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Menstruation: science and society

Open AccessPublished:July 21, 2020DOI:https://doi.org/10.1016/j.ajog.2020.06.004
      Women’s health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation—a fast, scarless healing process in healthy individuals—will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, “Menstruation: Science and Society” with an aim to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration—and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids—to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent “menstrual equity” and “period poverty” movements spreading across high-income countries.

      Key words

      Related editorial, page 617.

      Introduction

      Twenty-five years have passed since the National Institutes of Health (NIH) mandated that women and minorities be included in all government-funded clinical studies unless their exclusion could be justified. Clearly, this policy has led to numerous women’s health research programs. However, women and women’s health concerns continue to be underrepresented in research. Most recently, the 2019–2023 Trans-NIH Strategic Plan for Women’s Health Research was initiated to improve the health of women by advancing rigorous research relevant to advancing women’s health, including sexual and reproductive health (SRH). Despite focused initiatives such as these, diagnostic development for improving women’s reproductive health has been hampered by a lack of understanding of basic uterine and menstrual physiology. A PubMed search of the term “menstruation” yielded less the 1000 publications between 1941 and 1950, followed by a peak of more than 6000 publications between 1971 and 1980 (note: Our Bodies, Ourselves, a book addressing women’s health topics, including menstruation and birth control, was published in 1973), and then a stable trough with less the 4000 publications per decade over the past 3 decades spanning 1991 through 2019 (Figure 1, A). By contrast, a PubMed search of the term “menstrual blood” yielded 1 publication during 1941–1950, followed by a steady increase over time to more than 400 publications in the last decade (Figure 1, B). For reference, PubMed searches of “peripheral blood” and “semen” yielded almost 100,000 and 15,000 publications, respectively, over the past decade.
      16S rRNA gene: Encodes a component of the 30S small subunit of a prokaryotic ribosome. 16S rRNA gene sequencing is used for phylogenetic studies because its presence is highly conserved among bacteria, but its sequence is species-specific.
      Aromatase: An enzyme that transforms androgens into estrogens.
      AUB: Abnormal uterine bleeding.
      Biomass: Amount of living biological organisms in a given niche or ecosystem at a given time. The upper genital tract has a significantly lower amount of bacterial DNA than other human microbiomes and is therefore considered a low biomass microbiota.
      BMP-2: Bone morphogenetic protein 2.
      COEIN: Coagulopathy, Ovulatory, Endometrial, Iatrogenic, Not otherwise classified.
      Community state types (CST): Profile that defines the total bacterial community of a given body site based on the relative abundances of each bacterium. The human vaginal microbial communities were classified into 5 groups. Specifically, CSTs I, II, III, and V are dominated by L. crispatus, L. gasseri, L. iners, and L. jensenii, respectively, whereas CST IV has higher proportions of strictly anaerobic organisms.
      DCE-MRI: Dynamic contrast enhanced-MRI.
      Diversity (Beta diversity): Refers to the change in the number of taxa detected in 2 or more ecosystems. It is usually expressed as the total number of species that are unique to each of the ecosystems being compared.
      Dysbiosis: Shift in the physiologic microbiota resulting in an imbalance between commensal and pathogenic bacteria. Changes in microbial composition owing to the gain or loss of the community members or changes in the relative abundance of microbes may contribute to the initiation and/or persistence of many diseases.
      Epigenetics: Heritable phenotype changes without changes in genotype (DNA).
      Estrobolome: Represents the aggregate of enteric bacterial genes whose products are capable of metabolizing estrogens. Microbes in the estrobolome produce beta-glucuronidase, an enzyme that deconjugates estrogens into their active forms, which are capable of binding to estrogen receptors and influencing estrogen-dependent physiological processes.
      FIGO: International Federation of Gynecology and Obstetrics.
      GaP: Genotype and Phenotype Registry (registry of normal/control research subjects).
      GnRH: Gonadotrophin releasing hormone.
      Growth factor: A substance capable of stimulating cell growth, proliferation, and differentiation.
      Gut-brain axis: Consists of bidirectional neural processing of information between the central nervous system and digestive system. Recent research indicates that gut microbiota is a crucial part of the gut-brain network and communicates with the brain through the microbiota-gut-brain axis.
      HIF: Hypoxia inducible factor.
      HMB: Heavy menstrual bleeding.
      Hologenome: Theory that maintains that the physiology of any macroscopic organism derives from the integrated activities of the individual genomes contributing to the organism (holobiont).
      LNG-IUS: Levonorgestrel-releasing intrauterine system
      ME: Menstrual effluent.
      ME-SFCs: Menstrual effluent derived stromal fibroblast cells.
      mHealth: Mobile health.
      Microbiota and Microbiome: The human microbiota encompasses the group of microorganisms that live in association with the human body. Conversely, the microbiome refers to the genes and genomes of this microbiota as well as their products within the host environment.
      micro-RNA: Small noncoding RNA molecule regulating posttranscriptional gene expression.
      MRI: Magnetic resonance imaging.
      MSCs: Mesenchymal stem cells.
      MT-MRI: Magnetization transfer-MRI.
      Multipotent stem cell: A cell that can self-renew by division and can develop into multiple differentiated cell types.
      Natural killer (NK) cell: A type of lymphocyte that can bind to certain tumor cells and virus-infected cells without the stimulation of antigens and can kill them by the insertion of granules containing perforin.
      PA: Plasminogen activator.
      PAEC: Progesterone receptor-modulator-associated endometrial changes.
      PAI: Plasminogen activator inhibitor.
      PALM: Polyps, Adenomyosis, Leiomyoma, Malignancy.
      Paracrine signaling: Signaling involving hormone that has an effect only in the vicinity of the cell secreting it.
      PCOS: Polycystic ovary syndrome.
      Richness (Alpha diversity): Refers to the diversity within a particular area or ecosystem. It is usually expressed by the number of species (species richness) in a unique niche.
      ROSE: Research OutSmarts Endometriosis (research program dedicated to studying endometriosis).
      SPRM: Selective progesterone receptor modulator.
      TGF-β3: Transforming growth factor-beta 3.
      T2W: T-2 weighted.
      t-PA: Tissue plasminogen activator.
      uNK cells: Uterine natural killer cells.
      u-PA: Urokinase plasminogen activator.
      Figure thumbnail gr1
      Figure 1PubMed publications, 1941–2018
      A, Search term “Menstruation.” B, Search term “Menstrual Blood.”.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.
      In September 2018, the Gynecologic Health and Disease Branch (GHDB) of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) convened a 2-day meeting to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Leaders in the field with expertise in endometrial biology, omic analysis of the endometrium and menstrual effluent, new imaging or sampling modalities, smart technologies or applications (apps) and mobile health (mHealth) platforms, menstrual health, and health literacy and dissemination frameworks were invited to participate as speakers and discussants to critique and summarize new discoveries and avenues of future research surrounding menstruation. This meeting encompassed normal menstrual health and endometrial function and the potential of diagnostics for abnormal functioning and disease. To provide a broad perspective on menstruation science, this meeting included investigators and stakeholders across multiple disciplines, including population health and public health sectors, and carefully considered the broader societal implications of menstrual health. This manuscript summarizes the presentations and discussions that took place at the 2018 “Menstruation: Science and Society” meeting hosted by GHDB, NICHD.

