Publications by authors named "Francesca E Duncan"

70 Publications

Immature Follicular Origins and Disrupted Oocyte Growth Pathways Contribute to Decreased Gamete Quality During Reproductive Juvenescence in Mice.

Front Cell Dev Biol 2021 16;9:693742. Epub 2021 Jun 16.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States.

Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11-40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following maturation (IVM). However, eggs from the younger cohort (PND 16-20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.
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http://dx.doi.org/10.3389/fcell.2021.693742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244820PMC
June 2021

Fibroinflammatory Signatures Increase with Age in the Human Ovary and Follicular Fluid.

Int J Mol Sci 2021 May 5;22(9). Epub 2021 May 5.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. "Fibroinflammation" is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7-44.8 years). This signature (IL-3, IL-7, IL-15, TGFβ1, TGFβ3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFβ3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFβ3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.
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http://dx.doi.org/10.3390/ijms22094902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125514PMC
May 2021

Mitochondrial humanin peptide acts as a cytoprotective factor in granulosa cell survival.

Reproduction 2021 04 20;161(5):581-591. Epub 2021 Apr 20.

Instituto de Investigaciones Biomédicas (INBIOMED) UBA-CONICET, Facultad de Medicina, Uiversidad de Buenos Aires, Buenos Aires, Argentina.

Humanin (HN) is a short peptide involved in many biological processes such as apoptosis, cell survival, inflammatory response, and reaction to stressors like oxidative stress, between others. In the ovary, a correct balance between pro- and anti-apoptotic factors is crucial for folliculogenesis. In the follicular atresia, survival or death of granulosa cells is a critical process. The goal of this study was to evaluate the action of HN on granulosa cell fate. To explore endogenous HN function in the ovary, we used a recombinant baculovirus (BV) encoding a short-hairpin RNA targeted to silence HN (shHN). HN downregulation modified ovarian histoarchitecture and increased apoptosis of granulosa cells. HN was also detected in a granulosa tumor cell line (KGN). Transduction of KGN cells with BV-shHN resulted in HN downregulation and increased apoptosis. On the other hand, treatment of KGN cells with exogenous HN increased cell viability and decreased apoptosis. In summary, these findings indicate that HN is a cytoprotective factor in granulosa cells of antral follicles, suggesting that this peptide would be involved in the regulation of folliculogenesis. Also, this peptide is a cytoprotective factor in KGN cells, and therefore, it could be involved in granulosa tumor cell behavior.
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http://dx.doi.org/10.1530/REP-20-0197DOI Listing
April 2021

A decellularized oocyte-derived scaffold provides a "sperm safe" to preserve mammalian spermatozoa.

Andrology 2021 05 5;9(3):922-932. Epub 2021 Mar 5.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Background: Although only one spermatozoon is needed to create a zygote, a significant challenge is the storage and recovery of germ cells when sperm counts are extremely low.

Objectives: We engineered an oocyte-derived biomaterial-the zona pellucida (ZP)-as a "sperm safe" for storing spermatozoon. The ZP is a glycoprotein matrix that surrounds the mammalian oocyte.

Materials And Methods: We made a hole in the ZPs using a Piezo drill and mechanically separated them from the oocyte cytoplasm. A subset of ZPs were further purified through decellularization. Using a modified ICSI approach, we injected sperm heads into purified ZPs and tested the efficacy of cryopreservation and recovery of spermatozoon as well as function.

Results: Between 1-6 sperm heads were injected into purified ZPs (average 2.7 ± 1.7 sperm heads/ZP), which were then cryopreserved. Upon thawing, an average of 2.5 ± 1.4 sperm heads/ZP were observed, and in 11 of 12 thawed "sperm safes," we recovered all spermatozoa. Decellularized "sperm safes" maintained their three-dimensional structure and had a denser matrix relative to untreated controls as assessed by scanning and transmitted electron microscopy. The efficacy of "sperm safe" derived spermatozoon was evaluated by ICSI. Spermatozoon stored in either untreated or decellularized "sperm safes" elicited egg activation-associated calcium transients and zinc sparks when injected into eggs. Of the resulting zygotes, >80% of them formed pronuclei irrespective of the sperm source. 26.8 ± 4.6% and 18.1 ± 7.0% of the pre-implantation embryos generated from spermatozoon recovered from untreated or decellularized "sperm safes" developed to the blastocyst stage, respectively. Although this development was lower than that using fresh spermatozoon (59.3 ± 19.3%) or conventionally frozen-thawed spermatozoon (28.4 ± 1.7%), these differences were not significant.

