Knowledge, attitude and practice of emergency contraceptive among women who seek abortion care at Jimma University specialized hospital, southwest Ethiopia

Background

Uterine receptivity and implantation are complex processes requiring coordinated expression of molecules by zygote and uterus. Our objective was to evaluate the role of the endometrial expression of leukemia inhibitory factor (LIF) and its glycoprotein 130 (gp130) receptor molecules and their secretion in uterine flushing during the window of implantation in cases of primary unexplained infertility

Case presentation

The study was conducted on 25 infertile women with unexplained infertility for at least two years and 10 normal fertile women as a control group . Endometrial tissue and uterine flushing were obtained. Each tissue specimen was divided into two pieces; one piece was used for histological dating of the endometrium and for immunostaining of progesterone receptors, and the second was used for RNA extraction and PCR assay of LIF and gp130 mRNA expression. Serum estrogen and progesterone were measured for all subjects. LIF mRNA was expressed in the endometrium of all normal fertile women but significantly decreased in infertile women. LIF was not detectable in 88% of infertile women while it was fairly detectable in 12% of them. Gp130 mRNA was hardly detectable in both fertile and infertile women with no difference between them. Infertile women secreted significantly less LIF and gp130 molecules in the uterine flushing compared with normal fertile women.

Conclusions

Expression of LIF mRNA in endometrium could be used as a molecular marker of unexplained infertility. Assessment of secreted LIF and gp130 molecules in uterine flushing could be another useful and safe method for predicting successful implantation as well as for diagnosing and eventually treating women with impaired fertility using recombinant human LIF.     

The termination of early pregnancy in the mouse by β-momorcharin

β-Momorcharin is a basic glycoprotein found in the seeds of $\text{Momordica charantia}$ L. It differed immunologically from α-momorcharin also of the same source but possessed similar midterm abortifacient activity. Intraperitoneal administration of β-momocharin into mice on Days 4 and 6 of pregnancy led to an inhibition of pregnancy. $\text{In vitro}$ study showed that the protein disturbed peri-implantation development by: (a) blocking the hatching of embryos from the zona pellucida; (b) decreasing the incidence of successful attachment of the blastocyst; (c) reducing the trophoblast outgrowth; and (d) disrupting the development of inner cell mass. It is likely that β-momorcharin inhibited embryonic implantation by a similar biological action previously described for α-momorcharin.     

Monoclonal anti-leukemia inhibitory factor antibody inhibits blastocyst implantation in the rhesus monkey

INTRODUCTION: Leukemia inhibitory factor (LIF) appears important in the process of blastocyst implantation in primates. In the present study, we investigated the effect on pregnancy outcome of the administration of a monoclonal antibody (mAb) to recombinant human (rh) LIF (anti-LIF mAb) into the uterine cavity of mated female rhesus monkeys during the peri-implantation period. METHODS: Two milligrams of either mouse mAb to rhLIF or isotype-matched immunoglobulin (Ig) was administered into the uterine cavity on estimated Day 5 or Day 8 after ovulation either through the vagina (n=33) or through the oviduct (n=29) of successfully mated females. RESULTS: There was a significant (p<.04) decline in pregnancy outcome in groups treated with anti-LIF mAb (8 pregnancies from 32 animals) compared with control animals (24 pregnancies from 30 animals). There was, however, no significant difference in pregnancy inhibition between a group of animals subjected to vaginal route treatment and a group of animals subjected to oviductal route treatment, as well as between a group subjected to anti-LIF mAb on Day 5 after ovulation and a group subjected to anti-LIF mAb on Day 8 after ovulation. No significant change was seen in the number of viable pregnancy in animals treated with 6 mg of anti-LIF mAb (5 pregnancies from 16 animals) compared with animals treated with 2 mg of anti-LIF mAb (8 pregnancies from 32 animals). Serum profiles of estradiol, progesterone, monkey chorionic gonadotropin and mouse IgG did not show any difference among different treatment subgroups during the luteal phase. However, among animals treated with anti-LIF mAb, the mean area under the curve for serum mouse IgG in pregnant animals (234+/-55 microg/mL day) was significantly (p<.01) less than that of nonpregnant animals (1325+/-97 microg/mL day). CONCLUSION: The results of the present study put forward the proof of concept that LIF plays a critical role in the process of blastocyst implantation in the rhesus monkey.     

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.     

Potential and limitations of bovine-specific arrays for the analysis of mRNA levels in early development: preliminary analysis using a bovine embryonic array

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.     

