Phyllis C. Leppert

Anatomy and physiology of cervical ripening.

The uterine cervix is a unique organ composed predominately of the extracellular matrix proteins, collagen, elastin, and glycosaminoglycans. During pregnancy and labor, this organ is metabolically active, which is rare in adult tissue. The metabolism is under reproductive hormonal control and is more complex than previously appreciated. Smooth muscle cells, which comprise 10-15% of cervical tissue, undergo programmed cell death and play a role in cervical softening. Apoptosis is a genetically timed event and could explain the species-specific length of gestation. Further research in the next several years will reveal more completely the exciting process of cervical ripening. Only when this phenomenon is understood will rational therapy for preterm labor and post-term pregnancy with an unripe cervix be available. Specific defects in cervical ripening will then be diagnosed and treated. For example, if apoptosis is shown to play an important role in the process of cervical ripening, it could be inhibited. Conversely, it could be induced in the unripe cervix. If we would look for it, we would find that it is probably occurring today in the clinical use of cervical ripening agents. The most important contributor to cervical softening, however, is a rearrangement and realignment of the collagen, elastin, and smooth muscle cells, which occurs due to mechanical forces and to a rearrangement of the collagen that occurs as the content of glycosaminoglycans varies in the cervix with time. One form of dermatan sulfate, decorin, may help to separate the collagen fibrils and then open them up. This rearrangement also involves fiber shortening below the critical length for tensile strength, allowing for extensibility of the cervix. Because of its orientation in the cervix, elastin contributes to the ratchet-like mechanism of dilatation. Finally, the cervix undergoes change in two phases--softening, which involves collagen realignment, and dilatation. The proteolytic enzymes in the cervix degrade cross-linked, newly synthesized collagen, and they help activate other enzymes in a cascade. However, the predominate anatomic and physiologic change in ripening is the rearrangement of collagen.

Ovarian Preservation by GnRH Agonists during Chemotherapy: A Meta-Analysis

PURPOSE Treatment with cyclophosphamide (CYC) confers up to a 40% risk of ovarian failure in women of reproductive age. The use of GnRH agonists (GnRHa) to preserve ovarian function has been investigated in several small studies. We performed a systematic review of studies examining whether a GnRHa administered during chemotherapy is protective of ovarian function and fertility. METHODS We searched the English-language literature (1966-April 2007) using MEDLINE and meeting abstracts and included studies that reported an association between GnRHa and ovarian preservation in women receiving chemotherapy. Studies without a control group were excluded. Ovarian preservation was defined as the resumption of menstrual cycles and a premenopausal follicle-stimulating hormone (FSH) after chemotherapy. Fertility was determined by a womans ability to become pregnant. We estimated the summary relative risk (RR) and associated 95% confidence intervals (95% CI) using a random-effects model. RESULTS Nine studies included 366 women. Three studies included women with autoimmune disease receiving CYC; six included women with hematologic malignancy receiving combination chemotherapy. In total, 178 women were treated with GnRHa during chemotherapy, 93% of whom maintained ovarian function. Of the 188 women not treated with GnRHa, 48% maintained ovarian function. The use of a GnRHa during chemotherapy was associated with a 68% increase in the rate of preserved ovarian function compared with women not receiving a GnRHa (summary RR = 1.68, 95% CI 1.34-2.1). Among the GnRHa-treated women, 22% achieved pregnancy following treatment compared with 14% of women without GnRHa therapy (summary RR = 1.65, CI 1.03-2.6). CONCLUSIONS Based on the available studies, GnRHa appear to improve ovarian function and the ability to achieve pregnancy following chemotherapy. Several randomized trials are underway to define the role and mechanism of GnRHa in ovarian function preservation. In the meantime, premenopausal women facing chemotherapy should be counseled about ovarian preservation options, including the use of GnRHa therapy.

