Excess Death Estimates in Patients with End-Stage Renal Disease - United States, February-August 2020.

End-stage renal disease (ESRD) is a condition in which kidney function has permanently declined such that renal replacement therapy* is required to sustain life (1). The mortality rate for patients with ESRD in the United States has been declining since 2001 (2). However, during the COVID-19 pandemic, ESRD patients are at high risk for COVID-19-associated morbidity and mortality, which is due, in part, to weakened immune systems and presence of multiple comorbidities (3-5). The ESRD National Coordinating Center (ESRD NCC) supports the Centers for Medicare & Medicaid Services (CMS) and the ESRD Networks†,§ through analysis of data, dissemination of best practices, and creation of educational materials. ESRD NCC analyzed deaths reported to the Consolidated Renal Operations in a Web-Enabled Network (CROWNWeb), a system that facilitates the collection of data and maintenance of information about ESRD patients on chronic dialysis or receiving a kidney transplant who are treated in Medicare-certified dialysis facilities and kidney transplant centers in the United States. Excess death estimates were obtained by comparing observed and predicted monthly numbers of deaths during February 1-August 31, 2020; predicted deaths were modeled based on data from January 1, 2016, through December 31, 2019. The analysis estimated 8.7-12.9 excess deaths per 1,000 ESRD patients, or a total of 6,953-10,316 excess deaths in a population of 798,611 ESRD patients during February 1-August 31, 2020. These findings suggest that deaths among ESRD patients during the early phase of the pandemic exceeded those that would have been expected based on previous years' data. Geographic and temporal patterns of excess mortality, including those among persons with ESRD, should be considered during planning and implementation of interventions, such as COVID-19 vaccination, infection control guidance, and patient education. These findings underscore the importance of data-driven technical assistance and further analyses of the causes and patterns of excess deaths in ESRD patients.

The role of Notch signaling in the mammalian ovary.

The Notch pathway is a contact-dependent, or juxtacrine, signaling system that is conserved in metazoan organisms and is important in many developmental processes. Recent investigations have demonstrated that the Notch pathway is active in both the embryonic and postnatal ovary and plays important roles in events including follicle assembly and growth, meiotic maturation, ovarian vasculogenesis and steroid hormone production. In mice, disruption of the Notch pathway results in ovarian pathologies affecting meiotic spindle assembly, follicle histogenesis, granulosa cell proliferation and survival, corpora luteal function and ovarian neovascularization. These aberrations result in abnormal folliculogenesis and reduced fertility. The knowledge of the cellular interactions facilitated by the Notch pathway is an important area for continuing research, and future studies are expected to enhance our understanding of ovarian function and provide critical insights for improving reproductive health. This review focuses on the expression of Notch pathway components in the ovary, and on the multiple functions of Notch signaling in follicle assembly, maturation and development. We focus on the mouse, where genetic investigations are possible, and relate this information to the human ovary.

Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

The interactions between the stimulatory effect of follicle-stimulating hormone and the inhibitory effect of estrogen on mouse primordial folliculogenesis.

The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.

Point-of-care Lung Ultrasound Is More Sensitive than Chest Radiograph for Evaluation of COVID-19.

INTRODUCTION: Current recommendations for diagnostic imaging for moderately to severely ill patients with suspected coronavirus disease 2019 (COVID-19) include chest radiograph (CXR). Our primary objective was to determine whether lung ultrasound (LUS) B-lines, when excluding patients with alternative etiologies for B-lines, are more sensitive for the associated diagnosis of COVID-19 than CXR. METHODS: This was a retrospective cohort study of all patients who presented to a single, academic emergency department in the United States between March 20 and April 6, 2020, and received LUS, CXR, and viral testing for COVID-19 as part of their diagnostic evaluation. The primary objective was to estimate the test characteristics of both LUS B-lines and CXR for the associated diagnosis of COVID-19. Our secondary objective was to evaluate the proportion of patients with COVID-19 that have secondary LUS findings of pleural abnormalities and subpleural consolidations. RESULTS: We identified 43 patients who underwent both LUS and CXR and were tested for COVID-19. Of these, 27/43 (63%) tested positive. LUS was more sensitive (88.9%, 95% confidence interval (CI), 71.1-97.0) for the associated diagnosis of COVID-19 than CXR (51.9%, 95% CI, 34.0-69.3; p = 0.013). LUS and CXR specificity were 56.3% (95% CI, 33.2-76.9) and 75.0% (95% CI, 50.0-90.3), respectively (p = 0.453). Secondary LUS findings of patients with COVID-19 demonstrated 21/27 (77.8%) had pleural abnormalities and 10/27 (37%) had subpleural consolidations. CONCLUSION: Among patients who underwent LUS and CXR, LUS was found to have a higher sensitivity than CXR for the evaluation of COVID-19. This data could have important implications as an aid in the diagnostic evaluation of COVID-19, particularly where viral testing is not available or restricted. If generalizable, future directions would include defining how to incorporate LUS into clinical management and its role in screening lower-risk populations.

