Day 3 of Research Paper Analysis!
"Decreased Glycolysis at Menstruation is Associated with Increased Menstrual Blood Loss”
Glycolysis and Heavy Menstrual Bleeding:
In 2022, Mao et al. found that deficient glycolysis is associated with excessive menstrual bleeding. Heavy menstrual bleeding, abbreviated as HMB, is caused by faulty repair of the uterine lining after menstruation.
Fundamental Mechanics of a Period:
Each menstrual cycle elicits numerous external and internal changes that occur in a menstruator’s body, most notably physiologically. Upon each cycle, the uterine endometrium is repaired, which actually requires significant energy input. In each menstrual cycle, endometrial cells must decidualize to prepare for pregnancy and menstrual discharge. Aberrations in this cyclic renewal lead to abnormal menstrual bleeding (beavy menstrual bleeding), which afflict 1 ⁄ 3 of menstruators.
Methodology:
The aforementioned experiments utilized a murine model of menstruation, cell culture experiments, and studies of human samples. The goal of the investigation was to determine the role of glycolysis in the repair of the endometrium after menstruation.
You may be wondering how heavy menstrual bleeding can be assessed using mice? Obviously, mice do not menstruate, but human-like menses can be induced in mice using hormone stimulation.
Mice Treatments:
Estrogen: The mice were treated with estrogen to induce the proliferation of endometrial cells.
Progesterone: The mice were treated with progesterone to bring about the endometrial cell differentiation.
Inhibitor of glycolytic enzyme HK2 and immunohistochemistry:
Mao et al. demonstrated that inhibition of glycolysis ameliorated inflammation and impaired the uterine endometrium restoration to stable conditions.
Cell Culture Experiments:
Immunohistochemistry analysis of proteins and pro- and anti-inflammatory cytokines in the endometrium was performed. According to the Cleveland Clinic, immunohistochemistry uses antibodies to detect antigens in a tissue sample. Endometrial repair and menstrual blood loss (MBL) was assessed via staging endometrial morphology using histological scoring of regions of interest in the endometrium. Specifically, the CCK-8 and scratch assays were used to evaluate the impact of glycolysis regulation on cell viability.
About the CCK-8 and Scratch Assay:
The CCK-8 assay, of the cell counting kit 8, essentially utilizes water-soluble tetrazolium salt to quantify live cells. This assay is similar to the MTT colorimetric assay that is used to assess cell metabolism. The primary underlying difference lies in the fact that the CCK-8 assay involves most of the dehydrogenase in the cell, whereas the MTT assay only involves mitochondrial dehydrogenase.The scratch assay is a measure of cell migration, including particular parameters speed, persistence, and polarity. The scratch assay is performed by making a scratch on a cell monolayer and capturing images on a regular interval by time lapse microscope. This method has been characterized as convenient and inexpensive.
Ultimately, it was elucidated that endometrial repair was associated with increased glycolysis levels in vivo. Conversely, glycolysis repair was abated with glycolysis suppression, ultimately leading to an increase in heavy menstrual bleeding.
Human Samples:
Mao et al. then attained liquid biopsies of their patients. The patients were divided into 2 groups: excessive bleeding group and control group.
Those who had more than 100 mL of menstrual blood loss were delineated as the “excessive bleeding group.” Those who had less than 100 mL of menstrual blood loss were delineated as the “control group.”
The authors then performed immunostaining of Hexokinase 2 (HK2) on the samples. Women with adenomyosis, which is a condition in which endometrial cells penetrate into the myometrium, had lower HK2 expressions; this finding had major translational implications in humans as it demonstrated that abnormal glycolysis causes heavy menstrual bleeding in humans as well.
Hypoxia in the Endometrium During Menstruation:
Studies published prior to that of Mao et al. elucidated that hypoxia occurs in the endometrium during menstruation. Hypoxia is a condition in which oxygen is present in tissues at insufficient levels to maintain bodily equilibrium. The study showed that even when menstruation-induced hypoxia was maintained in the mice, hypoxia inducible factor 1 alpha (HIF-1alpha) levels were stabilized. HIF-alpha is an established hypoxia regulator and glycolytic gene inducer. As stated before, even in the presence of hypoxia-induced HIF-1alpha stabilization, glycolysis deficiency STILL impacted endometrium repair.
Avenues for Future Research
All in all, the findings from Mao et al.’s work are crucial as they pave the way for new treatment options for heavy menstrual bleeding. Currently, hormonal options such as progesterone and gonadotropin-releasing hormone agonists are employed to treat heavy menstrual bleeding. Mao et al.’s work opens the avenue for non-hormonal treatments such as enforcing the HIF pathway or glycolysis to ameliorate heavy bleeding, which often impairs the quality of life of menstruators.
*Disclaimer: The authors of “Decreased Glycolysis at Menstruation is Associated with Increased Menstrual Blood Loss” are Chenyu Mao, Xishi Liu, and Sun-Wei Guo.
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