Reduced Dermatopontin Expression Is a Molecular Link Between Uterine Leiomyomas and Keloids

Uterine leiomyomas are prevalent estrogen‐responsive clonal tumors, but the specific genetic alterations that contribute to their development have not been elucidated. To identify genes involved in the formation of leiomyomas, we used global expression profiling to compare clonal tumors with normal myometrium. Contrary to expectation, genes involved in estrogen action were not differentially expressed between leiomyoma and normal myometrium. Genes encoding extracellular‐matrix proteins were prominently featured, suggesting their involvement in formation of a myofibroblast phenotype. Analysis of the extracellular matrix in the leiomyomas revealed a disordered collagen fibril orientation. Expression of the collagen‐binding protein dermatopontin was found to be consistently decreased in leiomyoma by both reverse transcriptase–polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR (mean underexpression = 9.41‐fold) regardless of leiomyoma size, leiomyoma location, patient race, and patient age. This expression pattern was observed in 11 subjects and a total of 23 leiomyoma:myometrium pairs. Decreased expression of dermatopontin was also associated with keloid formation, a fibrotic disease that shares epidemiologic similarities with leiomyoma. Immunohistochemical studies of leiomyomas and keloids demonstrated reduced levels of dermatopontin in both tissues. In addition, ultrastructural analysis revealed that the orientation of the collagen fibrils in the keloid tissues strongly resembled that in the leiomyomas. Reduction in dermatopontin was associated with an increase in transforming growth factor–β3 (TGFB3) mRNA levels in leiomyomas, whereas other genes involved in dermatopontin signaling were not differentially expressed. These findings suggest that leiomyoma development involves a myofibroblast cell phenotype characterized by dysregulation of genes encoding extracellular‐matrix proteins. In particular, decreased expression of dermatopontin represents a molecular link between the leiomyoma and keloid phenotypes.

Transforming Growth Factor β3 Regulates the Versican Variants in the Extracellular Matrix-Rich Uterine Leiomyomas

Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor β (TGF-β) signaling. Here we characterized TGF-β3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-β3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription—polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor β3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-β RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 ± 1.12) and V1 (2.01 ± 0.27). Treatment of leiomyoma and myometrial cells with TGF-β3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-β3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-β signaling pathway and excessive production of GAG-rich versican variants.

Mechanical homeostasis is altered in uterine leiomyoma

OBJECTIVE Uterine leiomyoma produce an extracellular matrix (ECM) that is abnormal in its volume, content, and structure. Alterations in ECM can modify mechanical stress on cells and lead to activation of Rho-dependent signaling and cell growth. Here we sought to determine whether the altered ECM that is produced by leiomyoma was accompanied by an altered state of mechanical homeostasis. STUDY DESIGN We measured the mechanical response of paired leiomyoma and myometrial samples and performed immunogold, confocal microscopy, and immunohistochemical analyses. RESULTS Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was accompanied by alteration of the ECM, cell shape, and cytoskeleton in leiomyoma, compared with myometrial samples from the same uterus. Levels of AKAP13, a protein that is known to activate Rho, were increased in leiomyoma compared to myometrium. AKAP13 was associated with cytoskeletal filaments of immortalized leiomyoma cells. CONCLUSION Leiomyoma cells are exposed to increased mechanical loading and show structural and biochemical features that are consistent with the activation of solid-state signaling.

Strategy for elucidating differentially expressed genes in leiomyomata identified by microarray technology

OBJECTIVE cDNA microarray technology identifies genes that are differentially expressed between tissues. Our previous study identified several genes that might contribute to the fibroid phenotype. We therefore sought to confirm genes involved in three distinct signal transduction pathways. DESIGN Evaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium. University hospital. PATIENT(S) Five women undergoing medically indicated hysterectomy for symptomatic fibroids. INTERVENTION(S) Microarray analysis of up to 33000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. MAIN OUTCOME MEASURE(S) Expression of mRNA transcripts and protein in fibroid compared with myometrium.A more extensive microarray confirmed differential expression of Frizzled-2 and CD-24 but did not confirm Dlk overexpression. RT-PCR and real-time PCR demonstrated equivalent Dlk mRNA expression between fibroid and myometrium (ratio, 1.02), a slight Frizzled-2 overexpression (ratio, 2.09), and robust CD-24 overexpression in fibroids (ratio, 12.35). Western blot and immunohistochemistry confirmed Frizzled-2 overexpression, but did not confirm Dlk overexpression. CONCLUSION(S) Microarray technology is the first phase of tissue evaluation, but changes in gene expression must be confirmed. Confirmed genes can then be used to generate hypotheses testing their involvement in fibroid development.