      1. Toward a Better Understanding of Menstrual Health: Menstrual Health Literacy and Communication

      Kristen A. Matteson, MD, MPH; Missy Lavender, MBA; Erica E. Marsh, MD, MSCI; Kami Silk, PhD

      I. Introduction

      According to the World Health Organization, “health” is “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity.”
      World Health Organization
      Frequently asked questions. 2019.
      For women, menstrual health is an integral part of overall health because, between menarche and menopause, most women menstruate and menstruation can have a significant impact on the physical, mental, and social well-being.
      • Matteson K.A.
      • Raker C.A.
      • Clark M.A.
      • Frick K.D.
      Abnormal uterine bleeding, health status, and usual source of medical care: analyses using the Medical Expenditures Panel Survey.
      Normal menstruation is currently defined as cyclic bleeding that occurs from the uterine corpus between menarche and menopause. It can be described in terms of 4 simple domains: how frequently the woman has episodes of bleeding, the regularity or predictability of these episodes, the duration of bleeding episodes, and the volume or heaviness of bleeding.
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      • Sharp H.T.
      • Johnson J.V.
      • Lemieux L.A.
      • Currigan S.M.
      Executive summary of the reVITALize initiative: standardizing gynecologic data definitions.
      American College of Obstetricians and Gynecologists
      reVITALize: gynecology data definitions.
      Not all women experience “normal” menstrual bleeding; up to 30% of women will experience alterations in the volume or pattern of menstrual blood flow, which is defined as the symptom of abnormal uterine bleeding (AUB), which in turn can be caused by multiple etiologies and sometimes more than 1 etiology at the same time.
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      ,
      • Kjerulff K.H.
      • Erickson B.A.
      • Langenberg P.W.
      Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992.
      In addition, many women will have other symptoms such as pain, dysmenorrhea, anxiety, depression, and fatigue associated with their menstrual cycle that require attention for them to achieve early diagnosis of reproductive health issues such as endometriosis, premenstrual syndrome, and premenstrual dysphoric disorder and attain optimal health. In research and in clinical care, a better understanding of what the norms of menstrual health are and how a “lack” of menstrual health affects women’s quality of life is needed. Furthermore, for positive health and well-being outcomes, everyone—men and women, as well as clinicians—need to understand menstrual cycles and menstrual health, which can be achieved through menstrual health literacy initiatives and improved health communication.
      Menstrual health and menstrual health literacy are extremely broad topics with multiple stakeholders and diverse areas of active investigation and contributors. Adapted from the broader health literacy definition, menstrual health literacy refers to the level of capacity a person has to obtain, process, and understand basic information about menstruation so they can make appropriate health decisions.
      • Ratzan S.C.
      • Parker R.M.
      Introduction.
      This section of the manuscript summarizes the presentations and discussion that took place related to menstrual health and menstrual health literacy at the 2018 “Menstruation: Science and Society” meeting hosted by the GHDB of the NICHD. We summarize only areas of menstrual health and literacy that were part of the presentations and active discussions at the NICHD GHDB meeting, which were largely focused on the bleeding aspect of menstrual health.