Discussion And Conclusion: Purified ZPs represent a natural biomaterial for the efficient preservation and recovery of small sperm numbers.
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http://dx.doi.org/10.1111/andr.12986DOI Listing
May 2021

Sphingosine-1-phosphate and its mimetic FTY720 do not protect against radiation-induced ovarian fibrosis in the nonhuman primate†.

Biol Reprod 2021 May;104(5):1058-1070

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Oocytes are highly radiosensitive, so agents that prevent radiation-induced ovarian follicle destruction are important fertility preservation strategies. A previous study in rhesus macaques demonstrated that ovarian treatment with antiapoptotic agents, sphingosine-1-phosphate (S1P) and FTY720, its long-acting mimetic, preserved follicles following a single dose of 15 Gy X-ray radiation, and live offspring were obtained from FTY720-treated animals. However, it is unknown whether these antiapoptotic agents also protected the ovarian stroma from late effects of radiation, including vascular damage and fibrosis. Using ovarian histological sections from this study, we evaluated the vasculature and extracellular matrix in the following cohorts: vehicle + sham irradiation, vehicle + irradiation (OXI), S1P + irradiation (S1P), and FTY720 + irradiation (FTY720). One ovary from each animal was harvested prior to radiation whereas the contralateral ovary was harvested 10 months post-treatment. We assessed vasculature by immunohistochemistry with a PECAM1 antibody, hyaluronan by a hyaluronan binding protein assay, and collagen by picrosirius red and Masson's trichrome staining. Disorganized vessels were observed in the medulla in the OXI and S1P cohorts relative to the sham, but the vasculature in the FTY720 cohort appeared intact, which may partially explain fertoprotection. There were no differences in the hyaluronan matrix among the cohorts, but there was thickening of the tunica albuginea and fibrosis in the OXI cohort relative to the sham, which was not mitigated by either S1P or FTY720 treatment. Thus, the fertoprotective properties of S1P and FTY720 may be limited given their inability to protect the ovarian stroma against the late effects of radiation-induced fibrosis.
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http://dx.doi.org/10.1093/biolre/ioab012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111238PMC
May 2021

Dental resins used in 3D printing technologies release ovo-toxic leachates.

Chemosphere 2021 May 7;270:129003. Epub 2020 Dec 7.

Department of Obstetrics and Gynecology, Northwestern University, Chicago, IL, 60611, USA. Electronic address:

We recently engineered the first female reproductive tract on a chip (EVATAR), to enable sex-based ex vivo research. To increase the scalability and accessibility of EVATAR, we turned to 3D printing (3DP) technologies, selecting two biocompatible 3DP resins, Dental SG (DSG) and Dental LT (DLT) to generate 3DP microphysiologic platforms. Due to the known sensitivity of reproductive cells to leachable compounds, we first screened for toxicity of these biomaterials using an in vitro mammalian oocyte maturation assay. Culture of mouse oocytes in 3DP plates using conventionally treated DSG resin resulted in rapid oocyte degeneration. Oxygen plasma treatment of the surface of printed DSG resin prevented this degeneration, and the majority of the resulting oocytes progressed through meiosis in vitro. However, 57.0% ± 37.2% of the cells cultured in the DSG resin plates exhibited abnormal chromosome morphology compared to 19.4% ± 17.3% of controls cultured in polystyrene. All tested DLT resin conditions, including plasma treatment, resulted in complete and rapid oocyte degeneration. To identify the ovo-toxic component of DLT, we analyzed DLT leachate using mass spectroscopy. We identified Tinuvin 292, a commercial light stabilizer, as a major component of the DLT leachate, which resulted in a dose-dependent disruption of meiotic progression and increase in chromosomal abnormalities with oocyte exposure, showing significant ovo-toxicity in mammals. Severe reproductive toxicity induced by in vitro exposure to these 3D-printed resins highlights potential risks of deploying insufficiently characterized materials for biomedical applications and underscores the need for more rigorous evaluation and designation of biocompatible materials.
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http://dx.doi.org/10.1016/j.chemosphere.2020.129003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957323PMC
May 2021

Emerging Roles for the Nucleolus 2019.