Collagen Changes in Rat Cervix in Pregnancy—Polarized Light Microscopic and Electron Microscopic Studies:

Abstract The structural arrangement of collagen fibers in cervical ripening was studied in normal pregnant rats by picrosirius red staining and polarized light microscopy. The macromolecular arrangement of collagen fibers in the cervices of nonpregnant controls and in firm and rigid cervices of rats in early pregnancy (1–10 days of gestation) were optically anisotropic and had birefringence and a positive sign of elongation when examined by polarized light microscopy. The findings indicated that the structure of these collagen fibers was assembled from well-packed parallel collagen molecules. The direction of fibrous formation was arranged with regularity. In contrast, most of the collagen fibers in the soft cervices were optically isotropic. The fibers were fragmented and had a structure with discontinuous birefringence. Disarray and disorientation of the collagen fibers was found in the soft cervices. These collagen fibers changed their direction of formation. The disorganization of these collagen fibers might have a major impact on weakening the tensile strength of the cervix. Thus, we conclude that the processes of rearrangement of collagen fibers might be an important process in the cervical ripening. Electron microscopic studies suggest that in the focal hydrolytic processes of collagen and other matrix components degradation by lysosomal and phagosomal vesicles were associated with atrophic smooth muscle cells and fibroblasts of the cervices. Hydrolases released from lysosomes from these apoptotic cells may presumably be one of the processes in the remodeling of collagen structure.

Cervical Softening , Effacement, and Dilatation: A Complex Biochemical Cascade

The rearrangement of the extracellular matrix of the uterine cervix during pregnancy and parturition occurs by the progression of a complex, finely regulated biochemical cascade. In the past decade, major advances in our understanding of these molecular and biochemical changes have been made, yet the complete metabolic cascade is not understood. In this paper, current information regarding these metabolic pathways is reviewed and areas for further research are discussed.Term labor and preterm labor are etiologically heterogenous. The triggering mechanisms of the biochemical rearrangement of collagen, proteoglycans, and elastic fibers are likely different, however, depending on the presenting clinical situation. While the pathways of extracellular matrix rearrangement may be similar, studies focusing on term labor do not provide complete information regarding preterm labor. Animal models and in vitro studies are extremely useful, but do not in themselves explain the entire story of cervical softening, dila...

Decreased elastic fibers and desmosine content in incompetent cervix

Incompetence of the uterine cervix is a syndrome of painless, progressive dilatation and effacement occurring between the sixteenth and twenty-fourth weeks of gestation that represents abnormal functioning. It may serve as a model to elucidate normal function. Because the incompetent cervix results in painless opening of this organ without uterine contraction before term gestation, it is considered one of the causes of midtrimester spontaneous abortion, habitual spontaneous abortion, and early preterm labor. Untreated, it leads to rapid expulsion and often death of the fetus. We used light microscopy to compare decreased elastic fibers in incompetent cervices with those of normal nonpregnant and pregnant cervices. Morphologic analysis of this difference was extended to biochemical quantification of elastin content in one patient with cervical incompetence. The decrease in elastin suggests that one function of cervical elastin may be to maintain a closed and undilated cervix throughout gestation. There may be a relationship between changes in cross-linked elastin and the incompetent cervix; further studies are therefore indicated.

Proliferation and apoptosis of fibroblasts and smooth muscle cells in rat uterine cervix throughout gestation and the effect of the antiprogesterone onapristone

OBJECTIVE The purpose of this study was to determine the relative percentages of apoptosis and proliferation in fibroblasts and cervical smooth muscles throughout gestation and the effect of an antiprogesterone on these processes. STUDY DESIGN Rats were studied at days 5, 15, 18, and 21 and immediately postpartum (day 22). Apoptosis and proliferation as detected by specific immunohistochemistry quantitative morphometric analysis was performed. Onapristone, an antiprogesterone, was used to study effects of hormonal change on these processes in 16- and 19-day timed-pregnant rats. RESULTS Proliferation of fibroblasts and smooth muscle cells was highest in early pregnancy and decreased progressively, whereas apoptosis increased progressively in later pregnancy. Onapristone inhibited apoptosis. CONCLUSION Changes in cervical cellular turnover are initiated early in gestation and are under hormonal influence. Antiprogesterone inhibits cell death at days 16 and 19 of gestation.