      II. Progress in menstrual health terminology and menstrual health literacy and communication

      Progress in menstrual health terminology

      Standard terminologies related to menstrual bleeding, and specifically AUB, represent real progress for clinical care and research. Ill-defined terminologies to describe symptoms, signs, and diagnoses associated with AUB led to communication challenges in clinical care, difficulty interpreting populations included in published literature, and lost opportunities for multisite research collaboration for clinical research on treatments for AUB.
      In 2005, the Menstrual Disorders Working Group of the International Federation of Gynecology and Obstetrics (FIGO) embarked on a worldwide consensus-building process to generate and disseminate a simple symptom description system and a classification system for the etiologies associated with AUB.
      • Woolcock J.G.
      • Critchley H.O.
      • Munro M.G.
      • Broder M.S.
      • Fraser I.S.
      Review of the confusion in current and historical terminology and definitions for disturbances of menstrual bleeding.
      ,
      • Munro M.G.
      • Critchley H.O.
      • Fraser I.S.
      FIGO Menstrual Disorders Working Group
      The FIGO classification of causes of abnormal uterine bleeding in the reproductive years.
      The first system, “Terminologies and Definitions,” includes standard definitions for bleeding symptoms domains, which include regularity, frequency, duration, and volume.
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      The second system, “Classification of Causes of AUB in the Reproductive Years,” commonly referred to as the polyps/adenomyosis/leiomyoma/malignancy (PALM)–coagulopathy/ovulatory/endometrial/iatrogenic/not otherwise classified (COEIN) system, includes a list of etiologies that can be associated with AUB (polyps, adenomyosis, leiomyoma, malignancy and hyperplasia, coagulopathy, ovulatory, endometrial, iatrogenic, and not otherwise classified). Results of this work were published in peer-reviewed publications and were used by the American College of Obstetricians and Gynecologists (ACOG) during their process standardizing terminologies used across gynecologic specialties.
      • Sharp H.T.
      • Johnson J.V.
      • Lemieux L.A.
      • Currigan S.M.
      Executive summary of the reVITALize initiative: standardizing gynecologic data definitions.
      ,
      American College of Obstetricians and Gynecologists
      reVITALize: gynecology data definitions.
      ACOG and the members of the Women’s Health Registry alliance convened the revitalize Gynecology Data Definitions initiative in December 2013 to develop standardized data elements and definitions in gynecology. Throughout this process, ACOG engaged a broad range of stakeholders to identify priority topics and definitions and then worked with a core group of contributors to generate a total of 119 data elements, including 7 pain-related and 7 bleeding-related definitions.
      • Sharp H.T.
      • Johnson J.V.
      • Lemieux L.A.
      • Currigan S.M.
      Executive summary of the reVITALize initiative: standardizing gynecologic data definitions.
      ,
      American College of Obstetricians and Gynecologists
      reVITALize: gynecology data definitions.
      Although there is more work to be done in terms of evaluating these definitions across diverse populations of women, these standardized terminologies represent positive first steps to facilitate research data collection, collaboration for study participant recruitment, and identification of study cohorts with similar etiologies when investigating the prevalent symptom of AUB.
      Another area of progress has been increased emphasis on the patient experience of bleeding, quality of life, and related symptoms associated with the menstrual cycle in both research and national guidelines. The National Institute for Health and Clinical Excellence (NICE) Clinical Guidelines on Heavy Menstrual Bleeding (HMB) published a patient-centric definition of the symptom of HMB, which they define as “excessive menstrual blood loss that interferes with a woman’s physical, social, emotional, and/or material quality of life.”
      National Institute for Health and Clinical Excellence (NICE)
      Heavy menstrual bleeding: assessment and management. 2018.
      Qualitative and quantitative research with women to learn about their experiences with and knowledge of menstrual bleeding, fibroid-related symptoms, pain, and other associated symptoms has begun to inform research priorities, educational tools, and the need for outcome measures for AUB and uterine fibroids.
      • Matteson K.A.
      • Scott D.M.
      • Raker C.A.
      • Clark M.A.
      The Menstrual Bleeding Questionnaire: development and validation of a comprehensive patient-reported outcome instrument for heavy menstrual bleeding.
      • Matteson K.A.
      • Clark M.A.
      Questioning our questions: do frequently asked questions adequately cover the aspects of women’s lives most affected by abnormal uterine bleeding? Opinions of women with abnormal uterine bleeding participating in focus group discussions.
      • Ghant M.S.
      • Sengoba K.S.
      • Recht H.
      • Cameron K.A.
      • Lawson A.K.
      • Marsh E.E.
      Beyond the physical: a qualitative assessment of the burden of symptomatic uterine fibroids on women’s emotional and psychosocial health.
      • Marsh E.E.
      • Brocks M.E.
      • Ghant M.S.
      • Recht H.S.
      • Simon M.
      Prevalence and knowledge of heavy menstrual bleeding among African American women.
      Several studies have suggested that patient-reported outcome measures (PROMs), which include standardized interviews, questionnaires, charts, and surveys that assess the patient’s own evaluation of her health and symptoms, are the key to assessing the impact of illness and symptomatology among women with reproductive health issues including AUB.
      • Matteson K.A.
      • Raker C.A.
      • Clark M.A.
      • Frick K.D.
      Abnormal uterine bleeding, health status, and usual source of medical care: analyses using the Medical Expenditures Panel Survey.
      ,
      • Bernardi L.A.
      • Ghant M.S.
      • Andrade C.
      • Recht H.
      • Marsh E.E.
      The association between subjective assessment of menstrual bleeding and measures of iron deficiency anemia in premenopausal African-American women: a cross-sectional study.
      • Weldring T.
      • Smith S.M.
      Patient-reported outcomes (PROs) and patient-reported outcome measures (PROMs).
      • Rahn D.D.
      • Abed H.
      • Sung V.W.
      • et al.
      Systematic review highlights difficulty interpreting diverse clinical outcomes in abnormal uterine bleeding trials.
      • Clark T.J.
      • Khan K.S.
      • Foon R.
      • Pattison H.
      • Bryan S.
      • Gupta J.K.
      Quality of life instruments in studies of menorrhagia: a systematic review.
      • Warner P.E.
      • Critchley H.O.
      • Lumsden M.A.
      • Campbell-Brown M.
      • Douglas A.
      • Murray G.D.
      Menorrhagia II: is the 80-mL blood loss criterion useful in management of complaint of menorrhagia?.

