus having an anti-angiogenic effect. Mifepristone caused reduced formation of tubular structures in tube-formation assays involving HEECs co-cultured with stromal cells. Bisphenol A promoted tube formation in co-cultured HEECs which was related to changes in the expression of several angiogenesis-related genes as well as up-regulated expression of VEGF-D protein.In conclusion, we showed that EDCs have the ability to induce changes in endometrial angiogenic activity in vitro and may thus disturb normal endometrial functions related to fertility and pregnancy. HEECs grown in vitro may provide valuable information on the effects of EDCs on human endometrial functions. However, this model is not suitable as a large-scale screening tool.
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| 2. |
- Jansson, Caroline, 1963-
(author)
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Experiences of Miscarriage
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Experiences are unique and both men and women experience grief, anxiety, depressive symptoms after a miscarriage.The overall aim was to describe and measure experiences among women and men after a miscarriage.Study one was a qualitative study with interviews with Swedish midwives' and nurses' experiences of women's reactions after routine ultrasonographic diagnosis of a missed miscarriage.Study two concerned validation of "The revised impact of miscarriage scale" for Swedish conditions and a comparison of Swedish and American women's and men's experiences of miscarriage.Study three was a longitudinal study of Swedish women's and men's emotions.Study four was a longitudinal study, on women's feelings in relation to diagnosis and treatment.Scales about experiences, grief, and depressive symptom were used.The results showed that midwives perceived that the women had had a premonition of symptoms of a missed miscarriage and a follow-up was performed. The degree of consistency showed that the questionnaire can be used in a Swedish setting. The Swedish and American women and men scored similarly in two factors, and the women's experiences were more pronounced than the men's. Grief and depressive symptoms became reduced over time, while experiences persisted. No previous children, miscarriage or infertility treatment prior to miscarriage made the experience worse especially grief reaction. There was no difference between the two diagnosis groups in experiences one week after the miscarriage and their experience improved after four months. Women treated with misoprostol had more depressive symptoms than women treated with misoprostol and subsequent vacuum aspiration.In conclusion, care providers can confirm women's premonition of a missed miscarriage so a diagnosis can be set early in the pregnancy and they can do an individual follow-up. The high consistency between the countries in two factors show that RIMS is reliable for both women and men. Grief and depressive symptoms become reduced, while experiences persist. Previous miscarriage, lack of previous children and an infertility diagnosis can lead to negative feelings as grief. A diagnosis of miscarriage has a limited influence on experiences, and a shorter duration of treatment and treatment with misoprostol and subsequent vacuum aspiration led to a fewer depressive symptom.
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| 3. |
- Moberg, Christian, 1972-
(author)
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The Human Endometrium : Studies on Angiogenesis and Endometriosis
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Angiogenesis is thought to play a pivotal role in the cycling endometrium. Coordinated by oestrogen and progesterone, endometrial blood vessel development is primarily mediated by vascular endothelial growth factor-A (VEGF-A), which promotes endothelial cell (EC) proliferation and protects ECs from induced apoptosis. Studying changes at transcript level in human endometrial endothelial cells (HEECs) in response to mitogenic and inhibitory stimuli is one way towards understanding the regulation of physiological endometrial angiogenesis.Endometriosis, the presence of endometrial-like tissue outside the uterine cavity, is a common gynaecological disorder in women of reproductive age, often causing pelvic pain and reduced fertility. Chronic inflammation in the peritoneal environment and defective endometrial protein expression are some of the contributors to the complex pathophysiology of endometriosis. The aim of this work was to study the changes in the transcriptome induced by VEGF-A and partial serum deprivation in primary HEECs, and to investigate biochemical factors associated with subfertility and chronic pelvic pain in endometriosis patients.Exposing primary HEECs to VEGF-A, and serum withdrawal was found to regulate transcripts associated with survival, migration, apoptosis and progression through the cell cycle, when assessed using microarray technology and bioinformatic tools. A subset of 88 transcripts was reciprocally regulated under the two experimental conditions; thus probably important in HEEC biology.Higher endometrial epithelial staining scores of oestrogen receptor-α and reduced staining of progesterone receptors were seen in subfertile endometriosis patients. Lower levels of the receptivity biomarker leukaemia inhibitory factor (LIF) and its receptor, as well as signs of dysregulated αB-crystallin expression and increased peritoneal fluid concentrations of interleukin (IL)-1α and IL-6 were associated with reduced pregnancy rates.Endometriosis patients with chronic pelvic pain had higher levels of vasoactive intestinal peptide (VIP) in eutopic endometria and in endometriotic lesions compared with patients without chronic pain. The presence of chronic pelvic pain was also associated with increased concentrations of VIP and IL-6 in peritoneal fluid.The present results may constitute a basis for further investigation of regulatory pathways in endometrial angiogenesis as well as for studies of endometrial receptivity and pain in women with endometriosis.
