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Intraovarian mechanisms influencing the human corpus luteum
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction: The human corpus luteum (CL) is a transient endocrine gland, only functionally active for about 14 days. Its principal function is to produce and secrete progesterone and thereby support the endometrium for implantation of a blastocyst and prevent rejection of the developing embryo. In the event of a non-conceptive cycle, functional and structural demise of the CL follows and a new ovulatory cycle begins. The aims of the thesis were to study different mechanisms involved in the extrinsic and intrinsic regulation of the CL and correlate these findings to available clinical investigations tools.

Materials and methods: Sixty women volunteered to donate their CL prior to scheduled surgery due to benign conditions. They were grouped according to CL-age, based on the occurrence of a preovulatory luteinizing hormone (LH) surge where days 2-5 post LH surge were designated as early luteal phase, days 6-9 as mid luteal phase and days 11-14 as late luteal phase. The CL bearing ovary was investigated using transvaginal ultrasonographical B-mode and color Doppler imaging prior to surgery. Following excision, the CL was further characterized using anatomical and morphological measurements, in vivo and in vitro hormone synthesis, isolation and cultures of steroidogenic luteal cells. Moreover, active transcription of putative regulatory genes of interest was targeted using semi-quantitative reverse-transcription polymerase chain reaction, in situ hybridization and Northern blots, and these genes' respective translation products were characterized by immunocytochemistry.

Results: The bulk of progesterone is stimulated by human chorionic gonadotropin (hCG) in the peripheral (steroidogenic) layer of the CL, where the LH/hCG receptor, as well as progesterone receptor (PR) isoform A/B and estrogen receptor type β (ER-β), but not ER-α, was localized. The sesitivity towards hCG was highest during the mid luteal phase in concordance with the value of LH/hCG receptor coding mRNAs. During this phase, despite low levels of PR-B mRNA, hCG treatment initiated a significant rise in progesterone output which could be blunted by the PR antagonist mifepristone. Increased amounts of prostaglandin (PG) F and its respective receptor (FP) mRNA were detected during the later developmental stages of the CL. However, steroidogenic luteal cells were unresponsive to added PGF until late luteal phase, indicative of an acquisition of sensitivity to the proposed luteolytic signal during this stage. Intraluteal vascular density was highest in early luteal phase and dramatically decreased during the course of CL development, a finding which was inversely correlated to resistance to blood flow in intraovarian blood vessels supplying the CL. Furthermore, a high degree of agreement between ultrasonographical and anatomical measurements of the CL was found.

Conclusions: Based on the novel findings herein, the hypothesis of steroid influence, acting within or near the steroidogenic luteal cells is confirmed. Alongside the classical extrinsic signals (e.g. hCG) and locally produced factors (e.g. PGF) these findings may further explain their modulatory roles during luteolysis or very early pregnancy. Furthermore, transvaginal ultrasonography in combination with color Doppler measurements may serve as a clinical tool to evaluate CL function.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2000. , 44 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 695
Keyword [en]
Corpus luteum, steroidreceptors, progesterone, LH/hCG, PGF2α, ultrasonography
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
URN: urn:nbn:se:umu:diva-46975ISBN: 91-7191-915-5 (print)OAI: oai:DiVA.org:umu-46975DiVA: diva2:441645
Public defence
2000-10-27, Hörsal E04, by 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Available from: 2011-09-17 Created: 2011-09-17 Last updated: 2011-09-17Bibliographically approved
List of papers
1. Compartmentalization of human chorionic gonadotrophin sensitivity and luteinizing hormone receptor mRNA in different subtypes of the human corpus luteum
Open this publication in new window or tab >>Compartmentalization of human chorionic gonadotrophin sensitivity and luteinizing hormone receptor mRNA in different subtypes of the human corpus luteum
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1997 (English)In: Human Reproduction, ISSN 0268-1161, E-ISSN 1460-2350, Vol. 12, no 5, 1037-1042 p.Article in journal (Refereed) Published
Abstract [en]