      Progress in menstrual health literacy and communication

      There has been recent progress in the areas of menstrual health literacy, advocacy, and communication, in part facilitated by the rapid acceptance of mHealth apps, the use of mobile technologies to provide health-related services (tracking information and providing information or education to support an individual’s achievement of health objectives).
      World Health Organization Global Observatory for eHealth
      mHealth: new horizons for health through mobile technologies: second global survey on eHealth. 2011.
      ,
      • Radbron E.
      • Wilson V.
      • McCance T.
      • Middleton R.
      The use of data collected from mHealth apps to inform evidence-based quality improvement: an integrative review.
      At present, there are more than 300 reproductive health mHealth products in the IOS and Google stores, with the majority of the apps focused on women of childbearing age. The apps vary in their depth of pelvic health information, with the majority including cycle and fertility trackers. Two of the larger apps as far as global reach, CLUE and FLO, have expanded both their tracking (CLUE) or daily notifications and information (FLO) to include facts or daily tips, which, though brief, may be useful to assist in identifying women with the symptom of AUB. Additional data are needed to better understand the demographics of and reasons why women use these apps.

      III. Significant conceptual, practical, or technical challenges in the field of menstrual health research and menstrual health literacy

      The progress in the field of menstrual health outlined previously is remarkable given the multiple challenges and obstacles in the field of menstruation science. Menstruation is a physiological process that is experienced almost universally across cultures from the ages of menarche to menopause. What makes menstrual health and menstrual health literacy challenging to study is that for many, it is a normal process that is not associated with any distress or disability, but for some it can be associated with a significantly negative impact on the quality of life. Collecting data on a nearly universal process will require collaboration across the spectrum of disciplines and careful consideration of “who” to collect data from, “what” data elements to collect, and “how” to best collect data. Furthermore, the normalization of women’s pain and stigma surrounding menstrual bleeding and reproductive health represent significant barriers to women’s care seeking, diagnosis, and ability to conduct research in this area.
      • As-Sanie S.
      • Black R.
      • Giudice L.C.
      • et al.
      Assessing research gaps and unmet needs in endometriosis.
      Menstrual health and menstrual health literacy research is further complicated by a lack of standardization of tools and access to those tools, the multiple different etiologies of HMB, the multidimensional symptom complex surrounding bleeding, lack of clear diagnostic tests for reproductive health disorders that affect menstrual health, suboptimal norms for menstrual health and bleeding across the life span, and insufficient information related to cultural perceptions related to menstrual bleeding and health.
      Although awareness of the importance of patient experience with menstrual bleeding and menstrual symptoms has increased in research and clinical care, sustained reliance on “objective” laboratory measures for outcomes related to menstrual health represents an additional conceptual barrier to progress in this area. To provide an AUB-specific example, traditionally in research, bleeding was measured by volume of menstrual blood lost (>80 mL) as measured by the collection of used sanitary products and quantified using the alkaline hematin method.
      • Hallberg L.
      • Högdahl A.M.
      • Nilsson L.
      • Rybo G.
      Menstrual blood loss—a population study. Variation at different ages and attempts to define normality.
      However, research has highlighted that most women who seek treatment for HMB do not meet the objective mean blood loss criteria for heavy bleeding and clinical care objectively measured blood loss is not feasible.
      • Warner P.E.
      • Critchley H.O.
      • Lumsden M.A.
      • Campbell-Brown M.
      • Douglas A.
      • Murray G.D.
      Menorrhagia II: is the 80-mL blood loss criterion useful in management of complaint of menorrhagia?.
      As a result of these studies and others, NICE, the National Health Services (NHS) in the United Kingdom, stated in 2018 that “From the woman’s point of view objective reduction in mean blood loss are poor indicators of treatment effectiveness for heavy menstrual bleeding.”
      National Institute for Health and Clinical Excellence (NICE)
      Heavy menstrual bleeding: assessment and management. 2018.
      This lack of consistency between what has been prioritized as a measurement for research and what women prioritize in terms of desired outcomes represents a current obstacle for high-quality research and synergy between research on HMB, clinical care, and patient-centered care delivery.

      IV. Critical gaps in menstrual health literacy, advocacy, or communication and how they can be addressed to optimize women’s menstrual health

      Conceptual, practical, and technical challenges related to research on menstrual health and menstrual health literacy and communication have led to several critical gaps in the evidence base in this area. During the meeting, several gaps in the evidence base and opportunities to improve women’s health by addressing these gaps with high-quality research were discussed.

