Morphological and functional changes in human endometrium following intrauterine levonorgestrel delivery

The levonorgestrel releasing intrauterine system (LNG-IUS) provides a novel contraceptive method. Intrauterine LNG induces a dramatic transformation of the endometrium, characterized by extensive decidualization. This is associated with strong expression of local factors associated with decidualization, including prolactin receptor and insulin-like growth factor binding protein-1. A striking discovery was the down-regulation of oestrogen and progesterone receptors in all components of the endometrium after insertion of the LNG-IUS, with a gradual return between 6 and 12 months post-insertion. Preliminary findings suggest that androgen receptors are expressed during this time. Elevated leukocyte infiltrate is observed 1 month after insertion of the device, comprising large granular lymphocytes and macrophages. We examined a number of local mediators implicated in menstruation and breakthrough bleeding episodes. Expression of the chemokine interleukin-8 was enhanced after insertion of the device, with a notable decrease apparent 6 months post-insertion. Cyclooxygenase-2 was similarly strongly expressed in the first months after LNG-IUS insertion, in contrast to an initial suppression of prostaglandin dehydrogenase activity. By deduction it appears that higher local concentrations of prostaglandins are present in the initial period of local LNG exposure. Taken together these data suggest that in the first months following LNG-IUS insertion steroid receptor content is significantly decreased, resulting in the altered expression of many locally acting mediators which may be involved in breakthrough bleeding episodes.     

Uterus and endometrium: The effect of local intrauterine levonorgestrel administration on endometrial thickness and uterine blood circulation

To evaluate non-invasively the role of levonorgestrel releasingdevices in direct contact with the endometrium on menstrualspotting and endometrium inactivation, we inserted levonorgestrelreleasing devices (20 µg/24 h) either into the cervicalcanal or the uterine cavity of 30 fertile women. Both beforeinsertion and over the following 3 months, we used transvaginalsonography to measure the endometrial thickness in 20 of thewomen and Doppler flow to measure the uterine blood flow inthe remaining 10 women. The women were asked to keep recordsof menstrual bleeding and they gave blood samples for the measurementof serum oestradiol, progesterone and levonorgestrel. By 10weeks after insertion there was a significant decrease in endometrialthickness in both groups. Intracervical levonorgestrel releaseallowed the endometrium to maintain cyclic changes, whereasdirect intrauterine levonorgestrel release eliminated the cyclicalchanges. The total number of spotting days was significantlyless (P = 0.0249) in the intracervical release group at 3 months;1.2 ± 0.6 versus 8.1 ± 1.8 (mean ± SE).There were no significant differences in hormone concentrationsbetween the groups. The pulsatility index did not change significantlyduring the study. We concluded that the inactivation processof the endometrium can be monitored by transvaginal sonographyand that locally administered levonorgestrel does not changecirculatory conditions detectable by Doppler flow. Our resultsalso suggest that the inactivation process of the endometriumis different between intracervical and intrauterine levonorgestreladministration and may explain the difference in the numberof spotting days.     

Hormonally mediated disturbance of angiogenesis in the human endometrium after exposure to intrauterine levonorgestrel

BACKGROUND: The levonorgestrel intrauterine system (LNG-IUS) is a contraceptive device that is used for treatment of menorrhagia. The system induces inter-menstrual bleeding within the first few months after insertion. We hypothesized that this bleeding might be associated with a change in vascular development. METHODS: A randomized, controlled study was undertaken on 48 women. RESULTS: Hysterectomy specimens were obtained and immunocytochemistry was carried out with antibodies to CD31, alpha-smooth muscle actin and myosin. Stereological measurement of blood vessels was also undertaken. Most vessels appeared normal, including the arterioles. Large thin-walled vessels were present in the superficial endometrium of the treated group but were almost completely absent in the controls. The distribution of cytoskeletal markers revealed well-formed basal arterioles with more widespread expression in the superficial stroma than was found in untreated tissue. The volume fraction of blood vessels (P = 0.0001), the number of vessel cross-sections per unit area (P = 0.0003) and the cross-sectional diameters of the largest vascular lumens (P = 0.0001) were significantly increased following treatment with LNG-IUS. However, there was no difference in the median values of vessel diameter or the vascular surface density. CONCLUSION: These findings suggest that the LNG has a localized effect on some vessels within the superficial endometrium.     

