Progestin modulation of c-fos and prolactin gene expression in the human endometrium

Objective: To evaluate the influence of the menstrual cycle and the effects of medroxyprogesterone acetate (MPA) on the expression of the protooncogene c-fos and of prolactin (PRL) in the human endometrium in vivo.Design: Double-blind, placebo-controlled trial.Setting: Healthy volunteers in an academic research environment.Patient(s): Regularly cycling women who were not taking hormonal medication.Intervention(s): Medroxyprogesterone acetate (10 mg/d) or placebo was given for 10 days. Endometrial and blood samples were collected 8–12 hours after the last dose.Main Outcome Measure(s): Immunohistochemical localization of PRL and c-fos in the endometrium, PRL and c-fos messenger RNA levels in the endometrium, and E₂ and progesterone levels in the serum.Result(s): Immunoreactive c-fos was concentrated in the nucleus of stromal cells and was observed in a higher proportion of proliferative endometrial specimens compared with secretory specimens from placebo- or MPA-treated patients. The levels of c-fos messenger RNA were greatly reduced in the secretory endometrium regardless of treatment with placebo or MPA, compared with the proliferative endometrium. The c-fos gene expression correlated positively with the serum E₂ levels (r = 0.56) and inversely with the progesterone/E₂ ratio (r = −0.56). The endometrial PRL gene expression (messenger RNA and protein) was rare in the proliferative samples, increased from the early to the mid and late secretory samples, and was increased markedly after treatment with MPA compared with placebo.Conclusion(s): The differentiation of secretory endometrium is accompanied by decreased c-fos and increased PRL gene expression. The inhibition of c-fos gene expression may contribute to the antiproliferative effect of progestins on the endometrium.     

Ryudocan expression by luteinized granulosa cells is associated with the process of follicle atresia

Objective: To evaluate the presence of ryudocan in follicular fluid (FF) and its possible correlation with FF E₂ and P, and to study the levels of ryudocan in granulosa-lutein cells stimulated with hCG.

Design: Controlled clinical study and in vitro experiment.

Setting: University teaching hospital.

Patient(s): One hundred seven patients undergoing IVF.

Intervention(s): The FF and granulosa-lutein cells were aspirated from follicles 34 hours after an ovulatory gonadotropin bolus.

Main Outcome Measure(s): FF ryudocan, E₂, and P levels as well as hCG-mediated induction of ryudocan.

Result(s): Ryudocan was abundant in the FF; the concentration of ryudocan in human FF was estimated to be 305.5 ± 200.8 ng/mL (mean ± SD). Atretic follicles had higher concentrations of ryudocan (559.1 ± 156.5 ng/mL). FF ryudocan levels were inversely correlated with FF E₂ (r = −0.5023) and P concentrations (r = −0.4459). A detectable amount of ryudocan was found in pooled granulosa-lutein cells. Ryudocan production was augmented by surge levels of hCG.

Conclusion(s): Ryudocan is expressed in luteinized granulosa cells in vitro. The higher concentrations of ryudocan in FF of atretic follicles suggest an involvement of ryudocan in the process of atresia.     

Hyaluronan in follicular fluids and fertilization of oocytes

Objective: To determine the concentrations of hyaluronan, E₂, and progesterone in follicular fluids (FFs) and the incidence of apoptotic granulosa cells. Also, to examine the relationship between the concentration of hyaluronan and follicular steroids, the incidence of apoptotic cells, and the fertilizability of the oocyte in the same follicle.

Design: Samples of 130 follicles were retrospectively analyzed for hyaluronan and steroids and the incidence of apoptotic cells.

Setting: The reproductive center in Yamagata University Hospital.

Patient(s): Forty women infertile because of tubal damage or unknown causes undergoing IVF treatment were selected.

Intervention(s): The samples were collected from follicle aspirations.

Main Outcome Measurement(s): The concentrations of hyaluronan and steroids in FFs, the incidence of apoptotic granulosa cells, and oocyte fertilizability.

Result(s): The levels of hyaluronan in FF were found to correlate positively with P (r=0.444, P<0.0001) and the incidence of apoptotic cumulus granulosa cells (r=0.387, P=0.002) and inversely with E₂ (r = −0.601, P<0.0001) and free T (r = −0.344, P=0.001). The concentration of hyaluronan in FFs containing a subsequently fertilized oocyte after insemination was significantly lower than that in FFs containing a subsequently unfertilized oocyte (P=0.0005) (fertilized, 50.0 ± 2.6 ng/mL; triploidy, 59.1 ± 6.8; and unfertilized, 66.9 ± 5.9).