      Data to inform “norms” that hold across populations and span from menarche to menopause

      Generation of standard terminologies related to norms for uterine bleeding among adult women represents significant progress in the field of menstrual health, and there has been significant progress especially in describing symptom expectations in the later reproductive life stages and during the menopausal transition.
      • Harlow S.D.
      • Lin X.
      • Ho M.J.
      Analysis of menstrual diary data across the reproductive life span applicability of the bipartite model approach and the importance of within-woman variance.
      • Harlow S.D.
      • Mitchell E.S.
      • Crawford S.
      • et al.
      The ReSTAGE Collaboration: defining optimal bleeding criteria for onset of early menopausal transition.
      • Paramsothy P.
      • Harlow S.D.
      • Greendale G.A.
      • et al.
      Bleeding patterns during the menopausal transition in the multi-ethnic Study of Women’s Health Across the Nation (SWAN): a prospective cohort study.
      • Dasharathy S.S.
      • Mumford S.L.
      • Pollack A.Z.
      • et al.
      Menstrual bleeding patterns among regularly menstruating women.
      To ensure that menstrual bleeding norms represent bleeding patterns and other menstrual health symptoms across a racially and ethnically diverse and contemporary population (relative to comorbid conditions and body mass index) of women of all ages, further research is needed.
      • Harlow S.D.
      • Gass M.
      • Hall J.E.
      • et al.
      Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging.
      Prospective longitudinal cohort data on menstrual bleeding, menstrual symptoms, and reproductive health diagnoses could fill this critical gap.
      Developments in mHealth could also be used to inform norms and measure the personal impact menstruation and menstrual symptoms have on women across the life cycle. For population-based data outside of clinical care, data collected from mHealth and mobile device apps are starting to enable the analyses of population-level longitudinal menstrual symptom and cycle data.
      • Bull J.R.
      • Rowland S.P.
      • Scherwitzl E.B.
      • Scherwitzl R.
      • Danielsson K.G.
      • Harper J.
      Real-world menstrual cycle characteristics of more than 600,000 menstrual cycles.
      In addition, these mHealth data could facilitate investigation into cultural differences, knowledge, attitudes, and behaviors. By partnering with mHealth and app platforms, researchers, clinicians, and industry could generate data collection mechanisms and assist in generating research programs and interventions that could aid women in identifying when they are having a problem and address stigma and perceptions related to menstrual disorders, delays in diagnosis of reproductive health disorders, and delays in care seeking.

      Standardizing data collection in research, clinical care, and mobile health technologies to promote consistency and optimize comparative effectiveness research

      A shift in research to focus on measuring patient experiences with symptoms and chronic health problems, including reproductive health and menstrual health issues, represents significant progress in the arena of women’s health. However, although there are several validated PROMs for AUB, there is no single high-quality PROM that is considered “standard of care” or “standard for use across studies.”
      • Rahn D.D.
      • Abed H.
      • Sung V.W.
      • et al.
      Systematic review highlights difficulty interpreting diverse clinical outcomes in abnormal uterine bleeding trials.
      ,
      • Matteson K.A.
      Menstrual questionnaires for clinical and research use.
      ,
      • Matteson K.A.
      • Boardman L.A.
      • Munro M.G.
      • Clark M.A.
      Abnormal uterine bleeding: a review of patient-based outcome measures.
      This translates into hundreds of outcome measures, of varying quality, used across studies and an inability to combine data across studies to summarize patient experience. In a systematic review of patient-reported outcomes used across studies of AUB, authors found 80 studies that used at least 1 PROM and 77 different PROMs were used across studies.
      • Matteson K.A.
      • Boardman L.A.
      • Munro M.G.
      • Clark M.A.
      Abnormal uterine bleeding: a review of patient-based outcome measures.
      The Society of Gynecologic Surgeons, in a systematic review comparing treatments for HMB, identified that 114 different outcomes were collected and reported across 79 distinct clinical trials.
      • Rahn D.D.
      • Abed H.
      • Sung V.W.
      • et al.
      Systematic review highlights difficulty interpreting diverse clinical outcomes in abnormal uterine bleeding trials.
      The end result was that, because the method of assessing outcomes differed from study to study, data could not be combined or summarized for these outcomes (such as quality of life and bleeding-related quality of life), which prohibited the group from generating consensus on treatment effectiveness relative to patient-reported outcomes.
      • Rahn D.D.
      • Abed H.
      • Sung V.W.
      • et al.
      Systematic review highlights difficulty interpreting diverse clinical outcomes in abnormal uterine bleeding trials.
      Researchers across disciplines of menstrual health research have expressed challenges describing the menstrual symptom phenotype of patients involved in clinical research because of a lack of standardized structured menstrual history data elements. Finally, discussions at this meeting also highlighted the importance of a broader view of menstrual health that goes beyond bleeding to include other associated symptoms, which will need additional research and standardized data elements.
      The research community can collaborate to address this challenge and standardize outcomes and data elements for research and quality assessments. For example, the Core Outcomes in Women’s and Newborn Health, an international initiative led by journal editors and is endorsed by more than 80 peer-reviewed journals in women’s health, is working to stimulate the development of outcome sets that can be used across studies to ensure consistent outcome reporting, thereby improving the interpretability of study results and the feasibility of combining data across studies.
      • Khan K.S.
      • Romero R.
      Chief Editors of Journals participating in CROWN Initiative. The CROWN initiative: journal editors invite researchers to develop core outcomes in women’s health.
      ,
      CROWN Initiative
      Core Outcomes in Women’s and Newborn Health.
      Efforts to standardize data elements from a structured menstrual history describing frequency, regularity, duration, and patient-quantified volume of bleeding along with other associated menstrual symptoms are needed to facilitate consistent descriptions of populations in studies on menstrual health, AUB, uterine fibroids, and other reproductive health issues.
      • Matteson K.A.
      • Munro M.G.
      • Fraser I.S.
      The structured menstrual history: developing a tool to facilitate diagnosis and aid in symptom management.