J Biol Chem 2020 04;295(16):5535-5537

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.

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http://dx.doi.org/10.1074/jbc.MT120.013346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170534PMC
April 2020

A View from the past into our collective future: the oncofertility consortium vision statement.

J Assist Reprod Genet 2021 Jan 6;38(1):3-15. Epub 2021 Jan 6.

Division of Reproductive Endocrinology & Infertility, University of Pennsylvania, Philadelphia, PA, USA.

Purpose: Today, male and female adult and pediatric cancer patients, individuals transitioning between gender identities, and other individuals facing health extending but fertility limiting treatments can look forward to a fertile future. This is, in part, due to the work of members associated with the Oncofertility Consortium.

Methods: The Oncofertility Consortium is an international, interdisciplinary initiative originally designed to explore the urgent unmet need associated with the reproductive future of cancer survivors. As the strategies for fertility management were invented, developed or applied, the individuals for who the program offered hope, similarly expanded. As a community of practice, Consortium participants share information in an open and rapid manner to addresses the complex health care and quality-of-life issues of cancer, transgender and other patients. To ensure that the organization remains contemporary to the needs of the community, the field designed a fully inclusive mechanism for strategic planning and here present the findings of this process.

Results: This interprofessional network of medical specialists, scientists, and scholars in the law, medical ethics, religious studies and other disciplines associated with human interventions, explore the relationships between health, disease, survivorship, treatment, gender and reproductive longevity.

Conclusion: The goals are to continually integrate the best science in the service of the needs of patients and build a community of care that is ready for the challenges of the field in the future.
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http://dx.doi.org/10.1007/s10815-020-01983-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786868PMC
January 2021

Macrophage-derived multinucleated giant cells: hallmarks of the aging ovary.

Reproduction 2021 02;161(2):V5-V9

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Inflammaging is a state of chronic, low-grade inflammation associated with aging which contributes to age-related diseases. Recently, an age-associated increase in inflammation has been documented in the mammalian ovary, which is accompanied by a shift in the immune cell profile. In this Point of View article, we consider a unique population of macrophage-derived multinucleated giant cells, found in reproductively old mouse ovaries, as potential markers or functional drivers of inflammation in ovarian aging.
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http://dx.doi.org/10.1530/REP-20-0489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856073PMC
February 2021

Ovarian stiffness increases with age in the mammalian ovary and depends on collagen and hyaluronan matrices.

Aging Cell 2020 11 20;19(11):e13259. Epub 2020 Oct 20.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6-12 weeks) and old (14-17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme-mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical properties of tissues. The total HA content in the ovarian stroma decreased with age, and this was associated with increased hyaluronidase (Hyal1) and decreased hyaluronan synthase (Has3) expression. These gene expression differences were not accompanied by changes in ovarian HA molecular mass distribution. Furthermore, ovaries from mice deficient in HAS3 were stiffer compared to age-matched WT mice. Our results demonstrate that the ovary becomes stiffer with age and that both collagen and HA matrices are contributing mechanisms regulating ovarian biomechanics. Importantly, the age-associated increase in collagen and decrease in HA are conserved in the human ovary and may impact follicle development and oocyte quality.
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http://dx.doi.org/10.1111/acel.13259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681059PMC
November 2020

Unscrambling the oocyte and the egg: clarifying terminology of the female gamete in mammals.

Mol Hum Reprod 2020 11;26(11):797-800

Reproductive Medicine Group, National Institute of Environmental Health Sciences, Durham, NC, USA.