The relationship was investigated between different ultrasonographically defined subtypes of the human corpus luteum and progesterone production. Twenty-one women in the mid-luteal phase who underwent laparotomy for benign uterine conditions volunteered for this study. The corpus luteum was identified by preoperative ultrasound and classified into four types according to earlier established criteria, where types a and c were centrally hypoechoic, types b and d were centrally echogenic, types a and b had thin surrounding 'walls' (<3 mm) and types c and d had thick walls (<3 mm). After luteectomy, the theca externa capsule was removed and tissue from directly beneath the surface ('peripheral region') and the layer immediately beneath ('inner region') minced into 4-6 mg pieces. Following preincubation, pieces were incubated for 3 h at 37 degrees C in HEPES-minimal essential medium buffer with or without human chorionic gonadotrophin (HCG; 10 IU/ml), and subsequently progesterone accumulation in the medium was determined by radioimmunoassay. The highest progesterone production was consistently seen in the peripheral region. Type a had a significantly (P > 0.01) lower progesterone production (3.2 +/- 1.5 nmol/g tissue wet weight, mean +/- SEM, n = 4) than that of types b, c and d (17.7 +/- 3.5 nmol/g tissue wet weight, n = 9). All types responded to HCG with an almost two-fold increase in progesterone production. However, the maximal progesterone produced following stimulation by HCG in the type a corpus luteum was <50% of the basal (unstimulated) progesterone synthesis of any other type of corpus luteum. Using in-situ hybridization, with a primate RNA probe complementary to the region coding the extracellular part of the luteinizing hormone (LH) receptor, a highly localized expression of LH receptor mRNA to the peripheral region was found. Negligible or low levels of expression were found in the theca externa capsule and the inner region. No obvious correlations between the different subtypes of corpora lutea and LH receptor mRNA expression were seen. Thus, the ultrasonographic detection of a thin-walled and centrally hypoechoic corpus luteum correlates well with reduced progesterone secretion. The underlying cellular mechanism does not appear to involve a diminished sensitivity to the gonadotrophic stimulation by LH or HCG.

National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-41634 (URN)10.1093/humrep/12.5.1037 (DOI)9194662 (PubMedID)
Available from: 2011-03-30 Created: 2011-03-30 Last updated: 2017-12-11Bibliographically approved
2. Intraovarian blood flow measured with color doppler ultrasonography inversely correlates with vascular density in the human corpus luteum of the menstrual cycle
Open this publication in new window or tab >>Intraovarian blood flow measured with color doppler ultrasonography inversely correlates with vascular density in the human corpus luteum of the menstrual cycle
2004 (English)In: Fertility and Sterility, ISSN 0015-0282, E-ISSN 1556-5653, Vol. 81, no 1, 154-159 p.Article in journal (Refereed) Published
Abstract [en]

Objective

To evaluate the morphologic characteristics underlying the ultrasonographic appearance and blood flow dynamics in the human corpus luteum (CL) of the menstrual cycle.

Design

Cross-sectional study.

Setting

Umeå University Hospital, Umeå, Sweden.

Patient(s)

Twenty-six otherwise healthy women with proven fertility and a history of regular menstrual cycles, scheduled for elective hysterectomy or tubal sterilization.

Intervention(s)

An ovulatory LH rise in urine was established and the CL age was determined according to the day after the LH rise. Before surgery, a standardized ultrasonographic examination of the CL, including B-mode and color Doppler ultrasonography measurements, was performed. Upon commencing the minilaparotomy, the CL was excised and measured using a digital slide-caliper. The volume density (percentage of CL volume occupied by blood vessels) of factor VIII–related antigen immunostained endothelial cells was determined.

Main outcome measure(s)

Pulsatility index obtained from intraovarian blood vessels supplying the CL and volume density of blood vessels in CL tissue. The CL maximal and minimal outer and inner dimensions were measured in vivo by ultrasonography and ex vivo by a digital slide caliper.