Conclusion(s): The concentration of hyaluronan in FF is an indicator for estimation of oocyte viability for fertilization.     

Is there evidence for preferential delivery of ovarian estradiol to the endometrium?

Objective: To determine the direction of delivery of E₂ in the female pelvis by assessing the ratio of endometrial to serum E₂ in women whose ovaries were stimulated to produce E₂ with women who received exogenous E₂.

Design: Prospective comparative study.

Setting: University-based ART program.

Patient(s): Oocyte donors and recipients of donor oocytes.

Intervention(s): Micronized E₂ administered by the oral or vaginal route and oocyte donation.

Main Outcome Measure(s): Serum and endometrial levels of E₂.

Result(s): Serum E₂ levels were significantly higher in women who underwent controlled ovarian hyperstimulation (COH) and women receiving exogenous E₂ by the vaginal route than in those who received oral E₂. Levels of E₂ in endometrial tissue were similar in women who underwent COH and those receiving oral E₂. Endometrial E₂ levels in women who underwent vaginal administration were significantly higher than those in the oral E₂ or COH groups. The ratio of endometrial to serum E₂ was highest in women who underwent vaginal E₂ and lowest in those undergoing COH.

Conclusion(s): Vaginal administration of micronized E₂ results in preferential absorption of E₂ into the endometrium, consistent with a “uterine first pass” effect. Since endogenous E₂ produced the smallest ratio of E₂ between the endometrium and serum, E₂ produced by the ovaries is not preferentially delivered to the uterus.     

雌、孕激素对人子宫内膜Pbx2蛋白表达的影响

目的:探讨人子宫内膜腺上皮细胞和基质细胞中雌、孕激素对Pbx2蛋白表达的影响。方法:采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶(SP)连接法检测人增生期、分泌中期和蜕膜期子宫内膜中Pbx2的表达和分布;体外培养人子宫内膜基质细胞和高分化子宫内膜癌细胞Ishikawa,分别加入雌激素、孕激素、雌、孕激素联合刺激48 h,并以不加雌、孕激素的基质细胞核和Ishikawa细胞为对照组,采用免疫组织化学、免疫印迹法测定各种条件下Ishikawa细胞中Pbx2蛋白的表达。结果:①在人各期子宫内膜组织中,Pbx2表达于腺上皮细胞和基质细胞的细胞核中;Pbx2在分泌中期和蜕膜期基质细胞中的表达显著高于增生期,但在腺上皮细胞中增生期组织的表达高于分泌中期和蜕膜期,差异均具有统计学意义(P<0.05)。②Western blotting结果显示,在孕激素和雌、孕激素联合处理Ishikawa细胞组中,Pbx2的表达显著低于对照组(P<0.05),雌激素处理后Pbx2的表达与其他各组间的表达无显著性差异(P>0.05),在人子宫内膜基质细胞(ESC)中,Pbx2的表达在雌、孕激素处理各组间比较均无统计学差异(P>0.05)。结论:在人子宫内膜腺上皮Ishikawa细胞中孕激素对Pbx2的表达具有下调作用,在人子宫内膜基质细胞中,Pbx2的调控可能是非雌、孕激素依赖性的。     

Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients

Objective(s): To measure vascular endothelial growth factor (VEGF), nitric oxide (NO) and leptin levels in individual ovarian follicles and to examine their relationships with perifollicular blood flow, follicular metabolic indices, and the developmental potential of the corresponding oocyte and embryo.

Design: Prospective study.

Setting: Academic, tertiary care institution.

Patient(s): Unselected IVF patients.

Intervention(s): Color-pulsed Doppler analysis of perifollicular blood flow; determination of partial pressure of oxygen (pO₂), partial pressure of carbon dioxide (pCO₂), and pH and VEGF, leptin and NO levels in follicular fluid.

Main Outcome Measure(s): Fertilization and day 3 embryo morphology and cleavage.

Result(s): Fifty-five follicular fluid samples from 16 patients were studied. Mean follicular fluid levels were as follows: VEGF, 1,046 ± 863.7 pg/mL (range, <63–3,332.7 pg/mL); NO₃/NO₂, 34.2 ± 12 μM (range, 16.4–76.1 μM); and leptin, 20.1 ± 12.1 ng/mL (range, 3.3–52.2 ng/mL). Vascular endothelial growth factor had a negative correlation with embryo morphology (r = −0.28, P=.01). Leptin demonstrated a negative correlation with follicular pO₂ (r = −0.42, P=.005) and a positive correlation with follicular pCO₂ (r = 0.36, P=.02). Follicular leptin levels correlated positively with VEGF levels (r = 0.46, P=.008) and with NO₃/NO₂ levels (r = 0.39, P=.006).