Most reproductive biologists who study female gametes will agree with the 16th century anatomist William Harvey's doctrine: 'Ex Ovo Omnia'. This phrase, which literally translates to 'everything from the egg', recognizes the centrality of the egg in animal development. Eggs are most impressive cells, capable of supporting development of an entirely new organism following fertilization or parthenogenetic activation. Not so uniformly embraced in the field of reproductive biology is the nomenclature used to refer to the female germ cell. What is an oocyte? What is an egg? Are these terms the same, different, interchangeable? Here we provide functional definitions of the oocyte and egg, and how they can be used in the context of mammalian gamete biology and beyond.
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http://dx.doi.org/10.1093/molehr/gaaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648930PMC
November 2020

Zinc Dynamics during Drosophila Oocyte Maturation and Egg Activation.

iScience 2020 Jul 16;23(7):101275. Epub 2020 Jun 16.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.
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http://dx.doi.org/10.1016/j.isci.2020.101275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330606PMC
July 2020

Spatial Analysis of Growing Follicles in the Human Ovary to Inform Tissue Engineering Strategies.

Tissue Eng Part A 2020 07 26;26(13-14):733-746. Epub 2020 Jun 26.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Cancer survivorship has increased considerably, but common cancer treatments may threaten female reproductive health and fertility. In females, standard fertility preservation techniques include egg and embryo banking and ovarian tissue cryopreservation, but these methods are not suitable for all individuals. Emerging fertility preservation technologies include follicle growth and ovarian bioprosthetics. Although these platforms hold tremendous promise, they remain in the preclinical phase likely because of our inability to adequately phenocopy the complexity of the ovarian environment. The goal of this study was to use an established research archive of fixed human ovarian tissue established through the Oncofertility Consortium to better understand the dynamics and milieu of growing follicles within the human ovary. We performed a histological analysis of the immediate surroundings of primary and secondary stage follicles. We evaluated oocyte and follicle diameters of these growing follicles, analyzed their growth trajectories, and mapped their precise relationships to other stage follicles within a defined area. We also stratified our findings according to participant age and previous treatment history. Our results serve as benchmarks for follicles grown and provide insight into how follicles should be seeded spatially within bioprosthetic ovaries, potentially improving the efficacy and clinical translation of these emerging technologies. Impact statement Life-preserving cancer treatments have greatly increased survivorship. However, treatments often have off-target health consequences that threaten female reproductive health and fertility. Although several standard fertility preservation options exist, there is a constant need to explore and expand options for all populations follicle growth and ovarian bioprosthetics are new experimental procedures, which are currently limited to proof of concept. In this study, we analyzed human ovarian tissue from a deidentified biospecimen repository to characterize the growing follicle landscape with the ultimate goal of informing bioengineering practices. This spatial analysis pinpoints the geometry of growing follicles within the human ovary and provides a framework for paralleling this environment in platforms.
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http://dx.doi.org/10.1089/ten.tea.2020.0051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398444PMC
July 2020

Ovulation and ovarian wound healing are impaired with advanced reproductive age.

Aging (Albany NY) 2020 05 14;12(10):9686-9713. Epub 2020 May 14.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Aging is associated with reduced tissue remodeling efficiency and increased fibrosis, characterized by excess collagen accumulation and altered matrix degradation. Ovulation, the process by which an egg is released from the ovary, is one of the most dynamic cycles of tissue wounding and repair. Because the ovary is one of the first organs to age, ovulation and ovarian wound healing is impaired with advanced reproductive age. To test this hypothesis, we induced superovulation in reproductively young and old mice and determined the numbers of eggs ovulated and corpora lutea (CLs), the progesterone producing glands formed post-ovulation. Reproductively old mice ovulated fewer eggs and had fewer CLs relative to young controls. Moreover, reproductively old mice exhibited a greater number of oocytes trapped within CLs and expanded cumulus oocyte complexes within unruptured antral follicles, indicative of failed ovulation. In addition, post-ovulatory tissue remodeling was compromised with age as evidenced by reduced CL vasculature, increased collagen, decreased hyaluronan, decreased cell proliferation and apoptosis, impaired wound healing capacity, and aberrant morphology of the ovarian surface epithelium (OSE). These findings demonstrate that ovulatory dysfunction is an additional mechanism underlying the age-related loss of fertility beyond the reduction of egg quantity and quality.
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http://dx.doi.org/10.18632/aging.103237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288922PMC
May 2020

In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†.

Biol Reprod 2020 08;103(3):455-470

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980-2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.
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http://dx.doi.org/10.1093/biolre/ioaa073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442777PMC
August 2020

Zinc exocytosis is sensitive to myosin light chain kinase inhibition in mouse and human eggs.