Result(s)

A statistically significant decrease of blood vessel density and an increased resistance to blood flow, as indicated by pulsatility index, was established during the course of corpus luteum development. An inverse correlation between pulsatility index and volume density of blood vessels was found. A high degree of agreement between ultrasonographic and anatomic measurements of surgically removed CL was found.

Conclusion(s)

Transvaginal ultrasonography in combination with intraovarian color Doppler flow measurements is a simple and reliable method to evaluate the size and vascularization of the human CL.

Keyword
Angiogenesis; human corpus luteum; ultrasonography; Doppler imaging; vascularization; morphometry
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-15176 (URN)10.1016/j.fertnstert.2003.05.019 (DOI)14711559 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-14Bibliographically approved
3. Functional evidence for divergent receptor activation mechanisms of luteotrophic and luteolytic events in the human corpus luteum
Open this publication in new window or tab >>Functional evidence for divergent receptor activation mechanisms of luteotrophic and luteolytic events in the human corpus luteum
1999 (English)In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 5, no 5, 391-395 p.Article in journal (Refereed) Published
Abstract [en]

Using a dispersed human luteal cell culture model, progesterone synthesis following treatment by incremental doses of human chorionic gonadotrophin (HCG) and the stable prostaglandin F2alpha (PGF2alpha) analogue cloprostenol, alone or in combination, was related to corpora lutea (CL) mRNA transcript abundance coding for the luteinizing hormone (LH)/HCG receptor (LH-R) and PGF2alpha-receptor (FP) by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 33 otherwise healthy women, scheduled for surgery due to benign conditions. CL were grouped according to age, based on the occurrence of a preovulatory LH surge where post-LH days 2-5 were designated as early luteal phase, days 6-10 as mid-luteal phase and days 11-14 as late luteal phase. When exposed to HCG, maximal progesterone output was raised 2.2-fold (P = 0.08, n = 5) compared with untreated controls in the early CL, while it increased 5.7- and 4.6-fold in the mid- and late groups respectively (P<0.05, n = 4 mid-luteal phase, n = 3 late luteal phase). This stimulation pattern was found to be concordant with the value of mRNA coding for LH-R in all groups (n = 6 early luteal phase, n = 5 mid-luteal phase, n = 6 late luteal phase). The integrated response to HCG and cloprostenol showed a dose-dependent 60% inhibition of progesterone production; but only in late luteal phase luteal cells (P<0.01, n = 3). FP mRNA values were lowest in early luteal phase, and increased with the age of the CL. Interestingly, lowest CL tissue concentrations of the natural FP agonist PGF2alpha were found during mid-luteal phase while it increased again 1.6-fold during late luteal phase (P<0.05, n = 8 versus mid-luteal phase, n = 6). Collectively, these data demonstrate that (i) the extrinsic functional control (or rescue of CL in the event of pregnancy) occurs when the sensitivity towards LH/HCG is maximal; and (ii) the demise of CL function is mediated via an acquisition of sensitivity towards the intrinsic luteolytic signal, PGF2alpha in the ageing CL.

Keyword
corpus luteum, HCG, LH receptor, PGF2α receptor (FP), progesterone
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-41638 (URN)10.1093/molehr/5.5.391 (DOI)10338360 (PubMedID)
Available from: 2011-03-30 Created: 2011-03-30 Last updated: 2017-12-11Bibliographically approved
4. A putative stimulatory role of progesterone acting via progesterone receptors in the steroidogenic cells of the human corpus luteum
Open this publication in new window or tab >>A putative stimulatory role of progesterone acting via progesterone receptors in the steroidogenic cells of the human corpus luteum
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2000 (English)In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 62, no 3, 655-663 p.Article in journal (Refereed) Published
Abstract [en]