白血病抑制因子在女性生殖系统的表达及调控

白血病抑制因子是具有抑制多项生长潜能的胚胎细胞分化并保持其增殖能力的细胞因子。越来越多的研究发现白血病抑制因子影响生殖活动的各个环节,它表达于生殖系统的各个部位,是胚泡着床时所必须的。对白血病抑制因子的理化性质、分子量、生物活性进行综述,探讨其在生殖领域的作用。     

Target-oriented anti-implantation approaches for pregnancy interception: experiences in the rhesus monkey model

Blastocyst implantation is a critical process in the establishment of pregnancy in eutherian mammals and requires a harmonious symbiosis between the developing conceptus and the differentiating maternal uterus. A better understanding of this symbiotic relationship will provide novel approaches and interventions for realizing anti-implantation strategies for effective fertility regulation and reproductive health care management. We have been using the rhesus monkey (Macaca mulatta) as a nonhuman primate model to this end. In the present study, the process of progesterone-mediated regulation of endometrial receptivity for blastocyst implantation has been targeted by the use of mifepristone as an emergency contraceptive agent. Furthermore, based on cell-specific, temporal and spatial distribution of vasotropic cytokines and mediators in the "receptive" and periimplantation periods, the pregnancy interceptive potentials of (a) monoclonal antibody (MAb) to leukemia inhibitory factor (LIF); (b) inhibitors of nitric oxide synthase [e.g., N6-nitro-l-arginine (l-NAME) and aminoguanidine]; and (c) MAb to vascular endothelial growth factor (VEGF) were examined. LIF is a progesterone-responsive pleiotropic cytokine that functions as a proinflammatory cytokine, together with interleukins 1 and 6, during the process of implantation-placentation in primates, and its immunoneutralization with MAb resulted in inhibition (p<.04) of pregnancy establishment in the rhesus monkey. However, timed administration of l-NAME or aminoguanidine failed to inhibit blastocyst implantation in a significant manner. Also, no synergistic antinidatory action of antiprogestin combined with l-NAME was detected in the rhesus monkey. The application of MAb to VEGF during the periimplantation period, on the other hand, led to significant (p<.04) prevention of pregnancy without influencing steroid hormone levels in the circulation. Our data lend support to the hypothesis that VEGF is essential for pregnancy establishment and that trophoblast-derived VEGF, acting via its specific receptors Flt-1 and KDR, is necessary for blastocyst implantation. The use of cDNA-based expression arrays followed by differential display analysis has provided preliminary understanding of the nature of gene cluster networks operative in the receptive endometrium of potential conception cycles in the rhesus monkey. This knowledge may, in the future, lead to further innovative anti-implantation strategies for targeted pregnancy interception.     

Irisin对肥胖大鼠子宫内膜功能的影响

目的：观察鸢尾素（Irisin）对肥胖大鼠子宫内膜功能的影响。方法：选用成年雌性SD大鼠,采用高脂饲料建立肥胖模型;正常组给予正常饮食。将造模成功大鼠随机分为肥胖模型组（16只）和Irisin组（16只）,另设对照组（16只）。其中,Irisin组腹腔注射重组Irisin[100 ng/（g·d）×14 d],肥胖模型组和对照组腹腔注射生理盐水,并于注射前后记录大鼠体质量。后于动情期内合笼,于大鼠妊娠D5、D10天每组各处死8只大鼠,记录妊娠率及平均胚泡着床数,观察子宫内膜组织形态;检测大鼠子宫内膜白血病抑制因子（LIF）、整合素αvβ3蛋白及mRNA的表达。结果：Irisin组经腹腔注射Irisin 14 d后,大鼠体质量低于肥胖模型组,差异有统计学意义（P=0.035）。肥胖模型组平均胚泡着床数比对照组和Irisin组低,差异有统计学意义（均P＜0.05）。对照组大鼠子宫内膜宫腔饱满,胚胎发育好;肥胖模型组大鼠子宫腔间隙较大,胚胎发育不良;Irisin组大鼠子宫腔接近闭合,胚胎发育良好。肥胖模型组子宫内膜LIF、αvβ3的蛋白表达量和mRNA含量比对照组和Irisin组低,差异有统计学意义（均P＜0.05）。结论：Irisin可通过增加子宫内膜LIF、αvβ3的表达,从而改善肥胖大鼠子宫内膜容受性。     