Mol Hum Reprod 2020 04;26(4):228-239

Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Zinc dynamics are essential for oocyte meiotic maturation, egg activation, and preimplantation embryo development. During fertilisation and egg activation, the egg releases billions of zinc atoms (Zn2+) in an exocytotic event termed the 'zinc spark'. We hypothesised that this zinc transport and exocytosis is dependent upon the intracellular trafficking of cortical granules (CG) which requires myosin-actin-dependent motors. Treatment of mature mouse and human eggs with ML-7, a myosin light chain kinase inhibitor (MLCK), resulted in an 80% reduction in zinc spark intensity compared to untreated controls when activated with ionomycin. Moreover, CG migration towards the plasma membrane was significantly decreased in ML-7-treated eggs compared with controls when activated parthenogenetically with ionomycin. In sperm-induced fertilisation via intracytoplasmic sperm injection (ICSI), ML-7-treated mouse eggs exhibited decreased labile zinc intensity and cortical CG staining. Collectively, these data demonstrate that ML-7 treatment impairs zinc release from both murine and human eggs after activation, demonstrating that zinc exocytosis requires myosin light chain kinase activity. Further, these results provide additional support that zinc is likely stored and released from CGs. These data underscore the importance of intracellular zinc trafficking as a crucial component of egg maturation necessary for egg activation and early embryo development.
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http://dx.doi.org/10.1093/molehr/gaaa017DOI Listing
April 2020

Low Molecular Weight Hyaluronan Induces an Inflammatory Response in Ovarian Stromal Cells and Impairs Gamete Development In Vitro.

Int J Mol Sci 2020 Feb 4;21(3). Epub 2020 Feb 4.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

The ovarian stroma, the microenvironment in which female gametes grow and mature, becomes inflamed and fibrotic with age. Hyaluronan is a major component of the ovarian extracellular matrix (ECM), and in other aging tissues, accumulation of low molecular weight (LMW) hyaluronan fragments can drive inflammation. Thus, we hypothesized that LMW hyaluronan fragments contribute to female reproductive aging by stimulating an inflammatory response in the ovarian stroma and impairing gamete quality. To test this hypothesis, isolated mouse ovarian stromal cells or secondary stage ovarian follicles were treated with physiologically relevant (10 or 100 μg/mL) concentrations of 200 kDa LMW hyaluronan. In ovarian stromal cells, acute LMW hyaluronan exposure, at both doses, resulted in the secretion of a predominantly type 2 (Th2) inflammatory cytokine profile as revealed by a cytokine antibody array of conditioned media. Additional qPCR analyses of ovarian stromal cells demonstrated a notable up-regulation of the eotaxin receptor Ccr3 and activation of genes involved in eosinophil recruitment through the IL5-CCR3 signaling pathway. These findings were consistent with an age-dependent increase in ovarian stromal expression of Ccl11, a major CCR3 ligand. When ovarian follicles were cultured in 10 or 100 μg/mL LMW hyaluronan for 12 days, gametes with compromised morphology and impaired meiotic competence were produced. In the 100 μg/mL condition, LMW hyaluronan induced premature meiotic resumption, ultimately leading to in vitro aging of the resulting eggs. Further, follicles cultured in this LMW hyaluronan concentration produced significantly less estradiol, suggesting compromised granulosa cell function. Taken together, these data demonstrate that bioactive LMW hyaluronan fragments may contribute to reproductive aging by driving an inflammatory stromal milieu, potentially through eosinophils, and by directly compromising gamete quality through impaired granulosa cell function.
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http://dx.doi.org/10.3390/ijms21031036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036885PMC
February 2020

The long view: Considering spindle orientation in image analysis.

Mol Reprod Dev 2020 02 17;87(2):205. Epub 2020 Jan 17.

Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois.

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http://dx.doi.org/10.1002/mrd.23313DOI Listing
February 2020

Tissue-specific Fixation Methods Are Required for Optimal In Situ Visualization of Hyaluronan in the Ovary, Kidney, and Liver.