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| 4. |
- Murto, Tiina, 1975-
(author)
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Folate, Hormones and Infertility : Different factors affecting IVF pregnancy outcome
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Various hormones have been studied as regards prediction of pregnancy outcome after infertility treatment, but no ideal candidate has been found. Folate and genetic variations in folate metabolism have also been associated with infertility, but it remains unclear how these factors affect IVF pregnancy outcome. It is known that infertility is associated with active folic acid supplement use, but the effect of socioeconomic and lifestyle factors on folic acid supplement use in infertile women has not been well investigated. The overall aim of this work was to obtain information on the prediction of live birth, and to study factors affecting the role of folate and folic acid intake in relation to IVF pregnancy outcome. Infertile women with various infertility diagnoses were studied. Healthy, fertile non-pregnant women were used as controls in three of the studies. Blood samples were taken for assay of eight different hormones, folate and homocysteine, and for genomic DNA extraction. A questionnaire was used to assess background data and use of folic acid supplements. Twenty-four-hour recall interviews were performed for validation of the questionnaire. The studied hormones were not good predictors of live birth. The best predictor was age of the women, together with ovulatory menstrual cycles, and thyroid-stimulating hormone and anti-Müllerian hormone (AMH) status. Well-educated women, high-status employed women, and married and infertile women used the most folic acid supplements. Infertile women had better folate status than fertile women. However, pregnancy outcome after infertility treatment was not dependent on folic acid intake, folate status, genetic variation of 5,10-methylenetetrahydrofolate reductase or socioeconomic status. In conclusion, AMH levels vary less than those of other hormones during the menstrual cycle, and AMH could be used as a predictive marker of live birth together with age and ovulation. Folate might play a minor role in IVF pregnancy outcome, but the importance of folate as regards other health perspectives should not be forgotten.
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| 5. |
- Nordqvist, Sarah, 1962-
(author)
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Biological Markers of Fertility
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Infertility affects 15 % of couples, which corresponds to 60 - 80 million worldwide. The microenvironments in which the oocyte, embryo and fetus mature are vital to the establishment and development of a healthy pregnancy. Different biological systems, such as angiogenesis, the immune system and apoptosis need to be adequately regulated for pregnancy to occur and progress normally. The overall aim of this thesis was to investigate the impact of Histidine-rich glycoprotein (HRG) and Src homology 2 domain-containing adapter protein B (SHB) on human female fertility.HRG is a plasma protein that regulates angiogenesis, the immune system, coagulation/fibrinolysis and apoptosis, by building complexes with various ligands. The impact of HRG on fertility is studied here for the first time. HRG is present in follicular fluid, the Fallopian tube, endometrium, myometrium and placenta. HRG distribution within embryo nuclei depends on developmental stage. Blastocysts express and secrete HRG. The HRG C633T single nucleotide polymorphism (SNP) appears to affect the chance of pregnancy and, correspondingly, parameters associated with pregnancy in IVF. Additionally, this HRG genotype may increase the risk in IVF of only developing embryos unfit for transfer.SHB is an adaptor protein involved in intracellular signaling complexes that regulate angiogenesis, the immune system and cell proliferation/apoptosis. Shb knockout mice have altered oocyte/follicle maturation and impaired embryogenesis. The impact of three SHB polymorphisms (rs2025439, rs13298451 and rs7873102) on human fertility is studied for the first time. The SNP prevalences did not differ between infertile and fertile women. BMI, gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer seems to be affected by SHB genotype.In conclusion, HRG and SHB appear to influence female fertility. They are potential biomarkers that might be used for predicting pregnancy chance in infertile women. Knowledge of these genotypes may improve patient counseling and individualization of treatment.
