| 1. |
- Dahm-Kähler, Pernilla, 1964, et al.
(författare)
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An intravital microscopy method permitting continuous long-term observations of ovulation in vivo in the rabbit
- 2006
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Ingår i: Hum Reprod. ; 21:3, s. 624-31
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: A method for intravital microscopy of the rabbit ovary was developed to enable observations of real-time changes during ovulation in vivo. The aim was to correlate these events to biochemical events at specific stages of ovulation. METHODS: Virgin, female rabbits were primed with equine chorionic gonadotrophin (CG) (30-100 IU) then HCG (100 IU) 2 days later to induce ovulation. During anaesthesia, the right ovary was surgically exteriorized and submerged in an organ chamber with a microscopy lens positioned close to the ovary. Continuous video recordings were performed. RESULTS: Initial equine CG priming experiments revealed the highest ovulation rate, without premature luteinization, after 30 IU equine CG. This priming protocol subsequently demonstrated follicular ruptures 11.5-14 h after HCG. Numbers of ovulations from the exteriorized and contralateral non-exteriorized ovary were similar. The sequence of typical features of ovulation was: shutdown of microcirculation in the follicular apex, formation of petechiae in the follicular wall and a cone-shaped structure over the future rupture site, marked bleeding in connection with follicular rupture and a fairly steady extrusion velocity of granulosa cells and the oocyte. CONCLUSION: This method captured a sequence of structural changes during ovulation. It could be combined with blood and follicular fluid sampling for biochemical analysis and could be used in studies on biochemical reactions in relation to specific changes in the follicular structure during ovulation.
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| 2. |
- Dahm-Kähler, Pernilla, 1964, et al.
(författare)
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Monocyte chemotactic protein-1 in the follicle of the menstrual and IVF cycle
- 2006
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Ingår i: Mol Hum Reprod. ; 12:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Ovulation constitutes an inflammatory-like process, with macrophages migrating into the follicle. This study evaluates the production of two macrophage-specific chemokines, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), in the human follicle at ovulation. Blood samples, follicular fluids and follicular cells were collected during menstrual and IVF cycles. Levels of MCP-1 and MIP-1alpha were measured in follicular fluid, blood plasma and cultured media (granulosa, theca and granulosa-lutein cells [GLCs]). Cells were cultured with or without LH, FSH, interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor (TNF) alpha, progesterone or estradiol. The levels of MCP-1 were markedly higher in follicular fluid as compared with blood plasma in both menstrual and IVF cycles. The difference in MCP-1 levels between follicular fluid and plasma in menstrual cycles increased from the follicular phase (three-fold difference) to the late ovulatory phase (25-fold). Levels of MIP-1alpha were low in plasma and follicular fluid of both menstrual and IVF cycles. Theca cells from follicles of menstrual cycles secreted both MCP-1 and MIP-1alpha under basal conditions, and the secretion was increased by addition of IL-1beta (MCP-1 and MIP-1alpha) and IL-1alpha (MCP-1). GLCs secreted MCP-1 under basal conditions and also MIP-1alpha after IL-1beta stimulation. The macrophage-specific chemokine MCP-1 is highly expressed and is induced by IL-1 in the theca layer of the human follicle at ovulation.
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| 3. |
- Dahm-Kähler, Pernilla, 1964, et al.
(författare)
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Monocyte chemotactic protein-1 (MCP-1), its receptor, and macrophages in the perifollicular stroma during the human ovulatory process
- 2009
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Ingår i: Fertility and Sterility. - : Elsevier BV. - 1556-5653 .- 0015-0282. ; 91:1, s. 231-239
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To evaluate the expression and localization of the macrophage-specific chemokine monocyte chemotactic protein-1 (MCP-1), CC chemokine receptor 2 (CCR2), and macrophages (CD68) in the perifollicular stroma of different phases of the human ovulatory process and its relation to macrophage influx. DESIGN: Experimental study on patient-controlled material. SETTING: University hospital. PATIENT(S): Twenty-eight women planned to undergo laparoscopic sterilization. INTERVENTION(S): Surgery was performed at either of four distinct ovulatory phases, and a biopsy sample was obtained from the perifollicular stroma of the ovulatory follicle. MAIN OUTCOME MEASURE(S): Real-time polymerase chain reaction, immunoblotting, and immunohistochemistry were used for measurements of MCP-1, CCR2, and macrophages. RESULT(S): The messenger RNA levels of MCP-1 in the perifollicular stroma increased from the preovulatory to the late ovulatory phase and declined during the postovulatory phase. A higher density of macrophages was found in the preovulatory and early ovulatory phases compared with late and postovulatory phases. Monocyte chemotactic protein-1 and CCR2 were present in the stroma. Protein expression of CD68 and CCR2 were identified in the four ovulatory phases. CONCLUSION(S): This study demonstrates an upregulation of MCP-1 in the stroma compartment around the human follicle during the ovulatory process, and a high density of macrophages was found at earlier phases. Thus, inflammation-like reactions are integral in the ovulatory process and may be targeted to stimulate or inhibit this process.