J Histochem Cytochem 2020 01 12;68(1):75-91. Epub 2019 Nov 12.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin's Solution, Carnoy's Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson's Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy's Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson's Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.
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http://dx.doi.org/10.1369/0022155419884879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931168PMC
January 2020

Recombinant FSH glycoforms are bioactive in mouse preantral ovarian follicles.

Reproduction 2019 12;158(6):517-527

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn7 and Asn24 residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH21/18, glycans absent on either Asn24 or Asn7, respectively) or fully-glycosylated (FSH24, glycans present on both Asn7 and Asn24). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH21/18 in younger women to FSH24 in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18-20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles.
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http://dx.doi.org/10.1530/REP-19-0392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047736PMC
December 2019

Differential sensitivity of inbred mouse strains to ovarian damage in response to low-dose total body irradiation†.

Biol Reprod 2020 02;102(1):133-144

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Radiation induces ovarian damage and accelerates reproductive aging. Inbred mouse strains exhibit differential sensitivity to lethality induced by total body irradiation (TBI), with the BALB/cAnNCrl (BALB/c) strain being more sensitive than the 129S2/SvPasCrl (129) strain. However, whether TBI-induced ovarian damage follows a similar pattern of strain sensitivity is unknown. To examine this possibility, female BALB/c and 129 mice were exposed to a single dose of 1 Gy (cesium-137 γ) TBI at 5 weeks of age, and ovarian tissue was harvested for histological and gene expression analyses 2 weeks post exposure. Sham-treated mice served as controls. 1 Gy radiation nearly eradicated the primordial follicles and dramatically decreased the primary follicles in both strains. In contrast, larger growing follicles were less affected in the 129 relative to BALB/c strain. Although this TBI paradigm did not induce detectable ovarian fibrosis in either of the strains, we did observe strain-dependent changes in osteopontin (Spp1) expression, a gene involved in wound healing, inflammation, and fibrosis. Ovaries from BALB/c mice exhibited higher baseline Spp1 expression that underwent a significant decrease in response to radiation relative to ovaries from the 129 strain. A correspondingly greater change in the ovarian matrix, as evidenced by reduced ovarian hyaluronan content, was also observed following TBI in BALB/c mice relative to 129 mice. These early changes in the ovary may predispose BALB/c mice to more pronounced late effects of TBI. Taken together, our results demonstrate that aspects of ovarian damage mirror other organ systems with respect to overall strain-dependent radiation sensitivity.
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http://dx.doi.org/10.1093/biolre/ioz164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334620PMC
February 2020

Histologic analysis and lipid profiling reveal reproductive age-associated changes in peri-ovarian adipose tissue.

Reprod Biol Endocrinol 2019 Jun 12;17(1):46. Epub 2019 Jun 12.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 E. Superior Street, Lurie 7-117, Chicago, IL, 60611, USA.

Background: Reproductive aging is a robust phenotype that occurs in all females and is characterized by a significant reduction in gamete quantity and quality, which can have negative consequences on both endocrine function and fertility. Age-associated differences in the oocyte, follicle, and ovary have been well-documented, but how the broader environment changes with age is less well understood. Fat is one of the largest organs in the body, and peri-gonadal adipose tissue surrounds the rodent ovary and comprises a local ovarian environment. The goal of this study was to characterize how peri-ovarian adipose tissue changes with advanced reproductive age.

Methods: We isolated peri-gonadal adipose tissue from two cohorts of CB6F1 mice: reproductively young (6-12 weeks) and reproductively old (14-17 months). A comparative histological analysis was performed to evaluate adipocyte architecture. We then extracted lipids from the tissue and performed multiple reaction monitoring (MRM)-profiling, a mass spectrometry-based method of metabolite profiling, to compare the lipid profiles of peri-gonadal adipose tissue in these age cohorts.

Results: We found that advanced reproductive age was associated with adipocyte hypertrophy and a corresponding decrease in the number of adipocytes per area. Of the 10 lipid classes examined, triacylglycerols (TAGs) had significantly different profiles between young and old cohorts, despite quantitative analysis revealing a decrease in the total amount of TAGs per weight of peri-gonadal adipose tissue with age.

Conclusions: These findings pinpoint age-associated physiological changes in peri-gonadal adipose tissue with respect to adipocyte morphology and lipid profiles and lay the foundation for future studies to examine how these alterations may influence both adipocyte and ovarian function.
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http://dx.doi.org/10.1186/s12958-019-0487-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563378PMC
June 2019

Disruption of O-GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization.