      V. Additional future directions in menstrual health research

      Transforming comparative effectiveness research by incorporating patient-reported outcome measures into electronic health records

      Looking to the future, standardizing and harnessing the potential of patient-based outcomes assessment could transform comparative effectiveness research. Emerging technology developments may be paving the way to have PROM collection integrated into electronic health records, which would promote patient-centered comparative effectiveness research.
      • Gensheimer S.G.
      • Wu A.W.
      • Snyder C.F.
      PRO-EHR Users’ Guide Steering Group, PRO-EHR Users’ Guide Working Group. Oh, the places we’ll go: patient-reported outcomes and electronic health records.
      ,
      • Wu A.W.
      • Kharrazi H.
      • Boulware L.E.
      • Snyder C.F.
      Measure once, cut twice—adding patient-reported outcome measures to the electronic health record for comparative effectiveness research.
      Researchers, policy makers, and professional societies are currently working out best practices for integrating PROMs and electronic health records.
      • Gensheimer S.G.
      • Wu A.W.
      • Snyder C.F.
      PRO-EHR Users’ Guide Steering Group, PRO-EHR Users’ Guide Working Group. Oh, the places we’ll go: patient-reported outcomes and electronic health records.
      ,
      • Wu A.W.
      • Kharrazi H.
      • Boulware L.E.
      • Snyder C.F.
      Measure once, cut twice—adding patient-reported outcome measures to the electronic health record for comparative effectiveness research.
      This integration could mean substantially greater capabilities for patient-relevant comparative effectiveness research and health services research, which often relies on electronic health record or administrative datasets that rarely incorporated patient-reported data elements, particularly on reproductive health problems that affect the quality of life.
      Incorporating PROM collection into clinical care encounters may represent major opportunities to evaluate processes of healthcare delivery. Future research opportunities include assessing whether or not incorporating PROMs into electronic health records and clinical encounters for menstrual health disorders can improve physician-patient interactions and be used to monitor patient symptoms or progress over time. On the population level, incorporating PROMs into clinical care can assist with clinical care quality assessment and population surveillance. For example, in the UK NHS, PROMs are collected before and after certain surgical interventions to determine the quality of care delivery and to facilitate counseling for patients on what to anticipate in terms of the personal impact of the surgery.
      • Snyder C.F.
      • Jensen R.E.
      • Segal J.B.
      • Wu A.W.
      Patient-reported outcomes (PROs): putting the patient perspective in patient-centered outcomes research.

      Partnerships across diverse disciplines and stakeholder groups

      Innovative solutions to address comprehensive menstrual health across the life span will require collaboration across scientific disciplines, social science disciplines, and involvement of patient- and person-facing organization to ensure the relevance and success of these solutions for addressing the needs of the population. Menstrual health research in the future could be enhanced by developing collaborative interdisciplinary teams to investigate comprehensive menstrual health premenarche to menopause. In addition, including patient-facing groups in study design and beta testing of programs from the beginning and partnering with patient groups and advocacy groups to create and disseminate communication platforms and menstrual health educational initiatives could enhance the fields of menstrual health, menstrual health literacy, and menstrual health communication.

      VI. Conclusion

      Each year, 4.5 million women in the United States experience at least 1 gynecologic health problem, and many of these problems are related to menstrual health.
      • Kjerulff K.H.
      • Erickson B.A.
      • Langenberg P.W.
      Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992.
      Although significant progress has been made in menstrual health research in terms of emphasizing patient experience, standardizing terminologies related to menstrual bleeding, and use of PROMs for menstrual disorders, more work and research are needed to standardize data collection, generate longitudinal data on contemporary norms of menstrual bleeding and related symptoms, and optimize use of new technologies and educational interventions. Health communication strategies that are accessible to groups with low literacy and address potential stigma associated with menstruation will help to address barriers as well. Increasing the evidence base on menstrual health and menstrual health literacy will aid in the evolution of contemporary clinical care that meets the unique needs of women. Bringing women and advocacy groups to the table and bringing data collection and information directly to women through innovative technologies, smartphone apps, and mHealth has the potential to move the field of menstruation science away from treating problems and toward optimizing women’s overall health, and more specifically menstrual health. Continuing the recent momentum on patient-focused menstrual health research to sustain progress in the field of menstrual health, literacy, and communication has the potential to have a substantial impact on the lives of women.

      2. The Evolutionary History of Menstruation

      Günter P. Wagner, PhD

      Menstruation and its associated diseases such as HMB and AUB are a significant burden on women of reproductive age (see section Menstruation and abnormal uterine bleeding below), which raises the question of why women menstruate at all. This question is particularly pertinent given the fact that menstruation is dispensable for mammalian reproduction (see below). Answers require a review of the evolutionary history of mammalian reproduction, given that humans and great apes, that is, species that menstruate, evolved from ancestors that did not menstruate. What are the advantages menstruation affords humans and other primates that, from a biological point of view, could make the origin and biological role of menstruation understandable?

