The Reduction of the Cell Nuclear Size in the Cell Body Space During the Differentiation Might Be Cell Lineage Specific (A Retrospective Morphological Note).

The cell body space occupied by the nucleus decreased during the cell differentiation of the granulocytic cell lineage in CML (Chronic Myeloid Leukemia) patients. In contrary, in patients suffering from CLL (Chronic Lymphocytic Leukemia), the cell body space occupied by the nucleus during the cell differentiation of the lymphocytic lineage did not decrease despite the reduction of the cell size. Thus, the cell body space occupied by the cell nucleus during the differentiation was characteristic for each of these cell lineages.

Histone H2B.8 compacts flowering plant sperm through chromatin phase separation.

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state1,2. Sperm cells of flowering plants lack protamines, yet they have small, transcriptionally active nuclei with chromatin condensed through an unknown mechanism3,4. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin. This result demonstrates that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, which facilitates nuclear compaction without reducing transcription. Reciprocal crosses show that mutation of h2b.8 reduces male transmission, which suggests that H2B.8-mediated sperm compaction is important for fertility. Altogether, our results reveal a new mechanism of nuclear compaction through global aggregation of unexpressed chromatin. We propose that H2B.8 is an evolutionary innovation of flowering plants that achieves nuclear condensation compatible with active transcription.

Water transport regulates nucleus volume, cell density, Young's modulus, and E-cadherin expression in tumor spheroids.

Cell volume is maintained by the balance of water and solutes across the cell membrane and plays an important role in mechanics and biochemical signaling in cells. Here, we assess the relationship between cell volume, mechanical properties, and E-cadherin expression in three-dimensional cultures for ovarian cancer. To determine the effect of water transport in multi-cellular tumors, ovarian cancer spheroids were subjected to hypotonic and hypertonic shock using water and sucrose mixtures, respectively. Increased osmolality resulted in decreased nucleus volume, increased Young's modulus, and increased tumor cell density in ovarian cancer spheroids. Next, we looked at the reversibility of mechanics and morphology after 5 min of osmotic shock and found that spheroids had a robust ability to return to their original state. Finally, we quantified the size of E-cadherin clusters at cell-cell junctions and observed a significant increase in aggregate size following 30 min of hypertonic and hypotonic osmotic shocks. Yet, these effects were not apparent after 5 min of osmotic shock, illustrating a temporal difference between E-cadherin regulation and the immediate mechanical and morphology changes. Still, the osmotically induced E-cadherin aggregates which formed at the 30-minute timepoint was reversible when spheroids were replenished with isotonic medium. Altogether, this work demonstrated an important role of osmolality in transforming mechanical, morphology, and molecular states.

Nucleus size and its effect on nucleosome stability in living cells.

DNA architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

A deep hybrid learning pipeline for accurate diagnosis of ovarian cancer based on nuclear morphology.

Nuclear morphological features are potent determining factors for clinical diagnostic approaches adopted by pathologists to analyze the malignant potential of cancer cells. Considering the structural alteration of the nucleus in cancer cells, various groups have developed machine learning techniques based on variation in nuclear morphometric information like nuclear shape, size, nucleus-cytoplasm ratio and various non-parametric methods like deep learning have also been tested for analyzing immunohistochemistry images of tissue samples for diagnosing various cancers. We aim to correlate the morphometric features of the nucleus along with the distribution of nuclear lamin proteins with classical machine learning to differentiate between normal and ovarian cancer tissues. It has already been elucidated that in ovarian cancer, the extent of alteration in nuclear shape and morphology can modulate genetic changes and thus can be utilized to predict the outcome of low to a high form of serous carcinoma. In this work, we have performed exhaustive imaging of ovarian cancer versus normal tissue and developed a dual pipeline architecture that combines the matrices of morphometric parameters with deep learning techniques of auto feature extraction from pre-processed images. This novel Deep Hybrid Learning model, though derived from classical machine learning algorithms and standard CNN, showed a training and validation AUC score of 0.99 whereas the test AUC score turned out to be 1.00. The improved feature engineering enabled us to differentiate between cancerous and non-cancerous samples successfully from this pilot study.

Intraductal Carcinoma of the Prostate: Extreme Nuclear Size Is Not a Diagnostic Parameter.