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| 6. |
- Rajareddy, Singareddy, 1977-
(author)
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Studies of phosphatidylinositol 3 kinase (PI3K) signaling pathway in mammalian ovarian follicle activation and development
- 2007
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Doctoral thesis (other academic/artistic)abstract
- The intra-oocyte signaling pathways that control oocyte growth and early follicular development are largely unknown. The aim of this thesis was to investigate the regulation and functions of phosphatidylinositol 3 kinase (PI3K) pathway in the oocyte, focusing in the roles of Foxo3a, p27, and Pten (phosphatase and tensin homolog deleted on chromosome ten). The physiological significance of Foxo3a in oocytes had been investigated by generating a transgenic mouse, whereby constitutively active Foxo3a is maintained in oocytes using the oocyte-specific ZP3 (Zona pellucida) promoter. The expression of the constantly active “negative” molecule Foxo3a in mouse oocytes was found to cause retardation of oocyte growth, resulting in a significant reduction in oocyte volume in secondary follicles. The transgenic mice also showed arrested follicular development and were infertile. In addition, when Foxo3a was overexpressed in oocytes of primary follicles, oocyte growth and follicular development were retarded. One of the causes of this phenotype may be the retained expression of the cyclin-dependent kinase (Cdk) inhibitor 1B (Cdkn1b), commonly known as p27kip1 or p27, in the nuclei of oocytes. The role and related mechanisms of p27 in controlling early follicular development and oocyte growth were then investigated using wild-type and p27-deficient (p27-/-) mice, and we demonstrated that (i) p27 suppresses follicle endowment/formation and activation, (ii) p27 induces follicle atresia that occurs prior to sexual maturity, and (iii) the overactivated follicles in p27-/- ovaries are depleted in early adulthood, causing premature ovarian failure (POF). In this thesis, we also provide genetic evidence that in mice with conditional deletion of Pten a major negative regulator of PI3K in oocytes, the entire pool of primordial follicles becomes activated, and subsequently all activated follicles are depleted in young adulthood, causing POF. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling, which led to upregulation of both expression and activation of ribosomal protein S6 (rpS6) in oocytes. The results thus show that the mammalian oocyte serves as the headquarters of programming of the occurrence of follicle activation, and that the PI3K pathway of the oocyte governs follicle activation through control of initiation of oocyte growth.
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| 7. |
- Stavréus-Evers, Anneli
(author)
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Implantation : morphological and biochemical characterization of the receptive human endometrium
- 2002
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Doctoral thesis (other academic/artistic)abstract
- Our knowledge of the human implantation process, and the role of the endometrium. during this process, is very scarce. The endometrium is only receptive for implantation during a limited period, the so-called implantation window, after which it becomes refractory to the blastocyst. The ovarian steroid hormones regulate the cyclic changes in the endometrium. The epithelial cells form protrusions, the so-called pinopodes, on their apical side at the time of implantation. The function of the pinopodes has not been elucidated, but these structures might prevent the cilia from sweeping off an approaching blastocyst. It is possible that blastocysts adhere to adhesion molecules on the pinopodes. An endometrial biopsy was obtained from normal healthy women during the luteal phase of their menstrual cycle. The endometrial biopsies were divided and one piece was used for examination in scanning electron microscopy, while the other piece was used for immunohistochemistry and/or tissue culture. In the first study, a newly developed method was used to accomplish implantation in vivo by exposing the biopsy strips in culture to a blastocyst. In the second study it was established that the use of a culture insert in presence of Matrigel, and an oxygen concentration of 5%, is optimal for culturing endometrial strips. The epithelial cells were seen to cover the injured surface, suggesting a healing process. The third study showed that pinopodes develop when the progesterone levels were increasing and that the pinopodes regress when the progesterone levels were decreasing. The pinopodes were found mainly on days 6 to 9 after the LH surge. The progesterone receptor B level decreased in epithelial cells concomitantly with the appearance of pinopodes. This study suggests that progesterone is directly or indirectly involved in the development of pinopodes. The concomitant presence of Glutaredoxin (Grx) and pinopodes was investigated in the fourth study. The Grx staining was intense in the epithelial cells when the pinopodes were present and the most intense staining was found in pinopodes on the apical side of the luminal epithelial cells. The function of Grx is not known, but it is likely that it protects the endometrium from oxidative stress during the implantation process. The strong staining of Grx implies that it is possible to use Grx as a biochemical marker of pinopode appearance and thus the implantation window. Heparin binding epidermal growth factor-like growth factor (HB-EG17) exists both as a membrane-bound growth factor and as a free growth factor. In the fifth study, it was shown that the immunostaining of HB-EG17 was strongest when the pinopodes were present and it was also shown, by using confocal microscopy, that HB-EG17 is present on the surface of the pinopodes. In conclusion, a correct dating of the endometrium is important for implantation studies. Progesterone seems to be the main regulator, directly or indirectly, of changes in the receptive endometrium and thus the development of pinopodes and the up-regulation of HB-EGF and Grx. It is possible to achieve implantation in vitro. The healing process during the culture mimics the healing after blastocyst invasion. HB-EGF, known to be involved in healing processes, is likely to promote the healing process in the endometrium.
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