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| 4. |
- Dahm-Kähler, Pernilla, 1964
(författare)
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The Ovulatory Process. Studies in the human and the rabbit
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background and general summary: Ovulation is the cascade of events ending with follicular rupture and oocyte extrusion. This ovulatory process is triggered by the LH surge, which induces biochemical and biophysical alterations within the preovulatory follicle. The ovulatory process is an inflammation-like process and also involves alterations in hemodynamics and pressure within the follicle. A model for investigations of the events of ovulation in vivo in the rabbit was developed. The expression and regulation of two macrophage specific chemokines in the human ovary were also explored. To enable studies of the intrafollicular pressure (IFP) in the human ovary a new method was developed. The aims of these studies were to develop methodologies for studies of ovulation and to more specifically study one component of the inflammation-like response at ovulation. Methods and results: An intravital microscopy method, permitting long-term observation of the rabbit ovary in vivo, was developed. During anaesthesia the ovary of an eCG/hCG primed rabbit was submerged into a specially designed organ chamber with a microscopy lens close to the ovary. Video recordings documented ovulations, which occurred around 12 h after hCG. The sequence of typical features of ovulation were; vascular shut down in the follicular apex, petechiae in the follicular wall, formation of a cone-shaped structure over the rupture site, bulky bleeding at the site of follicular rupture and a steady extrusion velocity of granulosa cells and the oocyte (Paper I). The presence and regulation of two macrophage specific chemokines, monocyte chemoattractant protein-1 (MCP-1) and monocyte inflammatory protein-1£ (MIP-1£), were investigated in the human ovary during menstrual and IVF cycles. The levels of MCP-1 were markedly higher in follicular fluid as compared to blood plasma in both menstrual- and IVF-cycles. The follicular fluid to plasma difference in MCP-1 levels in menstrual cycles increased from the follicular phase to the late ovulatory phase. Theca cells from follicles of menstrual cycles secreted both MCP-1 and MIP-1 Ñ during basal conditions and the secretion increased by addition of IL-1. Granulosa-lutein cells secreted MCP-1 under basal condition and also MIP-1 Ñ after IL-1 (Paper II). MCP-1 expression and macrophage density were evaluated in the perifollicular stroma during precise phases of the ovulatory process in the human. Women, planned for laparoscopy, were monitored closely by transvaginal ultrasound and when the dominant follicle was 15-17mm in diameter 21 out of the 28 women received rhCG. Surgery was performed at four distinct ovulatory phases and the dominant follicle and its adjacent stroma was collected. The mRNA levels of MCP-1 in the perifollicular stroma increased from the preovulatory to late ovulatory phase and declined during post ovulatory phase. Immunoblot confirmed presence of macrophages and MCP-1 receptor CCR2 in the stroma. There was a tendency to higher macrophage density during the two earlier ovulatory phases (Paper III). A methodology for in vivo measurements of the IFP in the human ovary was developed. A pressure sensor was inserted into the follicular antrum, in ovaries of women undergoing laparotomy. The ovarian arteries and veins were dissected free to enable manipulation during pressure measurement. The baseline IFP was positive and stable. When the ovarian veins were blocked the IFP increased and then declined after clamping of the ovarian artery. The pressure inside ovarian non-follicular cysts was considerably lower and no alterations were seen during vascular manipulation (Paper IV).Taken together, the present studies present two new methodologies that enable in vivo examinations of the ovulatory process and do also show that the chemokine MCP-1 is expressed and under cytokine regulation during the ovulatory process of the human.
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| 5. |
- Dahm-Kähler, Pernilla, 1964, et al.
(författare)
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Transplantation of the uterus in sheep: methodology and early reperfusion events.