High-grade prostatic adenocarcinoma involving duct/acinar structures is labeled intraductal carcinoma of the prostate (IDCP). As numerous studies have shown that IDCP is associated with high stage disease with a significant negative impact on cancer-specific survival, accurate diagnosis is crucial to ensure appropriate patient management. The definition of IDCP recommended by 2016 World Health Organization (WHO) classification suggests that cases of IDCP with micropapillary or loose cribriform architecture without comedonecrosis should have cells with ≥6× nuclear enlargement. It is unclear how this size criterion was derived and which of the parameters of nuclear size (nuclear diameter, nuclear surface area, or nuclear perimeter) it relates to. To evaluate the extent of nuclear enlargement in IDCP, we performed morphometric analyses relating to each of these parameters in 100 radical prostatectomy specimens. One hundred nuclei from foci of IDCP and 50 nuclei from foci of normal luminal epithelium were examined for each patient. Diagnosis of IDCP was based on cells with definite features of carcinoma present within duct/acinar structures. Comparing the means of each of the parameters between IDCP cells and benign luminal cells, there was a statistically significant enlargement in nuclear perimeter (P<0.0005), nuclear area (P<0.0005), and nuclear diameter (P<0.0005); however, the difference in mean nuclear size was limited to factors of 1.3×, 1.6×, and 1.3×, respectively. Three patients each had rare large nuclei (largest perimeter 45, 45, and 44 µm; maximum nuclear area 135, 136, and 136 µm2; and the largest diameter 18 µm in each). For these rare cells, the nuclear size difference, when compared with benign nuclei was; nuclear perimeter 2.0×, 2.1×, and 2.1×; nuclear area 3.6×, 3.8×, and 3.8×; and nuclear maximum diameter 3.0×, 2.5×, and 2.5×. The definition of nuclear enlargement of ≥6× was not reached in any of our cases, all of which clearly showed features of duct invasive carcinoma. In these cases, reliance on nuclear size criteria would have resulted in underdiagnosis of IDCP. This is of concern as failure to recognize IDCP, particularly in needle biopsies, could lead to delays in the timely treatment of aggressive high-grade prostate cancer, resulting in cancer progression and suboptimal patient oncological outcomes.

VCP maintains nuclear size by regulating the DNA damage-associated MDC1-p53-autophagy axis in Drosophila.

The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.

Frequent blood flow restricted training not to failure and to failure induces similar gains in myonuclei and muscle mass.

The purpose of the present study was to compare the effects of short-term high-frequency failure vs non-failure blood flow-restricted resistance exercise (BFRRE) on changes in satellite cells (SCs), myonuclei, muscle size, and strength. Seventeen untrained men performed four sets of BFRRE to failure (Failure) with one leg and not to failure (Non-failure; 30-15-15-15 repetitions) with the other leg using knee-extensions at 20% of one repetition maximum (1RM). Fourteen sessions were distributed over two 5-day blocks, separated by a 10-day rest period. Muscle samples obtained before, at mid-training, and 10-day post-intervention (Post10) were analyzed for muscle fiber area (MFA), myonuclei, and SC. Muscle size and echo intensity of m.rectus femoris (RF) and m.vastus lateralis (VL) were measured by ultrasonography, and knee extension strength with 1RM and maximal isometric contraction (MVC) up until Post24. Both protocols increased myonuclear numbers in type-1 (12%-17%) and type-2 fibers (20%-23%), and SC in type-1 (92%-134%) and type-2 fibers (23%-48%) at Post10 (p < 0.05). RF and VL size increased by 5%-10% in both legs at Post10 to Post24, whereas the MFA of type-1 fibers in Failure was decreased at Post10 (-10 ± 16%; p = 0.02). Echo intensity increased by ~20% in both legs during Block1 (p < 0.001) and was ~8 to 11% below baseline at Post24 (p = 0.001-0.002). MVC and 1RM decreased by 5%-10% after Block1, but increased in both legs by 6%-11% at Post24 (p < 0.05). In conclusion, both short-term high-frequency failure and non-failure BFRRE induced increases in SCs, in myonuclei content, muscle size, and strength, concomitant with decreased echo intensity. Intriguingly, the responses were delayed and peaked 10-24 days after the training intervention. Our findings may shed light on the mechanisms involved in resistance exercise-induced overreaching and supercompensation.