Conclusion(s): Vascular endothelial growth factor, NO and leptin appear to be markers of follicular hypoxia and suboptimal embryo development. Whether fluctuations of these regulatory factors determine or reflect changes in the follicular microenvironment affecting oocyte developmental potential remains to be elucidated.     

Expression of aminopeptidase N in human endometrium and regulation of its activity by estrogen

Objective: To determine whether aminopeptidase N (APN) regulates the cycle-dependent bioavailability of interleukin-8 (IL-8) in the endometrium.

Design: Prospective study.

Setting: University medical center.

Patient(s): Women without endometrial pathology from the proliferative (n = 25) or secretory (n = 18) phase of the menstrual cycle.

Intervention(s): We first immunolocalized APN in the endometrium using an anti-APN antibody. We then determined the regulation of APN kinetic activity by sex steroids in endometrial stromal cell cultures.

Main Outcome Measure(s): Expression of APN in human endometrium throughout the menstrual cycle. Regulation of APN activity by estradiol and progesterone in cultured endometrial stromal cells.

Result(s): Immunohistochemistry of endometrial sections revealed staining of endometrial stroma throughout the menstrual cycle. There was no detectable staining in glandular cells. The expression of APN as detected by immunohistochemistry was significantly lower in the early proliferative phase. In cultured cells, estradiol inhibited APN activity in a concentration-dependent manner. Progesterone did not have a significant effect.

Conclusion(s): Stromal localization of APN in endometrium may explain the epithelial rather than stromal presence of IL-8 in vivo. Decreased expression of APN may increase IL-8 bioavailability thus contributing to angiogenesis and polymorphonuclear leukocyte chemotaxis in early proliferative phase.     

In Vivo Assessment of the Regulation of Transforming Growth Factor Alpha,Epidermal Growth Factor (EGF), and EGF Receptor in the Human Endometrium by Medroxyprogesterone Acetate

Purpose: The present study evaluated the in vivo effect of medroxyprogesterone acetate (MPA) on the localization of immunoreactive transforming growth factor alpha (TGF), epidermal growth factor (EGF), and their common receptor (EGF-R) in the human endometrium.Methods: The study design was a randomized clinical trial enrolling 36 healthy women with regular menstrual cycles. The participants were randomly assigned into three groups: groups 1 (n = 11) and 2 (n = 17) received placebo and were submitted to endometrial biopsy during the proliferative and secretory phases of menstrual cycle, respectively; group 3 (n = 8) received MPA (10 mg/day) for 10 days followed by endometrial biopsy, which was performed during the secretory phase. Immunohistochemistry was used to localize TGF, EGF, and EGF-R in the endometrial tissue.Results: TGF was present markedly in the luminal and glandular epithelia but also in the periglandular stroma, with a distribution pattern similar in the three experimental groups. EGF immunostaing was equally distributed in epithelial and stromal layers of the endometrium and remained unchanged in endometrial samples from women treated with MPA compared to placebo. EGF-R was expressed only in the epithelium. The intensity of EGF-R immunostaining was higher in secretory than in proliferative endometrium and was further increased by administration of MPA (p < 0.05, chi-square test).Conclusion: The present results suggest that the progestogen-induced in vivo differentiation of secretory endometrium does not require dramatic changes in the expression of EGF or TGF, whereas EGF-R may be up regulated.     

Immunohistochemical localization of insulin-like growth factor-binding protein-3 in eutopic and ectopic endometrial tissues

Objective: To evaluate the expression of insulin-like growth factor–binding protein-3 (IGFBP-3) in the eutopic endometrium and in endometriotic lesions.

Design: Retrospective immunohistochemical study.

Patient(s): Twenty-five normal women and 39 women with endometriosis.

Intervention(s): Endometrial and endometriotic tissue biopsies obtained at laparoscopy.

Main Outcome Measure(s): Expression of IGFBP-3 assessed by immunohistochemistry.