      I. Menstruation is rare among animals

      Menstruation is defined as the shedding of the upper (the so-called “functionalis”) layer of the uterine lining after the luteal phase of the ovarian cycle. Although menstruation is a normal part of the life of a woman during her fertile years, it is only found in a small minority of animals. Because menstruation is a function of the female reproductive organs, one would expect to find menstruation in animals with a similar mode of reproduction as humans, that is, the so-called placental mammals (technically called “eutherian mammals”). Eutherian mammals are all the species that descended from the most recent common ancestor of humans and elephants, meaning all the mammals that we are most familiar with: apes, monkeys, farm animals, cats, dogs, seals, hedgehogs, and others (Figure 2). All of these animals have a placenta and a gestational period that is longer than their ovarian cycle, so-called trans-cyclic gestation,
      • Chavan A.R.
      • Bhullar B.A.
      • Wagner G.P.
      What was the ancestral function of decidual stromal cells? A model for the evolution of eutherian pregnancy.
      with the exception of animals that have pseudopregnancy in the absence of fertilization, such as the dog.
      • Kowalewski M.P.
      • Gram A.
      • Kautz E.
      • Graubner F.R.
      The dog: nonconformist, not only in maternal recognition signaling.
      Figure thumbnail gr2
      Figure 2Phylogenetic distribution of menstruating species among eutherian mammals
      Lineages in black are from menstruating species, and lineages in white from nonmenstruating species. The lineages with red outline are the lineages where menstruation originated. Note that there are at least 4 independent originations of menstruation.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.
      Despite the substantial similarities, with respect to female reproductive biology, between humans and all other eutherian mammals (eg, compared with reptiles and birds), menstruation only occurs in a small minority of eutherian species. The largest cluster of menstruating species is found among our closest relatives, the primates. In particular, apes, old world monkeys, and most but not all of new world monkeys have menstruation. More basally diverging primate lineages do not (lemurs and tarsiers, where in the latter conflicting evidence has been reported, summarized in the study by Emera et al
      • Emera D.
      • Romero R.
      • Wagner G.
      The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.
      ). Outside the primates, menstruating species are rare. Among the rodents, only one species has been described as menstruating: the spiny mouse, Acomys cahirinus.
      • Bellofiore N.
      • Ellery S.J.
      • Mamrot J.
      • Walker D.W.
      • Temple-Smith P.
      • Dickinson H.
      First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).
      This is surprising, given the large number of rodent species (2277 species). Then there is a small number of bat species belonging to 2 groups of bats, 1 molossid bats and 3 phyllostomid bats.
      • Rasweiler J.J.
      • Badwaik N.K.
      Anatomy and physiology of the female reproductive tract.
      Most distantly related menstruating species to humans is the elephant shrew (Elephantulus myurus
      • van der Horst C.
      • Gillman J.
      The menstrual cycle in Elephantulus.
      ,
      • van der Horst C.
      Elephantulus going into anoestrus; menstruation and abortion.
      ) related to elephants and other afrotherian mammals. These menstruating species add up to 84 species, or about 1.6% of the 5149 recognized extant eutherian species. This estimate could be a slight undercount because it is not easy to diagnose menstruation in species that have not been kept in laboratories or zoos and have been closely monitored.
      If we put the menstruating species on the phylogeny of mammals (Figure 2), we see a rather dispersed distribution. Clearly, all the primate species that menstruate are relatively closely related, but the spiny mouse, bats, and the elephant shrew are not. The conclusion that follows from these facts is that menstruation must have evolved at least 4 times independently during the evolutionary history of mammals. This conclusion is also supported, for instance, by differences in the exact location and nature of the endometrial changes in the elephant shrew (summarized in the study by Carter
      • Carter A.M.
      Classics revisited: C. J. van der Horst on pregnancy and menstruation in elephant shrews.
      ). The rarity and repeated evolution of menstruation raise the question about its biological role. Menstruation is clearly not necessary for a mammal because it is rare, but it might have a specific role, rather being there accidentally, because it originated at least 4 times independently.
      Before we turn our attention to the question of why some mammals menstruate and others do not, we should mention that not every case of vaginal bleeding by a healthy animal is menstruation. The best known example is the vaginal bleeding of the dog, which is not a sign of menstruation.
      • Kowalewski M.P.
      • Gram A.
      • Kautz E.
      • Graubner F.R.
      The dog: nonconformist, not only in maternal recognition signaling.
      The main difference between what is happening in dogs and in menstruating species is that the vaginal bleeding in dogs happens in proestrus, that is, in preparation for mating, rather than after the fertile phase is over, as it is the case in women. The bleeding in dogs is caused by extravasation during the growth of the uterine lining, which can break through the epithelium leading to a vaginal efflux.