To further explore the proposed auto-regulatory role of progesterone action in the human corpus luteum (CL), the expression and functional roles of progesterone receptor (PR) isoforms A and B during the luteal phase (LP) of the menstrual cycle were investigated. A total of 27 otherwise healthy patients previously scheduled for surgery were recruited after informed consent. An LH rise was detected, and CL were grouped according to age (Days 2-5 post-LH-rise, early LP; Days 6-10, mid LP; Days 11-14, late LP). Using a semiquantitative reverse transcription-polymerase chain reaction assay, the PR-B mRNA levels, which were 100- to 1000-fold lower than PR-A/B mRNA, were 46% lower (P < 0.05, n = 24) in mid LP, compared to early and late LP. CL tissue levels of progesterone and PR-A/B protein levels were inversely correlated to increasing CL age; i.e., significantly reduced levels were observed in the late LP (r(2) = 0.34, P < 0.01, n = 23). Expression of PR-A/B mRNA as well as PR-A/B protein were detected by in situ hybridization and immunohistochemistry, respectively. Both methods revealed a clear and distinct localization to cells in the steroidogenic layer of the CL. Freshly obtained mid-luteal CL cells were cultured in vitro, and media were analyzed for progesterone concentrations after treatment by incremental doses of hCG and the stable PR antagonist mifepristone, alone or in combination. Mifepristone did not per se alter progesterone synthesis, but when it was added in conjunction with hCG, a dose-related inhibitory response was seen, with a maximal 47% reduction in progesterone output at a 10 microM addition (P < 0.05, n = 3). Collectively, these data implicate a stimulatory role of progesterone receptor-mediated action in the steroidogenic cells of the human CL, which may serve as an important pathway for maintaining functional homeostasis during early pregnancy.

National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-41640 (URN)10.1095/​biolreprod62.3.655 (DOI)10684807 (PubMedID)
Available from: 2011-03-30 Created: 2011-03-30 Last updated: 2017-12-11Bibliographically approved
5. Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum
Open this publication in new window or tab >>Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum
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2001 (English)In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 7, no 2, 137-145 p.Article in journal (Refereed) Published
Abstract [en]

To investigate the potential importance of oestrogen as a local regulator of human corpus luteum function, the mRNA expression pattern and cellular localization of oestrogen receptors (ERs), ER-alpha and ER-beta, were studied in corpora lutea grouped according to age, where days 2-5 post-LH rise were designated as the early luteal phase, days 6-10 as mid-luteal and days 11-14 as the late luteal phase respectively. Northern blot analysis using an ER-beta probe in samples from whole ovarian tissue and isolated corpora lutea, revealed a major band at 7.5 kb and several minor bands between 4-10 kb, while no signals for ER-alpha mRNA were obtained. However, using a semi-quantitative reverse transcription-polymerase chain reaction followed by Southern blotting, ER-beta mRNA levels were found to be 63% lower (P: < 0.05, n = 39) in the mid-luteal phase compared with the early luteal phase, while ER-alpha mRNA expression showed no statistical differences between the different age groups. Using in-situ hybridization, ER-beta mRNA expression was localized to the steroidogenic luteal cells as well as perivascular cells and fibroblasts in the corpus luteum. Immunohistochemistry confirmed the localization of ER-beta protein, but no clear staining of luteal cells was found using antibodies against ER-alpha. Collectively, the findings of low to moderate expression of ER-beta mRNA and protein in the steroidogenic cells, and also in vascular endothelial cells of the corpus luteum, as opposed to diminutive amounts of ER-alpha mRNA, suggest that oestrogen activity is primarily transduced via ER-beta in the human corpus luteum.

Keyword
17β-oestradiol, human corpus luteum, oestrogen receptors, steroids
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-41642 (URN)10.1093/molehr/7.2.137 (DOI)11160839 (PubMedID)
Available from: 2011-03-30 Created: 2011-03-30 Last updated: 2017-12-11Bibliographically approved

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