Result(s): In the endometrium, positive immunostaining of IGFBP-3 was observed both in the stroma and the epithelial glands. The intensity of staining in the glands during the secretory phase was significantly higher in women with endometriosis compared with controls (P=.018). An increased expression of IGFBP-3 over controls was found in stages I and II of the disease (P=.018), whereas in stages III and IV, the difference between controls and women with endometriosis was not significant (P=.300). In endometriotic tissues, a much-marked immunostaining of IGFBP-3 was noted in 90% of the glands and 67% of the stroma without apparent differences related to cycle phase.

Conclusion(s): These data show intense staining of IGFBP-3 in endometriosis lesions and increased expression of the protein in the endometrium of patients with endometriosis compared to controls. This marked expression of IGFBP-3 could be related to its previous finding in the peritoneal fluid and to its potential involvement in the pathophysiology of endometriosis.     

PTEN与bcl-2、FasL在异位子宫内膜的表达及临床意义

目的:研究PTEN与bcl-2、FasL的表达与异位子宫内膜的关系。方法:用免疫组化法检测PTEN与bcl-2、FasL在正常子宫内膜,异位内膜及在位内膜的表达。结果:(1)正常子宫内膜组:bcl-2在增生期的表达显著高于分泌期(P0.05),FasL及PTEN在增生期的表达显著低于分泌期(P0.05),即三者在正常子宫内膜细胞的表达均有明显周期性;(2)异位内膜组(OEM及AM):bcl-2、FasL及PTEN的表达在增生期和分泌期的表达均无统计学差异(P0.05)。即在异位子宫内膜中的bcl-2、FasL及PTEN呈持续表达且失去周期性;(3)在位内膜组(OEM及AM):与正常子宫内膜组相似,三者在增生期和分泌期的表达均有差异,其中,FasL与PTEN在分泌期的表达高于增生期,bcl-2在增生期的表达则高于分泌期,差异均有统计学意义(P0.05);(4)3组内膜中的表达比较:FasL及bcl-2在异位内膜组的表达均高于在位内膜组及正常子宫内膜(P0.05),PTEN在异位内膜组的表达均低于在位内膜组及正常子宫内膜(P0.05);FasL、bcl-2及PTEN在正常内膜组的表达与在位内膜组无统计学差异(P0.05)。结论:bcl-2、FasL及PTEN的表达变化在子宫内膜异位症的发生发展过程中起着重要作用。     

乙酰肝素酶基因在子宫内膜异位症患者在位与异位子宫内膜组织中的表达

目的　研究乙酰肝素酶基因在子宫内膜异位症发病中的作用。方法　应用原位杂交技术 ,检测腹腔镜手术中证实为子宫内膜异位症患者 (EM组 ,2 3例 )的在位和异位子宫内膜组织乙酰肝素酶mRNA的表达 ,并与正常妇女 (对照组 ,2 5例 )子宫内膜组织比较。结果　 ( 1)EM组在位与异位子宫内膜乙酰肝素酶mRNA阳性表达率一致 ,但异位子宫内膜组织中 ,阳性细胞着色较深。在位子宫内膜腺上皮细胞较间质细胞胞浆染色深 ,异位子宫内膜腺上皮细胞与间质细胞胞浆着色基本一致 ;EM组增生期与分泌期子宫内膜均有乙酰肝素酶mRNA的表达 ,增生期阳性表达率为 83 3%( 10 / 12 ) ;分泌期为 72 7% ( 8/ 11) ,两者比较 ,差异也无显著性 (P >0 0 5 ) ;( 2 )对照组增生期子宫内膜乙酰肝素酶mRNA的阳性表达率为 4 1 7% ( 5 / 12 ) ,分泌期子宫内膜阳性表达率为 7 7% ( 1/ 13) ,两者比较 ,差异有显著性 (P <0 0 5 )。 ( 3)EM组子宫内膜乙酰肝素酶mRNA阳性表达率为 78 3% ( 18/2 3) ;对照组为 2 4 0 % ( 6 / 2 5 ) ,两者比较 ,差异也有显著性 (P <0 0 5 )。结论　乙酰肝素酶基因的表达与子宫内膜异位症的发病相关 ,其可能成为子宫内膜异位症治疗的一个有价值的靶点。     

Effects of the route of estrogen administration and exercise on hormonal levels in postmenopausal women

Objective: To examine the effects of exercise on serum estrogens, growth hormone, insulin, cortisol, lactate, and glucose levels in postmenopausal women receiving two routes of administration of estrogen replacement therapy (ERT).

Design: Prospective, randomized, crossover study.

Setting: The general clinical research center of an academic medical center.

Patient(s): Eleven active, postmenopausal women.