种植窗期子宫内膜类型和白血病抑制因子的关系

目的:研究种植窗期子宫内膜类型和白血病抑制因子(LIF)的关系。方法:对94例不孕患者于排卵后7~9天行B超测量子宫内膜的厚度、宫腔镜观察子宫内膜血管网及腺体的形态分布、酶连免疫吸附实验测定宫腔冲洗液中LIF浓度。结果:Ⅰ组(腺体开口小或内膜血管呈点状或片状分布)不孕患者的平均年龄高于Ⅱ组(腺体开口大、极度扩张呈指环状,且内膜血管呈网状分布)(P<0.01),不孕年限无差异(P>0.05);原发不孕和继发不孕患者子宫内膜类型无差异(P>0.05);Ⅰ组和Ⅱ组患者的子宫内膜厚度无差异(P>0.05);Ⅰ组宫腔冲洗液中LIF浓度为(15.367±16.285)pg/ml,Ⅱ组中LIF浓度为(71.583±54.233)pg/ml,高于Ⅰ组(P<0.01)。结论:种植窗期子宫内膜类型与不孕患者的年龄及宫腔液中LIF含量有关,推测可能内膜发育差的不孕患者子宫内膜上LIF基因表达发生变异,不能分泌正常量的LIF蛋白,导致子宫内膜腺体和血管网发育不良,胚胎到达宫腔内无法着床或早期流产,这可能是影响种植窗期子宫内膜发育的分子生物学基础之一。     

Wnt信号途径及其调节因子Dkk 与子宫内膜容受性

Wnt信号通路是存在于生物体内的一条极其保守的信号转导通路,其相关基因在胚胎种植前后的子宫表达,且受标准的种植相关基因调控,如LIF 和HOXA鄄10。Dkk（Dickkopf）是一种分泌蛋白,通过调节Wnt信号转导途径发挥作用,其在子宫内膜表面表达有时空特异性,种植期表达增高,引起子宫内膜分泌期容受性发生改变。Wnt信号和Dkk 参与调节子宫内膜容受性,是其关键因素之一。     

Analysis of the effects of overexpression of metallothionein-I in transgenic mice on the reproductive toxicology of cadmium.

Exposure to low levels of cadmium reduces fertility. In male mice spermatogenesis is highly sensitive to cadmium, whereas in females the peri-implantation period of pregnancy is sensitive. To examine the potential roles of the cadmium-binding protein, metallothionein (MT), in the reproductive toxicology of cadmium, we examined a transgenic mouse strain that overexpresses metallothionein-I (MT-I). These mice had dramatically increased steady-state levels of MT-I mRNA and MT in the testes and in the female reproductive tract during the peri-implantation period of pregnancy, and this overexpression occurred in a cell-specific and temporally regulated manner similar to that of the endogenous MT-I gene. Transgenic and control males were injected with cadmium, and the histology of the testes was examined. An injection of 7.5 mumol Cd/kg had no effect on histology of the testes in either transgenic or control mice. In contrast, an injection of 10 mumol Cd/kg caused rapid changes in the histology of the testes and resulted in pronounced testicular necrosis in both control and transgenic mice. Female transgenic and control mice were mated and then injected with cadmium (30-45 mumol Cd/kg) on the day of blastocyst implantation (day 4). In both of these groups, injection of cadmium reduced pregnancy rate, and no dramatic protection was afforded by maternal and/or embryonic overexpression of MT. Thus, overexpression of MT-I does not significantly protect against either of these cadmium-induced effects on fertility.     

Wnt信号途径及其调节因子Dkk与子宫内膜容受性

Wnt信号通路是存在于生物体内的一条极其保守的信号转导通路,其相关基因在胚胎种植前后的子宫表达,且受标准的种植相关基因调控,如LIF和HOXA-10。Dkk(Dickkopf)是一种分泌蛋白,通过调节Wnt信号转导途径发挥作用,其在子宫内膜表面表达有时空特异性,种植期表达增高,引起子宫内膜分泌期容受性发生改变。Wnt信号和Dkk参与调节子宫内膜容受性,是其关键因素之一。     

Mifepristone regulation of leukemia inhibitory factor and uterine receptivity in rabbits

The effects of mifepristone on production of leukemia inhibitory factor (LIF) and uterine receptivity in rabbits was studied. In ovariectomized rabbits, LIF protein was at an undetectable level in control (score = 0), and upregulated by progesterone alone (score = 4). Estrogen had no additive effect, and may even have had a slightly negative effect when the rabbits were treated with both estrogen and progesterone (score = 3). Meanwhile, mifepristone obviously inhibited the stimulation of progesterone on the production of LIF in rabbit uterus (score = 1). The results also showed that LIF protein has a beneficial effect on uterine receptivity and mifepristone prevents this effect. The transfer of embryos to LIF-treated recipients significantly increased pregnancy (70%) and implantation rate (31%) as compared with control (pregnant rate = 50% and implantation rate = 17%). The transfer of embryos to LIF and mifepristone-treated recipients significantly decreased pregnancy (30%) and implantation rate (9%). The results of this study suggest that mifepristone prevented the effects of progesterone on LIF production and the beneficial effect of LIF on uterine receptivity.     