Mol Reprod Dev 2019 05 21;86(5):543-557. Epub 2019 Feb 21.

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois.

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O-GlcNAcylation-the addition of a single sugar residue (O-linked β-N-acetylglucosamine) on proteins-is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O-GlcNAcylation is mediated by O-GlcNAc transferase (OGT), which adds O-GlcNAc onto proteins, and O-GlcNAcase (OGA), which removes it. Here we investigated O-GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O-GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O-GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O-GlcNAc function, we disrupted O-GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet-G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O-GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O-GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.
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http://dx.doi.org/10.1002/mrd.23131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510634PMC
May 2019

Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation.

Theriogenology 2019 Mar 24;127:41-48. Epub 2018 Dec 24.

The Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA; Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA; Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA. Electronic address:

Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.
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http://dx.doi.org/10.1016/j.theriogenology.2018.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647840PMC
March 2019

Oocyte-specific deletion of Hdac8 in mice reveals stage-specific effects on fertility.

Reproduction 2019 03;157(3):305-316

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Eighteen histone deacetylases exist in mammals. The class 1 histone deacetylases HDAC1 and HDAC2 are important for oogenesis and fertility in mice, likely via their effects on histones. The reproductive function of HDAC8, another class 1 enzyme, has not been explored. One key target of HDAC8 is the SMC3 subunit of cohesin, an essential complex mediating sister chromatid cohesion and chromosome segregation. In current models, HDAC8 activity is required for SMC3 recycling, but this function should be dispensable in oocytes since cohesion is established during pre-meiotic S phase and maintained until meiotic resumption during ovulation. Whether other oocyte-specific HDAC8-mediated deacetylation events are required for oogenesis and female fertility is unknown. We used two Cre drivers to remove Hdac8 at specific stages of oocyte development to address whether HDAC8 is required for female fertility in mice. When HDAC8 was knocked out in oocytes in primary and later stage follicles (Zp3-Cre), oogenesis and folliculogenesis appeared normal and mice were fertile. However, females were subfertile when HDAC8 was knocked out prior to pre-meiotic S phase and cohesion establishment (Vasa-Cre). This subfertility was independent of chromosome segregation errors during meiosis but rather appeared to be the result of defects in oogenesis that resulted in smaller fully grown oocytes with a reduced ability to resume meiosis. In all cases, we did not observe compensatory changes in HDAC1, HDAC2 and HDAC3 levels. Thus, although oocyte-specific expression of HDAC8 is not essential for mouse oogenesis after meiotic S phase, it contributes to optimal fertility. We infer that oocyte-specific expression of the deacetylase HDAC8 is required early in oogenesis for optimal fertility.
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http://dx.doi.org/10.1530/REP-18-0560DOI Listing
March 2019

The National Physicians Cooperative: transforming fertility management in the cancer setting and beyond.

Future Oncol 2018 Dec 26;14(29):3059-3072. Epub 2018 Nov 26.

Department of Urology, University of Washington School of Medicine, Seattle, WA 98195, USA.

Once unimaginable, fertility management is now a nationally established part of cancer care in institutions, from academic centers to community hospitals to private practices. Over the last two decades, advances in medicine and reproductive science have made it possible for men, women and children to be connected with an oncofertility specialist or offered fertility preservation soon after a cancer diagnosis. The Oncofertility Consortium's National Physicians Cooperative is a large-scale effort to engage physicians across disciplines - oncology, urology, obstetrics and gynecology, reproductive endocrinology, and behavioral health - in clinical and research activities to enable significant progress in providing fertility preservation options to children and adults. Here, we review the structure and function of the National Physicians Cooperative and identify next steps.
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http://dx.doi.org/10.2217/fon-2018-0278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331694PMC
December 2018

The use of a virtual journal club to promote cross-cultural learning in the reproductive sciences.

J Assist Reprod Genet 2018 Dec 24;35(12):2141-2147. Epub 2018 Sep 24.

Departamento de Biología Celular e Histología, Facultad de Medicina 30100, Universidad de Murcia, IMIB, Murcia, Spain.