- 2008
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Ingår i: The journal of obstetrics and gynaecology research. - : Wiley. - 1341-8076 .- 1447-0756. ; 34:5, s. 784-93
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Tidskriftsartikel (refereegranskat)abstract
- AIM: Uterine transplantation is developing into a clinical treatment for uterine factor infertility. An animal model with a similar uterus size and vessels to humans and with pregnancy extending over several months would be beneficial for research on uterine transplantation. The aim of this study was to develop and evaluate autotransplantation of the sheep uterus to an orthotopic position in the pelvis. METHODS: Female sheep (n=7) were subjected to laparotomy with the uterus and its vascular supply and drainage being surgically isolated. The excised uterus was kept ex vivo at +4 degrees C for 60 min and then autotransplanted with vascular end-to-side anastomoses to the external iliac vessels. The effects of uterine blood-reperfusion were assessed by measurements of pCO(2), pO(2), lactate and pH in uterine venous blood. Uterine contractility and histology was assessed after 3 h of reperfusion. RESULTS: Reperfusion of blood was observed in five out of seven transplanted uteri. The pCO(2)/pO(2)-ratio and the lactate level were initially elevated but decreased and became normal after 60 min. After 3 h of reperfusion there was a visible tissue blood flow and spontaneous uterine contractions were seen. Histological analysis revealed a mild inflammation, but no edema or stasis. CONCLUSIONS: This study demonstrates that the sheep uterus can successfully be autotransplanted to an orthotopic position with novel vascular connections. This model is suitable for future experiments studying long-term results concerning uterine viability and pregnancy using a transplanted uterus of similar size to the human uterus.
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| 6. |
- Lind, Anna Karin, 1962, et al.
(författare)
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Collagens in the human ovary and their changes in the perifollicular stroma during ovulation
- 2006
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Ingår i: Acta Obstet Gynecol Scand. ; 85:12, s. 1476-84
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Remodeling of the collagens around the follicle is a major event in ovulation. The aim of the present study was to investigate the distribution of collagen I, III, and IV in the human ovary. METHODS: Biopsies of the perifollicular stroma were obtained at sterilization during the preovulatory phase (follicle size >14 mm) or at any of three intervals (12-18 h after human chorionic gonadotrophin: early ovulatory phase; >18-24 h: late ovulatory phase; 44-77 h: postovulatory phase) after human chorionic gonadotrophin. Excised dominant follicles and whole ovarian sections were also obtained. Immunohistochemistry using antibodies against collagen I, III, IV, vimentin, and CD 45 was performed. RESULTS AND CONCLUSIONS: Collagens I and III were distributed in concentric layers in the capsular stroma with bundles of collagens connecting these layers to form a mesh. Collagen I was present in larger quantities in the outer layers and collagen III showed the inverse distribution. In the theca, collagen I was present in the externa and collagen III in the entire layer. The staining intensity of collagens I and III in the perifollicular stroma decreased from the preovulatory stage. Collagen IV was present in the basal lamina separating granulosa and theca cells. This study shows that collagen I and III are abundant in and around the ovulating human follicle with typical patterns of distribution. Collagen IV is present in the basal membrane that separates the granulosa from the theca cells. Taking into account the abundance of collagens in the follicular wall and their specific localization, major site-directed degradation of collagens seems to be necessary for follicular rupture to occur.
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| 7. |
- Lind, Anna Karin, 1962, et al.
(författare)
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Gelatinases and their tissue inhibitors during human ovulation: increased expression of tissue inhibitor of matrix metalloproteinase-1
- 2006
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Ingår i: Mol Hum Reprod. ; 12:12, s. 725-36
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Tidskriftsartikel (refereegranskat)abstract
- Remodelling of the extracellular matrix (ECM) of the follicular wall by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) has been suggested to be crucial in ovulation. To investigate the expression of the gelatinases, MMP-2 and MMP-9, together with their inhibitors, TIMP-2 and TIMP-1, in the perifollicular ovarian stroma from women just before and during ovulation, we obtained biopsies of the stroma adjacent to the leading follicle. Laparoscopic surgery was performed either before the LH peak or at any of three intervals after ovulation triggering by hCG. Immunoblotting, immunohistochemistry and quantitative RT-PCR were performed. All four proteins were expressed by immunoblots, with no detectable changes in the expression of MMP-2, MMP-9 and TIMP-2. Scattered immunostaining for MMP-9 and TIMP-2 was seen, and MMP-2 was demonstrated in a concentric layer. A significant increase in TIMP-1 protein and mRNA was seen during the three ovulatory phases, and a strong and patchy immunostaining for TIMP-1 was shown. This is the first study that has demonstrated an ovulation-associated expression of these ECM-remodelling enzymes around the human follicle at ovulation. The increased expression of TIMP-1 may reflect a specific temporal inhibition of collagenolysis and thereby a time-dependent regulation of ECM breakdown in areas surrounding the apex of the follicle.
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| 8. |
- Wallin, Ann, 1948, et al.