Focused vascular access education to reduce the use of chronic tunneled hemodialysis catheters: results of a network quality improvement initiative.

Tunneled hemodialysis catheters (TDCs) carry the highest mortality risk for chronic hemodialysis patients of any access modality. Recent data have emphasized that mortality risk decreases when these devices are discontinued. Herein, we present the results of a gap-reduction assisted catheter elimination strategy that Network 7 employed as its quality improvement initiative to reduce the use of TDCs. Hemodialysis facilities with high catheter rates (>90 days) were identified. Interventions included focused vascular access education, monthly follow-up and site visits to assist the facility catheter reduction program. The "goal" of interventions was defined as the gap-reduction of 50% from the baseline catheter rate to the Network mean plus sustainability of catheter reduction for at least 3 consecutive months. Fifteen facilities (n = 891) were identified with high catheter rates (31.5 +/- 5.3%) in May 2006. Interventions resulted in a catheter reduction to 12.2 +/- 8.5% in May 2007 (p = 0.0001). Five of the 15 facilities (n = 280) achieved the goal (preintervention = 31.7 +/- 5.3%, postintervention = 8.7 +/- 2.8%, p = 0.001). In May 2007, eight additional facilities (n = 438) with high catheter rates (31.7 +/- 7.8%) were added to the 10 that failed to achieve the goal previously. Interventions employed in these 18 facilities (n = 1,049) resulted in catheter reduction in all (preintervention = 31.5 +/- 5.5%, postintervention = 16.2 +/- 5%, p = 0.01). Five of these 18 met the goal (preintervention = 32 +/- 8%, postintervention = 5.9 +/- 4.3%). Overall, all 23 facilities (n = 1,329) demonstrated catheter reduction postintervention (preintervention = 31.6 +/- 6%, postintervention = 13.9 +/- 6%, p = 0.001), and 10/23 (43%) met the project goal (preintervention = 31.9 +/- 6%, postintervention = 7.3 +/- 4%, p = 0.002). Medical director's involvement had a positive impact in achieving the goal (p = 0.003). The presence or absence of a vascular access coordinator did not affect catheter reduction. The results of this analysis reveals that an organized approach implemented by an ESRD Network can have a significant impact in reducing catheter use.

Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells.

The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway activation in granulosa cells is significantly suppressed when germ cells are reduced. We further demonstrate that disruption of the gene for the Notch ligand Jag1 in oocytes similarly impacts Notch activation and that recombinant JAG1 enhances Notch target gene expression in granulosa cells. These data are consistent with the hypothesis that germ cells provide a ligand, such as Jag1, that is necessary for activation of Notch signaling in the developing ovary.

Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid.

BACKGROUND: The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model. METHODS: To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection. RESULTS: Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight. CONCLUSION: Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.

Pituitary-specific expression and Pit-1 regulation of the rat growth hormone-releasing hormone receptor gene.

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells.

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary.

In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.

Ovarian epithelial inclusion cysts in chronically superovulated CD1 and Smad2 dominant-negative mice.

Chronic ovulation as a contributing factor for the development of epithelial ovarian cancer in women has long been an outstanding hypothesis. To test the incessant ovulation hypothesis, mice were superovulated using weekly ip injections of pregnant mare serum gonadotropin (5 IU/animal), followed 48 h later by human chorionic gonadotropin (5 IU/animal). Wild-type CD1 mice were used along with CD1 mice expressing a Smad2 dominant-negative (Smad2DN) transgene under the control of the Müllerian inhibiting substance promoter that targets expression to the ovary and enhances cyst formation. After chronic injections, ovaries were analyzed from animals 6 months of age for the total adjusted number of cysts, cyst area, cyst location, and key signaling pathways. All observed cysts were confirmed to be of epithelial origin. The number of cysts was not significantly different between superovulated and control mice in either the wild-type or Smad2DN groups. However, the combination of the Smad2DN transgene and superovulation resulted in an increase in cyst formation compared with normal littermates that were unstimulated. Rapid proliferation of the cells lining the cysts was detected using bromodeoxyuridine and phospho-histone 3 immunohistochemistry but was not different in the ovarian surface epithelium or in the cyst lining between groups. These data suggest that chronic superovulation in Smad2DN mice results in a higher incidence of cyst formation compared with unstimulated controls, but the epithelial lined cysts did not progress to cancer over the course of this study.