Purpose: Scientific literacy and communication are critical skills in the biological sciences. Journal clubs, in which peer-reviewed academic literature is discussed, are traditionally used to teach students to evaluate the literature, review scientific findings, and learn about historical, controversial, or current topics.

Methods: We used a virtual journal club to facilitate the international interaction between two universities with master's degree programs in the reproductive sciences: the University of Murcia (Spain) and Northwestern University (USA). The virtual journal club occurred over a 2-hour period and was held using Blue Jeans Conferencing Service software and involved a total of 29 students. During this event, the students who were separated physically by thousands of miles discussed and exchanged ideas about a high-impact publication in real time. A survey assessment was administered to students at the University of Murcia following the event.

Results: Positive perceptions included the establishment of cross-institutional interactions and the ability to practice scientific communication in another language. Areas noted for improvement included preparation time and engagement opportunities.

Conclusion: Overall, the virtual journal club is an innovative technology that can easily be broadened and has the potential to foster collaboration, ameliorate multilingual communication, improve cultural competencies, and expand professional global networks.
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http://dx.doi.org/10.1007/s10815-018-1309-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289918PMC
December 2018

Use of a Small Animal Radiation Research Platform (SARRP) facilitates analysis of systemic versus targeted radiation effects in the mouse ovary.

J Ovarian Res 2018 Aug 30;11(1):72. Epub 2018 Aug 30.

Center for Reproductive Science, Northwestern University, Chicago, IL, 60611, USA.

Background: Radiation exposure is known to cause accelerated aging and damage to the ovary, but the contribution of indirect versus direct effects is not well understood. We used the Small Animal Radiation Research Platform (SARRP) (Xstrahl) to deliver radiation to precise fields equivalent to clinical practice, allowing us to investigate systemic versus targeted damage in a structure as small as the mouse ovary. The X-ray dose was kept constant at 1 Gy, but the field varied. Mice either received total body irradiation (TBI), radiation targeted to both ovaries (T2), or radiation targeted to one ovary (left) while the contralateral ovary (right) was spared (T1). Sham mice, handled similarly to the other cohorts but not exposed to radiation, served as controls. Two weeks post-exposure, ovaries were harvested and analyzed histologically to identify and count follicles within each ovary.

Results: Radiation significantly reduced primordial follicles in the TBI and T2 cohorts compared to the Sham cohort. There were no significant differences between these two irradiated groups. These findings suggest that at 1 Gy, the extent of damage to the ovary caused by radiation is similar despite the different delivery methods. When investigating the T1 cohort, targeted ovaries showed a significant decrease in primordial and growing follicles compared to non-targeted contralateral ovaries.

Conclusions: These findings demonstrate that the SARRP is an effective strategy for delivering precise ionizing radiation to small organs such as mouse ovaries. Such tools will facilitate identifying the relative risks to ovarian function associated with different radiation fields as well as screening the efficacy of emerging fertoprotective agents.
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http://dx.doi.org/10.1186/s13048-018-0442-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116356PMC
August 2018

Quantifying the growth of oncofertility.

Biol Reprod 2018 08;99(2):263-265

Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

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http://dx.doi.org/10.1093/biolre/ioy068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190656PMC
August 2018

Revisiting the scientific method to improve rigor and reproducibility of immunohistochemistry in reproductive science.

Biol Reprod 2018 10;99(4):673-677

Center for Reproductive Science, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Immunohistochemistry (IHC) is a robust scientific tool whereby cellular components are visualized within a tissue, and this method has been and continues to be a mainstay for many reproductive biologists. IHC is highly informative if performed and interpreted correctly, but studies have shown that the general use and reporting of appropriate controls in IHC experiments is low. This omission of the scientific method can result in data that lack rigor and reproducibility. In this editorial, we highlight key concepts in IHC controls and describe an opportunity for our field to partner with the Histochemical Society to adopt their IHC guidelines broadly as researchers, authors, ad hoc reviewers, editorial board members, and editors-in-chief. Such cross-professional society interactions will ensure that we produce the highest quality data as new technologies emerge that still rely upon the foundations of classic histological and immunohistochemical principles.
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http://dx.doi.org/10.1093/biolre/ioy094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203879PMC
October 2018
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