Regulation of matrix metalloproteinase-9 in endometrium during the menstrual cycle and following administration of intrauterine levonorgestrel

Remodelling of endometrial tissues is fundamental to the cyclical changes that occur during the menstrual cycle, implantation and, in the absence of pregnancy, at menstruation. The enzyme matrix metalloproteinase-9 (MMP-9) is recognized as important in these processes but its regulation is not well defined. These studies have demonstrated that MMP-9 activity is present in the endometrium and exhibits cyclical changes in its distribution in the glandular and stromal cells. MMP-9 protein is present throughout the cycle with highest expression, as determined by semiquantitative analysis of specific MMP-9 immunoreactivity, in glandular cells during the mid secretory phase. A similar distribution was observed in first trimester decidua. In women with a levonorgestrel intrauterine system (LNG-IUS), which delivers high local concentrations of progestagen to the uterine cavity, MMP-9 is highly expressed in both endometrial glandular and stromal cells, and in the vasculature (in endothelial and perivascular cells). It can be concluded that MMP-9 is stimulated directly or indirectly by progesterone. Furthermore, MMP-9 may play a role in the remodelling of the endometrium that occurs during the menstrual cycle and in the aetiology of the morphological changes and breakthrough bleeding associated with long-term progestagen administration via a LNG-IUS.     

Insulin-like growth factors and insulin-like growth factor binding proteins in the endometrium. Effect of intrauterine levonorgestrel delivery

Insulin-like growth factor (IGF) system is one of the growth factor systems that are believed to modulate steroid hormone actions in the endometrium through autocrine/paracrine mechanisms. IGF-I and IGF-II stimulate proliferation and differentiation, and maintain differentiated cell functions in several cell types in vitro. Endometrial stromal cells produce IGF-I and IGF-II as well as the high affinity IGF-binding proteins (IGFBP), whereas epithelial cells and, in a lesser amount, stromal cells contain cell membrane receptors for IGF. Oestrogen stimulates IGF-I gene expression, and IGF-II gene expression is associated with endometrial differentiation. The mRNA of six high affinity IGFBPs, which can modulate IGF actions, are expressed in human endometrium. The most abundant IGFBP in human endometrium is IGFBP-1, which is secreted by predecidualized/decidualized endometrial stromal cells in late secretory phase and during pregnancy. The primary negative regulator of IGFBP-1 production is insulin. IGFBP-1 competes with type I IGF receptor for binding of IGF in the endometrium and in cultured human trophoblastic cells. IGF-I mRNA is suppressed and mRNA encoding IGF-II and IGFBP-1 are consistently up-regulated in decidualized endometrium in women treated with the intrauterine levonorgestrel system (LNG-IUS). Strong cytoplasmic staining for IGFBP-1 was detected in decidualized endometrium in women using LNG-IUS for contraception or for endometrial protection during post-menopausal oestrogen replacement therapy. Simultaneously, oestrogen receptors were present, while progesterone receptors were hardly detectable in the endometrium by immunohistochemistry. The latter findings suggest that suppression of IGF-I action by IGFBP-1 may be one of the molecular mechanisms accounting for progestagenic and anti-oestrogenic effects of LNG-IUS in the endometrium. Consequently, examination of local IGF-I, IGF-II and IGFBP-1 expression might provide additional information when evaluating the effect of different progestins on the endometrium at the molecular level.     

Local levonorgestrel regulation of androgen receptor and 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrium

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, unscheduled breakthrough bleeding (BTB), leads to discontinuation in a proportion of users. The LNG-IUS down-regulates endometrial progesterone and estrogen receptors and this may play a role in the mechanism responsible for BTB. LNG is an androgenic progestogen and so we examined the regulation of the androgen receptor (AR) in endometrium exposed to intrauterine LNG. Furthermore, as the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) regulates intracellular levels of estrogens, progestins and androgens, we evaluated the changes in expression of 17betaHSD2 in the same tissue endometrial samples. METHODS: Immunohistochemistry and real time quantitative RT-PCR were used to compare protein and mRNA expression of AR and 17betaHSD2 in endometrial biopsies from women with normal menstrual cycles and those using a LNG-IUS. RESULTS: Immunohistochemistry showed that AR and 17betaHSD2, which were immunolocalized to the stroma and glands of endometrium respectively, were both suppressed by LNG-IUS treatment, though moderate staining of 17betaHSD2 was evident 1 month after insertion of the LNG-IUS. AR mRNA expression was down-regulated in LNG-exposed endometrium when compared with the proliferative phase of the menstrual cycle. 17betaHSD2 mRNA was significantly increased 3 months (but not 6-12 months) after LNG-IUS insertion. CONCLUSIONS: Endometrial intracellular estradiol levels would have been suppressed by 17betaHSD2 during the first few, but not the later, months of LNG-IUS action, and the lowered endometrial estradiol level may contribute to the frequent BTB evident in the early months of LNG-IUS use. The subsequent decline in 17betaHSD2 would lead to elevated local intracellular estradiol in the later months, when the BTB tends to subside. The suppression of AR by the LNG-IUS may also play a role in BTB, as elevated AR has been associated with amenorrhoea.     