High-content imaging-based pooled CRISPR screens in mammalian cells.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software µManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex.

The novel human betacoronavirus SARS-CoV-2 has caused an unprecedented pandemic in the 21st century. Several studies have revealed interactions between SARS-CoV-2 viral proteins and host nucleoporins, yet their functions are largely unknown. Here, we demonstrate that the open-reading frame 6 (ORF6) of SARS-CoV-2 can directly manipulate localization and functions of nucleoporins. We found that ORF6 protein disrupted nuclear rim staining of nucleoporins RAE1 and NUP98. Consequently, this disruption caused aberrant nucleocytoplasmic trafficking and led to nuclear accumulation of mRNA transporters such as hnRNPA1. Ultimately, host cell nucleus size was reduced and cell growth was halted.

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

The Paris System "atypical urothelial cells" category: can the current criteria be improved?

INTRODUCTION: The Paris System (TPS) for reporting urine cytology was developed for standardization of diagnosis focusing on the detection of high-grade urothelial carcinoma (HGUC). Probably the most challenging task for TPS is to provide criteria for the atypical urothelial cell (AUC) category. The TPS criteria for AUC include increased nuclear/cytoplasmic (N/C) ratio (>0.5) and 1 of the 3 minor criteria including nuclear hyperchromasia (NH), coarse chromatin (CC) and irregular nuclear membrane (INM). We evaluated TPS-AUC diagnostic value and investigated whether other morphologic parameters can improve its criteria. MATERIALS AND METHODS: Urine samples with diagnoses of AUC collected during a 6-month period were re-reviewed. Data captured included N/C ratio >0.5, NH, CC, INM, and 2 additional criteria including enlarged nuclear size (ENS) and the presence of nucleolus (N). ENS was considered when the nucleus was 2 times larger than the urothelial cell or 3 times larger than lymphocyte. RESULTS: By applying the TPS-AUC criteria, the rate of atypia diagnosis reduced in comparison to Pre-TPS (9% versus 13%, P = 0.02). Among the AUC minor criteria, NH was the best criterion with the highest interobserver agreement (IOA) and correlation with HGUC (k = 0.342, r = 0.61, P < 0.001) and strong PPV (93.6%). ENS had the highest PPV (95.8%) and, after NH, had the highest IOA and correlation with HGUC (k = 0.29, r = 0.52, P < 0.001). CONCLUSION: TPS improves the diagnostic value of urine cytology, particularly in cases with atypia. ENS is a strong criterion for increasing the diagnostic value of AUC and potentially can improve TPS performance as a minor criterion.

DNA content contributes to nuclear size control in Xenopus laevis.

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.

A Cytokinin Signaling Type-B Response Regulator Transcription Factor Acting in Early Nodulation.

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.

Control of cellular organization and its coordination with the cell cycle.

Cells organize themselves to maintain proper shape, structure, and size during growth and division for their cellular functions. However, how these cellular organizations coordinate with the cell cycle is not well understood. This review focuses on cell morphogenesis and size of the membrane-bound nucleus in the fission yeast Schizosaccharomyces pombe. Growth polarity, an important factor for cell morphogenesis, in rod-shaped fission yeast is restricted to the cell tips and dynamically changes depending on the cell cycle stage. Furthermore, nuclear size in fission yeast is proportional to the cell size, resulting in a constant ratio between nuclear volume and cellular volume (N/C ratio). This review summarizes the signaling pathway(s) involved in growth polarity control and key factors involved in N/C ratio control and provides their roles in coordination between cell organization and the cell cycle.

Intrauterine exposure to chronic hypoxia in the rat leads to progressive diastolic function and increased aortic stiffness from early postnatal developmental stages.