The structures that underlie normal reproductive function.

The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This 'cutting edge' editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

Switching of NR5A proteins associated with the inhibin alpha-subunit gene promoter after activation of the gene in granulosa cells.

The inhibin alpha-subunit gene is transcriptionally activated by FSH in ovarian granulosa cells during follicular growth. We have investigated the roles of the NR5A family nuclear receptors steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) in transcriptional activation of the inhibin alpha-subunit gene. Transfection assays using an inhibin alpha-subunit promoter reporter in GRMO2 granulosa cells show that LRH-1 and SF-1 act similarly to increase promoter activity, and that the activity of both transcription factors is augmented by the coactivators cAMP response element-binding protein-binding protein and steroid receptor coactivator 1. However, chromatin immunoprecipitation experiments illustrate differential dynamic association of LRH-1 and SF-1 with the alpha-subunit inhibin promoter in both primary cells and the GRMO2 granulosa cell line such that hormonal stimulation of transcription results in an apparent replacement of SF-1 with LRH-1. Transcriptional stimulation of the inhibin alpha-subunit gene is dependent on MAPK kinase activity, as is the dynamic association/disassociation of SF-1 and LRH-1 with the promoter. Inhibition of the phosphatidylinositol 3-kinase signaling pathway influences promoter occupancy and transcriptional activation by SF-1 but not LRH-1, suggesting a possible mechanistic basis for the distinct functions of these NR5A proteins in inhibin alpha-subunit gene regulation.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Sequence-specific deoxyribonucleic acid (DNA) recognition by steroidogenic factor 1: a helix at the carboxy terminus of the DNA binding domain is necessary for complex stability.

Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-alpha gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

A dominant-negative human growth hormone-releasing hormone (GHRH) receptor splice variant inhibits GHRH binding.

GHRH is a hypothalamic peptide that stimulates the synthesis and secretion of GH from pituitary somatotroph cells. The GHRH receptor is a seven-transmembrane G protein-coupled receptor that localizes to the surface of somatotroph cells and binds GHRH. Alternative splicing of the GHRH receptor primary transcript at the intron/exon boundary 3' of exon 11 results in inclusion of sequence that is normally intronic. In the human, this inclusion has an in-frame premature stop codon, and this variant mRNA encodes a protein truncated just before the sixth transmembrane domain. To identify the effects of the truncated receptor on signaling of the wild-type receptor and the mechanisms by which its effects are produced, the full-length and truncated receptor constructs were epitope tagged and transfected into HeLa T4 cells to examine signaling and expression. Results show that the truncated GHRH receptor cannot signal through the cAMP pathway and acts as a dominant inhibitor of wild-type receptor signaling. The wild-type and truncated GHRH receptor proteins form a complex. Stably transfected cell lines were generated to examine the mechanism of signal inhibition by the truncated receptor. The data show that receptor cell surface expression is not altered when the wild-type and truncated receptors are cotransfected, but that truncated receptor coexpression substantially reduces GHRH binding by the wild-type receptor. The results support an important role for alternative splicing in mediating the effects of G protein-coupled receptors in general, and suggest that the GHRH receptor can form multimers, which may be important to its signaling properties.

Gonadotropin-induced superovulation drives ovarian surface epithelia proliferation in CD1 mice.

The ovarian surface epithelium (OSE) is a monolayer of cells that surround the ovary and accommodate repeated tear and repair in response to ovulation. OSE cells are thought to be the progenitors of 90% of ovarian cancers. Currently, the total amount of proliferation of the OSE has not been reported in response to one ovulatory event. In this study, proliferation of the OSE was quantified in response to superovulation induced by ip injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in immature 27-d-old CD1 mice using bromodeoxyuridine (BrdU). BrdU incorporation into the OSE cells was measured from the time of hCG injection for a total cumulative label of 12 h. BrdU incorporation was also measured from the time of PMSG injection for a total label of 60 h to correlate proliferation with specific gonadotropin stimulation. The OSE proliferation was significantly higher in superovulated animals compared with control mice at all time points. Proliferation was also analyzed in discrete anatomical sections and indicated that OSE covering antral follicles and corpora lutea proliferated more rapidly than OSE distal to follicular growth. Finally, apoptosis was assessed in response to ovulation, and virtually no cell death within the OSE was detected. These data demonstrate that the OSE, especially near antral follicles and corpora lutea, proliferates significantly in response to the gonadotropins PMSG and hCG. Therefore, ovarian surface cell division in response to ovulation could contribute to ovarian cancer by proliferation-induced DNA mutations and transformed cell progression.