米非司酮对分泌早期人子宫内膜超微结构的影响

目的:探讨米非司酮对分泌早期人子宫内膜超微结构的影响。方法:子宫内膜组织取自10例排卵后1周内因非子宫内膜原因的疾病而接受子宫切除的育龄妇女。任选5例在手术前24 h口服米非司酮25 mg(米非司酮组), 其余5例未服药者为对照组。子宫内膜组织经常规电镜样品制备后, 进行电镜观察。结果:与对照组相比, 米非司酮组的子宫内膜出现下列明显的形态改变:(1)在腺上皮未见核仁管道系统及巨大线粒体, 核下糖原聚集少见, 但是, 巨大溶酶体常见; (2)腺上皮细胞间隙窄直, 侧膜褶迭少见; (3)基质细胞常出现溶解或核固缩, 并有红细胞渗出。结论:米非司酮引起的上述子宫内膜分泌早期形态改变势必造成胚泡植入的困难, 因而达到紧急避孕的效果。     

The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium

Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endometrium were studied in Norplant users and normal controls, using immunohistochemistry on formalin-fixed paraffin-embedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stages of the menstrual cycle and between normal and Norplant endometria were compared. Norplant VEGF staining index was analysed for correlation with microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period up to 90 days prior to biopsy, and the length of time since the last bleeding/spotting episode. The results showed that immunoreactive VEGF was detected predominantly in endometrial glands but weakly expressed in the stroma throughout the menstrual cycle, and also in Norplant users. Large variation in the VEGF staining index between individuals was observed and no significant difference in the VEGF staining index was detected between stages of the menstrual cycle for the glands and stroma. The glandular and stromal VEGF staining indices were significantly higher in Norplant than in normal endometrium (P<1x10(-4)). No correlation was found between the Norplant VEGF staining index and endometrial microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period, and the length of time since the last bleeding/spotting episode. The VEGF staining index was higher in glands than stroma for both normal and Norplant endometrium. The results suggest a differential control of endometrial glandular versus stromal VEGF expression, and possible positive effects of levonorgestrel on VEGF expression.     

Limited hormonal responsiveness of ectopic endometrium: histologic correlation with intrauterine endometrium

In order to assess the hormonal responsiveness of ectopic endometrium, 438 unselected endometrial implants and corresponding intrauterine endometrium from 196 patients were evaluated and classified by standard endometrial dating criteria. Only 13% of the endometrial implants were histologically synchronous with the corresponding intrauterine endometrium. Both proliferative and secretory implants were present in relatively constant proportions throughout the menstrual cycle, demonstrating a lack of correlation with cyclic endogenous hormones. A significant percentage (range, 25% to 49%) of endometrial implants displayed some form of local hemorrhage irrespective of the menstrual cycle timing. Sixty percent of the patients had evidence of hemorrhage in at least one implant. In women receiving hormonal therapy at the time of surgery, the proportion of endometrial implants that were histologically in concert with the corresponding endometrium ranged from 0% to 33%. In early pregnancy and menopause, 50% and 31% of endometrial implants were histologically similar, respectively. These data indicate that the hormonal responsiveness of endometrial implants is unpredictable and inconsistent.     

The effects of the levonorgestrel intrauterine system (Mirena coil) on endometrial morphology

AIMS: The Mirena coil is a levonorgestrel releasing intrauterine device that is in widespread use. This study aims to document the endometrial morphology associated with this device. METHODS: Endometrial specimens from 75 women with the Mirena coil were reviewed and the histological features detailed. RESULTS: Morphological features found in most of the endometria were decidualisation of stroma (72 of 75 cases), atrophy of endometrial glands (65 of 75 cases), a surface papillary pattern (38 of 75 cases), and a stromal inflammatory cell infiltrate (59 of 75 cases). Additional common histological features were the presence of foci of stromal myxoid change (29 of 75 cases) and stromal haemosiderin pigment (24 of 75 cases). Reactive atypia of surface glands, glandular metaplastic changes, stromal necrosis, and stromal calcifications were found in small numbers of cases. CONCLUSION: The endometrial features are characteristic and relatively constant and are in keeping with the effects of both a progestogenic compound and a mechanical device. Pathologists should be aware of these histological features because the Mirena coil is in widespread use.     

The receptive endometrium is characterized by apoptosis in the glands

Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation - a key feature of apoptosis - detected by using the terminal deoxynucleotidyl transferase- mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15-19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19-20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment comprising the large granular lymphocytes - during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation.      