AIM: We sought to explore whether fetal hypoxia exposure, an insult of placental insufficiency, is associated with left ventricular dysfunction and increased aortic stiffness at early postnatal ages. METHODS: Pregnant Sprague Dawley rats were exposed to hypoxic conditions (11.5% FiO2 ) from embryonic day E15-21 or normoxic conditions (controls). After delivery, left ventricular function and aortic pulse wave velocity (measure of aortic stiffness) were assessed longitudinally by echocardiography from day 1 through week 8. A mixed ANOVA with repeated measures was performed to compare findings between groups across time. Myocardial hematoxylin and eosin and picro-sirius staining were performed to evaluate myocyte nuclear shape and collagen fiber characteristics, respectively. RESULTS: Systolic function parameters transiently increased following hypoxia exposure primarily at week 2 (p < .008). In contrast, diastolic dysfunction progressed following fetal hypoxia exposure beginning weeks 1-2 with lower early inflow Doppler velocities, and less of an increase in early to late inflow velocity ratios and annular and septal E'/A' tissue velocities compared to controls (p < .008). As further evidence of altered diastolic function, isovolumetric relaxation time was significantly shorter relative to the cardiac cycle following hypoxia exposure from week 1 onward (p < .008). Aortic stiffness was greater following hypoxia from day 1 through week 8 (p < .008, except week 4). Hypoxia exposure was also associated with altered nuclear shape at week 2 and increased collagen fiber thickness at week 4. CONCLUSION: Chronic fetal hypoxia is associated with progressive LV diastolic dysfunction, which corresponds with changes in nuclear shape and collagen fiber thickness, and increased aortic stiffness from early postnatal stages.

Nuclear size changes caused by local motion of cell boundaries unfold the nuclear lamina and dilate chromatin and intranuclear bodies.

The mechanisms by which mammalian nuclear shape and size are established in cells, and become abnormal in disease states are not understood. Here, we tracked motile cells that underwent systematic changes in cell morphology as they moved from 1-D to 2-D micro-patterned adhesive domains. Motion of the cell boundaries during cell motility caused a dynamic and systematic change in nuclear volume. Short time scales (∼1 h) distinguished the dilation of the nucleus from the familiar increase that occurs during the cell cycle. Nuclear volume was systematically different between cells cultured in 3-D, 2-D and 1-D environments. Dilation of the nuclear volume was accompanied by dilation of chromatin, a decrease in the number of folds in the nuclear lamina, and an increase in nucleolar volume. Treatment of 2-D cells with non-muscle myosin-II inhibitors decreased cell volume, and proportionately caused a decrease in nuclear volume. These data suggest that nuclear size changes during cell migration may potentially impact gene expression through the modulation of intranuclear structure.

Constitutive expression of a fluorescent protein reports the size of live human cells.

Cell size is important for cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of cell size, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement with the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle and reaffirm that there is a negative correlation between size at cell birth and G1 duration. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple wide-field fluorescence imaging. Thus, we expect our method will be of use to researchers interested in how dynamically changing protein concentrations control cell fates.

Unravelling nuclear size control.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

Effect of Progestin Usage on the Interpretation of Cervical High-grade Squamous Intraepithelial Lesion.

Progestin usage can alter the histologic and cytologic features of HSIL, which may potentially lead to the under-diagnosis of this precancerous lesion. The objective of the present study was to assess the relationship between the usage of progesterone-based contraceptives and the cytologic features of high-grade squamous intraepithelial lesion (HSIL) of the cervix. In this case-control study, we assembled 46 cases of cervical HSIL (CIN 3), including samples from 26 patients with known history of progestin usage (study group) and 20 samples from patients with no history of any exogenous hormone usage (control group). Cell image analysis was performed on all samples using a proprietary software. Immunohistochemical studies for Ki67, p16, estrogen receptor and progesterone receptor were performed on all cases, as was RNA in situ hybridization for HPV subtypes 16 and 18. Compared with the control group cases, the average nuclear size (21.5±1.80 µm) and the nuclear: cytoplasmic ratio (0.28±0.015) of HSILs in the study group cases were significantly smaller (P=0.026) and reduced (P=0.005), respectively. In addition, the study group cases showed reduced nuclear atypia and pleomorphism and significantly reduced mitotic figures (1.74±1.86/mm in study group versus 5.94±1.3/mm in control group, P<0.0001). The later was likely a consequence of the significantly reduced mitotic figures in the superficial and middle epithelial layers of the study group cases compared with their control group counterparts (0.73±0.88/mm vs. 7.3±2.6/mm, P<0.0001). Progestin usage has no discernible effects on p16 immunoreactivity, Ki67 proliferative index, hormone receptor expression, and HPV RNA levels of HSIL lesions. Being aware of progestin induced morphologic changes on those cervical precancerous lesions will be ultimately improve patient care quality in our daily practice.