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.     

Uterine expression of implantation serine proteinase 2 during the implantation period and in vivo inhibitory effect of its antibody on embryo implantation in mice

The aim of the present study was to examine the uterine expression pattern of implantation serine proteinase 2 (ISP2) protein during early pregnancy in mice and the effects of anti-ISP2 antibody on embryo implantation. Expression of ISP2 protein was found to be specifically up-regulated in mouse uterine endometrial glands following the initiation of embryo implantation. Similarly, ISP2 protein expression was observed during pseudopregnancy, indicating that its expression is not embryo dependent. In other experiments, rabbit anti-ISP2 IgG was infused into the mouse uterine lumen on Day 3 or 4 of pregnancy to examine its effects on embryo implantation, whereas vehicle (saline) or unspecific rabbit IgG served as controls. The mean number of implanted embryos from anti-ISP2-IgG-treated mice was significantly lower than that from control mice. These results suggest that ISP2 may play an important role during embryo implantation.     

补肾安胎方对胚泡着床障碍小鼠子宫LIF及其受体表达的影响

目的:观察补肾安胎方对胚泡着床障碍小鼠子宫白血病抑制因子(Leukaemia inhibitory factor,LIF)及其受体(Leukaemia inhibitory factor receptor,LIFR)表达的影响。方法:将雌鼠随机分为未孕组、正常组、模型组、中药组,每组40只,在妊娠4、5、6天(Pd4、5、6)均取妊娠子宫-80℃保存。采用免疫荧光双标和免疫印迹法分别观察LIF及其受体的表达。结果:免疫印迹显示正常组和中药组Pd4LIF蛋白为表达高峰,高于模型组(P<0.01),此后下降;模型组LIF蛋白表达高峰延迟至Pd5,高于正常组和中药组(P<0.01),此后下降。免疫荧光双标显示正常组和中药组LIF及其受体Pd4荧光较强,LIF及其受体表达基本同步;Pd4模型组LIF及LIFR均有较少表达,但LIFR表达更弱,其范围及强度不如LIF广泛,二者表达强度不完全同步;Pd5为模型组LIF及LIFR表达高峰,此后表达强度降低。结论:补肾安胎方可能通过促进LIF及其受体的同步表达来促进胚泡着床。     

Effect of interferon-gamma treatment on the expression of interleukin-1beta at the maternal-fetal interface of pregnant rats

In the present study, the possible mechanisms by which interferon (IFN)-gamma affects pregnancy were investigated using the cytokine network model. The IFN-gamma-induced expression of interleukin (IL)-1beta was examined using western blotting, immunohistochemistry and immunofluorescence. The results showed that IFN-gamma treatment significantly decreased the expression of uterine IL-1beta protein during the preimplantation, post-implantation and mid-gestation periods. The expression of IL-1beta protein was increased after IFN-gamma treatment compared with the control group in late pregnancy. In the placenta, IL-1beta protein levels were significantly increased after IFN-gamma treatment in early and mid-pregnancy. In late pregnancy, IFN-gamma treatment significantly decreased placental IL-1beta protein levels. IL-1beta was mainly expressed in the myometrium, uterine arteries, decidua basalis, trophospongium of the junctional layer and trophoblastic epithelium of the labyrinthine layers. IL-1beta was mainly located in the cytoplasm of in vitro cultured endometrial stromal cells (ESCs). IFN-gamma treatment did not affect the distribution of IL-1beta, only the expression of IL-1beta. The effects of IFN-gamma on the proliferation of ESCs were determined using an MTS (a novel tetrazolium compound) assay. IFN-gamma treatment inhibited the proliferation of ESCs and decreased the weight of the fetus and placenta. These results indicate that exogenous IFN-gamma affects the expression of IL-1beta and inhibits ESC proliferation.     

白血病抑制因子及其受体在早孕蜕膜和绒毛组织中表达的研究

目的探讨正常早孕蜕膜和绒毛组织白血病抑制因子(LIF)及其受体(LIFR)的表达. 方法采用原位杂交和免疫组织化学染色技术对18例正常早孕妇女蜕膜和绒毛组织LIF-mRNA 、LIF和LIFR蛋白的表达进行研究. 结果全部早孕蜕膜组织和绒毛滋养细胞中有LIF-mRNA、LIF和LIFR蛋白表达,同一组织和细胞LIF和LIFR基因表达强弱基本一致,LIFR在绒毛滋养细胞的表达强于蜕膜. 结论人类蜕膜和绒毛滋养细胞LIF和LIFR基因表达可能与胚泡着床、维持胎盘功能和促进胚胎发育有关.