(författare)
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Viability and function of the cryopreserved whole ovary: in vitro studies in the sheep
- 2009
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Ingår i: Hum Reprod. ; 24:7, s. 1684-94
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cryopreservation of whole ovaries followed by vascular transplantation may improve long-term function in comparison to conventional cryopreservation of ovarian cortex and avascular transplantation. The aim of this study was to assess methods for the evaluation of viability and function of frozen-thawed whole ovaries. METHODS: Ewe ovaries were flushed with either cryoprotectant (propandiol: FROZEN-PROH) or Ringer Acetate (FROZEN-RA) followed by slow freezing. Some ovaries were assessed fresh after flushing with Ringer Acetate (FRESH-RA). Assessment was done by light microscopy, biochemical response (cyclic adenosine 3',5'-monophosphate (cAMP) and steroids) during in vitro perfusion with forskolin, viability assay and cell culture. RESULTS: Microscopy showed well-preserved morphology with the presence of small follicles in all groups before perfusion. Stromal oedema was seen after in vitro perfusion of FROZEN ovaries, and shrunken small follicles were seen only in FROZEN-RA at the end of perfusion. During in vitro perfusion, FRESH-RA ovaries responded with large increase in levels of cAMP after stimulation with forskolin. FROZEN-PROH and FROZEN-RA ovaries exhibited lower production of cAMP. Progesterone concentrations in cell cultures of dispersed ovarian cells were higher in FRESH-RA when compared with FROZEN groups. Addition of hCG to cell cultures resulted in higher progesterone levels in the FROZEN-PROH compared with FROZEN-RA. Cell viability assay showed overall viability of 60-75% with no significant difference between groups. CONCLUSION: In vitro perfusion may prove to be a suitable method to test viability and function of frozen-thawed whole ovaries contributing to the optimization of current cryopreservation protocols.
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| 9. |
- Wranning, Caiza, 1963, et al.
(författare)
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Auto-transplantation of the uterus in the domestic pig (Sus scrofa): Surgical technique and early reperfusion events
- 2006
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Ingår i: J Obstet Gynaecol Res. ; 32:4, s. 358-67
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Tidskriftsartikel (refereegranskat)abstract
- AIM: To develop a method for auto-transplantation of the uterus in the pig and to evaluate the early reperfusion events after short-term cold ischemia. METHODS: The bicornate uterus, with the cervix but without ovaries, was dissected and isolated with its bilateral feeding and draining vessels. The uterine arteries were cannulated in situ and the uterus was flushed with heparinized Ringer Acetate. It was stored at 4 degrees C for 1-2 h during continuous flushing. The uterus was then placed in its original pelvic position and the uterine arteries and veins were anastomosed end-to-end to their origin. During approximately 100 min of reperfusion, blood samples and tissue biopsies were taken for monitoring of reperfusion events and detection of ischemia-reperfusion injuries. RESULTS: Out of 19 auto-transplanted pigs, seven were considered well flushed and were kept for cold ischemia. Of these seven, four showed satisfactory reperfusion judged by change in gross appearance and presence of appropriate venous blood flow. Analysis of blood-gas and metabolite parameters and histology indicated that at least two of these transplants were well reperfused, with no severe ischemia-reperfusion injuries. CONCLUSION: In this first report ever on auto-transplantation of the pig uterus it is demonstrated that an acceptable reperfusion can be achieved. Furthermore, it is suggested that because of the large total size of the pig uterus with long uterine horns and the small size of the vessels available for re-anastomosis, the pig is a fairly difficult model for further studies on transplantation of the uterus.
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| 10. |
- Wranning, Caiza, 1963, et al.
(författare)
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Transplantation of the uterus in the sheep: oxidative stress and reperfusion injury after short-time cold storage
- 2008
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Ingår i: Fertility and Sterility. - : Elsevier BV. - 1556-5653 .- 0015-0282. ; 90:3, s. 817-26
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the effects of cold ischemia and reperfusion after transplantation of the sheep uterus and to compare the preservation solution Perfadex (Vitrolife, Molndal, Sweden) with Ringer's acetate. DESIGN: Experimental animal study. SETTING: University hospital. ANIMAL(S): Adult, female sheep. INTERVENTION(S): One uterine horn with the common uterine cavity and cervix of sexually mature ewes was auto-transplanted after 1 hour of cold ischemic storage in either Perfadex (n = 5) or Ringer's acetate (n = 5). During 3 hours of reperfusion, uterine venous blood was collected and analyzed for several parameters that were indicative of oxidative stress and reperfusion injury. A biopsy was taken for histological analysis at the end of the experiment. MAIN OUTCOME MEASURE(S): Lipid peroxidation and ascorbyl radicals in uterine venous blood during reperfusion. Light microscopy and quantification of neutrophils in tissue after 3 hours of reperfusion. RESULT(S): A decline in pH and a rise in lactate and pCO(2)-pO(2), as well as an elevation of antioxidant capacity, lipid peroxidation, and intensity of ascorbyl radical electron spin resonance signal, was found that was more prominent after storage in Ringer's acetate. The histological analysis revealed mild inflammation in both study groups. CONCLUSION(S): Short-time cold ischemic storage of the sheep uterus does not induce any severe reperfusion damage, but the use of the protective buffer Perfadex decreases oxidative stress and inflammation when compared with a more simple solution.
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