子宫内膜微创术及宫腔内人工授精治疗不明原因不孕的疗效观察

目的观察子宫内膜微创术及宫腔内人工授精治疗不明原因不孕患者的疗效。方法选择2008年3月至2009年9月因不明原因不孕在不孕不育门诊就诊的患者108例。所有患者均进行过3次及以上正规宫腔内人工授精未孕。以上患者随机分为研究组47例,对照组61例。研究组于月经周期第8-10天行子宫内膜微创术,用5号刮匙酌情轻微搔刮子宫内膜壁,当周期于卵泡成熟破裂前后行IUI。对照组仅行IUI。结果研究组47例共进行82周期治疗,对照组61例共进行125周期治疗,研究组临床妊娠率20.73%,累积妊娠率36.17%。对照组妊娠率12%,累积妊娠率24.59%。结论子宫内膜微创术联合IUI治疗,可明显提高不明原因不孕患者的妊娠率,无毒副作用,易于操作,值得基层推广应用。     

Cytopathological changes in human cervix and endometrium following prolonged retention of copper-bearing intrauterine contraceptive devices

Since 1971, cytological evaluation of cervical smears and endometrial aspirates was carried out in 604 women wearing CuT200 intrauterine contraceptive devices (IUDs) for periods ranging from 6 mo to 15 yr. No cases of cervical neoplasia or endometrial carcinoma were encountered, even after continuous use of the device for 15 years. Dysplastic cervical smears were, however, found in 45 postinsertional smears, and endometrial hyperplasia was detected in seven aspirates; in no cases was the dysplasia or hyperplasia higher than of moderate degree. Thirty-nine of the 45 women with postinsertional dysplastic smears were followed for 3-4 yr; in no case did the lesion progress to a higher grade or to frank malignancy. However, persistence and recurrence of dysplasia were seen in 10 women, necessitating removal of the IUDs. The incidence of cervical dysplasia and endometrial hyperplasia was found to be much higher when the IUDs had been changed than when the original devices were worn continuously. The rate of removal of IUDs because of persistent or recurring dysplasia was also much higher in the former group. Since no pregnancies were reported in any of the women wearing the original device for as long as 15 yr, we do not advocate the practice of changing the device at the end of 3 yr for maintaining contraceptive efficacy as recommended by the manufacturers; instead, we recommend the uninterrupted retention of the original device for periods not longer than 5 yr in view of occurrence of endometrial hyperplasia in two 6-yr wearers.     

The allocation of attention during locomotion is altered by anxiety

We tested the hypotheses that: (1) anxiety regarding the possibility of falling alters the attentional demands of gait; and (2) this alteration in the attentional requirements of gait occurs independently of gait pattern accommodations. Sixteen younger and 15 older adults participated in this study. Subjects walked at a self-determined velocity along a 7.2-m walkway under four conditions of postural threat; the walking conditions varied depending on the width constraints of the walkway (60 cm vs 15 cm) and the height of the walking surface (0 cm vs 60 cm). Attentional demands of locomotion in each condition of testing were assessed using the dual-task methodology, in which participants verbally responded to an auditory cue as quickly as possible while continuing to walk. Our findings revealed that: (1) participants were successful in verbally responding to the auditory cue without modifying their gait pattern; (2) reaction times increased for all subjects when walking in the condition of greatest postural threat; (3) the attentional demands for locomotion varied with the phase of the gait cycle for younger adults but not for older adults; (4) the phase-dependent effect for younger adults disappeared in the condition of greatest postural threat, while reaction time scores for older adults systematically increased; and (5) increases in reaction time persisted despite significant changes in gait kinematics. Our findings confirm that anxiety increases the attentional demands for locomotion and provide further explanation for age-dependent increases in the attentional demands of gait. Furthermore, our findings confirm that fall-related anxiety predicates an increase in the allocation of attention to locomotor control that is independent of gait pattern adjustments. Electronic Publication     

Morphological and functional features of endometrial decidualization following long-term intrauterine levonorgestrel delivery

Irregular bleeding remains a common reason for the discontinuation of progestin-only contraception. The levonorgestrel releasing intrauterine system (LNG-IUS) has profound morphological effects upon the endometrium. Specific features are gland atrophy and extensive decidual transformation of the stroma. Morphological changes in the endometrium may be associated with perturbation of mechanisms regulating normal endometrial function. This study describes endometrial stromal and glandular features prior to and up to 12 months following insertion of the LNG-IUS. Comparison is made with first trimester decidua. In order to elucidate further mechanisms governing endometrial function with local intrauterine delivery of LNG, we here report histological features consistent with decidualization; a significant increase in granulocyte-macrophage colony stimulating factor (GM-CSF) immunoreactivity in decidualized stromal cells; glandular and stromal prolactin receptor expression and an infiltrate of CD56 + large granular lymphocytes and CD68 + macrophages. We are unaware of previous reports which have documented longitudinally both morphological and functional observations in endometrium exposed to local intrauterine levonorgestrel delivery. These studies demonstrate that long-term administration of intrauterine levonorgestrel results in features of altered morphology and function. No correlation was apparent between the end points in the study and the bleeding patterns described by the subjects. Further evaluation of these features in the context of menstrual bleeding experience may contribute to a better understanding of this troublesome side-effect which often leads to dissatisfaction and discontinuation of the intrauterine system.