Intervention(s): The patients were screened with exercise stress testing, then oral micronized estradiol or transdermal estradiol was administered, followed by two 45-minute submaximal exercise tests. Dietary intake before the tests was standardized.

Main Outcome Measure(s): The study measured maximal heart rate and aerobic power ( ₂max), and serum levels of estradiol (E₂), estrone (E₁), cortisol, growth hormone (GH), insulin, glucose, and lactate.

Result(s): Growth hormone, cortisol, and insulin all changed significantly in response to the 45-minute exercise bouts, but no differences were observed between the oral micronized estradiol and transdermal estradiol responses. E₂ levels increased significantly during the transdermal estradiol 45-minute exercise bout; this change did not occur during the oral estradiol exercise bout. In the transdermal estradiol treatment group, the E₂ levels at +30 and +45 minutes of exercise were elevated compared to the post-exercise levels at −15, 0, and 30 minutes. E₁ was not significantly changed during the 45-minute exercise bouts in either group.

Conclusion(s): During exercise, serum E₂ levels rise significantly higher with transdermal but not oral routes of E₂ administration. However, the elevated levels are not prolonged and normalize by 30 minutes after exercise.     

Elevated Levels of Basal Estradiol-17β Predict Poor Response in Patients with Normal Basal Levels of Follicle-Stimulating Hormone Undergoing In Vitro Fertilization

Objective: To evaluate whether the predictive ability of a normal FSH level on cycle day 3 can be enhanced by levels of estradiol-17β (E₂) on cycle day 3.

Design: Prospective cohort study.

Setting: University hospital–based, tertiary care infertility center.

Patient(s): Two hundred thirty-one consecutively seen patients who attended the center for their first IVF attempt.

Intervention(s): Blood samples were collected on day 3 of the cycle preceding IVF; IVF was performed in all patients.

Main Outcome Measure(s): Patient’s age, number of ampules of hMG, cancellation rate, number of oocytes, fertilization rate, and clinical pregnancy rate.

Result(s): In patients with elevated FSH levels on cycle day 3, a low oocyte yield was achieved (7 versus 11) and a high number of ampules of hMG was necessary (56 versus 33). Their cancellation rate was high (67% versus 16%). In patients with normal basal FSH levels, high E₂ levels predicted a high cancellation rate (56%, versus 13% in patients with low E₂ levels) and a low oocyte yield (9, versus 11 in patients with low E₂ levels). Patients with both normal FSH levels and low E₂ levels on cycle day 3 fared best.

Conclusion(s): The basal E₂ level on cycle day 3 is a useful prognosticator of response to stimulation in IVF patients with normal basal FSH levels.     

Apoptosis and Ki-67 expression in adenomyotic lesions and in the corresponding eutopic endometrium.

OBJECTIVE: To examine biologic and proliferative properties of adenomyotic lesions and to determine whether adenomyotic lesions originate in the basal layer of the eutopic endometrium. METHODS: We examined eutopic and ectopic endometria from 23 patients with adenomyosis. To obtain evidence for the induction of programmed cell death, apoptotic cells were identified using a modified terminal deoxynucleotidyltransferase-biotin nick end-labeling method. To evaluate cell death repressor activity, bcl-2 gene expression was examined using immunohistochemical staining. As a proliferative marker, Ki-67 expression was also examined immunohistochemically. RESULTS: In the eutopic endometrium, apoptosis was most frequently observed in epithelial cells during mid- to late secretory phases, although it was rarely found during early proliferative through early secretory phases (P<.01). In contrast, bcl-2 gene expression inversely correlated with the appearance of apoptosis. A similar tendency was observed in stromal cells. In the ectopic endometrium of adenomyosis, endometrial dating revealed that secretory change was rare, even in the secretory phase, and that induction of apoptotic cells as well as bcl-2 gene expression showed no cyclic change. In stromal cells of the ectopic endometrium, apoptosis was more frequent than was seen in the eutopic endometrium, in all menstrual phases (P<.05). Ki-67 was constantly expressed in the glandular epithelium of the ectopic endometrium, irrespective of the menstrual phases, whereas in the secretory phase it was less expressed in the eutopic endometrium of functional and basal layers (P<.01). CONCLUSION: The induction of apoptosis seems to be regulated by hormonal changes in the eutopic endometrium and has an inverse correlation with bcl-2 gene expression. The ectopic endometrium in adenomyosis is rarely influenced by hormonal change and has different biologic and proliferative properties than events observed in the eutopic endometrium findings, which strongly suggest that the adenomyotic lesion does not originate in the basal endometrium.