      II. Why did menstruation evolve?

      The fact that menstruation plays a major role in the life of a woman and that it is rare among animals has inspired many scientists, anthropologists, and medical researchers to speculate about its biological role.
      • Ramos-Goyette S.
      • Craton L.G.
      Evolution of the menstrual cycle.
      • Brosens J.J.
      • Parker M.G.
      • McIndoe A.
      • Pijnenborg R.
      • Brosens I.A.
      A role for menstruation in preconditioning the uterus for successful pregnancy.
      • Jarrell J.
      The significance and evolution of menstruation.
      • Alvergne A.
      • Högqvist Tabor V.
      Is female health cyclical? Evolutionary perspectives on menstruation.
      • Strassmann B.I.
      The evolution of endometrial cycles and menstruation.
      • Bellofiore N.
      • Cousins F.
      • Temple-Smith P.
      • Dickinson H.
      • Evans J.
      A missing piece: the spiny mouse and the puzzle of menstruating species.
      • Finn C.A.
      Menstruation: a nonadaptive consequence of uterine evolution.
      This is not the place to review all the ideas that have been proposed to explain the evolution of menstruation but note that the most honest and shortest answer to this question is “we do not know.”
      • Maybin J.A.
      • Critchley H.O.
      Menstrual physiology: implications for endometrial pathology and beyond.
      Nevertheless, there has been some progress in reframing the question that points to two plausible answers.
      An important breakthrough in understanding the evolution of menstruation was the realization that menstruation itself may not be the direct biological trait that was shaped by natural selection, but rather that menstruation could be a secondary consequence of an underlying biological trait: spontaneous decidualization.
      • Finn C.A.
      Menstruation: a nonadaptive consequence of uterine evolution.
      Decidualization is the process by which the uterine lining prepares for pregnancy. This is a complex process including proliferation of the endometrial stroma, the traffic of various kinds of white blood cells into the endometrium, and the differentiation of the endometrial fibroblasts into so-called decidual stromal cells (DSCs).
      • Gellersen B.
      • Brosens J.J.
      Cyclic decidualization of the human endometrium in reproductive health and failure.
      Decidualization in the narrow sense refers to the differentiation of DSC, rather than to the whole organ-level process. In most animals, decidualization occurs in the estrogen- and progesterone-primed uterus in response to the presence of the embryo. This is induced decidualization. However, in humans, decidualization occurs even in the absence of an embryo and is therefore called spontaneous decidualization. It turns out that all menstruating species undergo spontaneous decidualization,
      • Emera D.
      • Romero R.
      • Wagner G.
      The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.
      ,
      • Bellofiore N.
      • Ellery S.J.
      • Mamrot J.
      • Walker D.W.
      • Temple-Smith P.
      • Dickinson H.
      First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).
      ,
      • Finn C.A.
      Menstruation: a nonadaptive consequence of uterine evolution.
      suggesting that the evolved trait is not menstruation per se, but spontaneous decidualization. In humans, it has been shown that the proximate cause for menstruation (see section on Menstruation and abnormal uterine bleeding) is the decrease in progesterone levels owing to the degeneration of the corpus luteum. An experimental model of artificial decidualization in a nonmenstruating species, the mouse, Mus musculus, shows that in fact progesterone withdrawal after decidualization is sufficient to cause menstruation-like symptoms, that is, degeneration of part of the endometrium and vaginal bleeding.
      • Finn C.A.
      • Pope M.
      Vascular and cellular changes in the decidualized endometrium of the ovariectomized mouse following cessation of hormone treatment: a possible model for menstruation.
      • Rudolph M.
      • Döcke W.D.
      • Müller A.
      • et al.
      Induction of overt menstruation in intact mice.
      • Cousins F.L.
      • Murray A.
      • Esnal A.
      • Gibson D.A.
      • Critchley H.O.
      • Saunders P.T.
      Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.
      • Maybin J.A.
      • Murray A.A.
      • Saunders P.T.K.
      • Hirani N.
      • Carmeliet P.
      • Critchley H.O.D.
      Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.
      There are several versions of this experiment but the cleanest model is the one published by Rudolph et al
      • Rudolph M.
      • Döcke W.D.
      • Müller A.
      • et al.
      Induction of overt menstruation in intact mice.
      in 2012: intact female mice were mated with sterile, vasectomized males, which in mice causes pseudopregnancy, meaning that the female maintains a high level of progesterone even though no fetus is developing in her uterus. After copulation, the pseudopregnant mice were injected with a small droplet of oil into the lumen of the uterus. It is known that this treatment causes the uterine lining of the mouse to decidualize, leading to a so-called “deciduoma,” which is a condition that, in many respects, mimics fetus-induced decidualization. The key observation of this experiment then was that as progesterone levels were decreasing toward the end of the pseudopregnancy, menstruation ensued. This result supports a model according to which menstruation is an inevitable consequence of spontaneous decidualization if fertilization and pregnancy do not occur (Figure 3).
      Figure thumbnail gr3
      Figure 3Schematic outline of the experiment by Rudolph et al
      • Rudolph M.
      • Döcke W.D.
      • Müller A.
      • et al.
      Induction of overt menstruation in intact mice.
      testing the idea that menstruation is a secondary consequence of spontaneous decidualization
      The experiment is conducted with the laboratory mouse, which is a species that under normal conditions is neither decidualizing nor menstruating. In this species, clitoral or vaginal stimulation during copulation leads to the maintenance of the corpus luteum even if no pregnancy ensued, leading to pseudopregnancy, as is the case by copulation with a vasectomized male. Furthermore, it is known that injection of a small droplet of oil into the uterine lumen causes decidualization. The experiment starts with mating a female to a vasectomized male to induce a pseudopregnancy. At the morning of the following day, the females are checked for a copulatory plug to verify that copulation has taken place. Then at day 4 after copulation, a small droplet of oil is injected into the uterus to induce decidualization. Day 4 is the normal day of implantation in mice. Then the mice are monitored for their level of progesterone and signs of vaginal bleeding. Progesterone starts to decrease after day 7, and bleeding ensues at about day 9. This experiment shows that differentiation of the endometrium (decidualization) is sufficient to cause menstruation-like symptoms in a species that normally does not menstruate.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.
      Both, the comparative evidence, namely, the association between menstruation and spontaneous decidualization among mammals, and the experimental evidence with artificial decidualization lead to the conclusion that the real question thus is not “why some species menstruate?” but “why do some species show spontaneous decidualization and menstruate as a consequence?” There are 2 plausible answers, but no definite consensus on this issue has been reached.
      One model assumes that spontaneous decidualization is a protective device for the mother against an aggressive fetus.
      • Emera D.
      • Romero R.
      • Wagner G.
      The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.
      ,
      • Brosens J.J.
      • Parker M.G.
      • McIndoe A.
      • Pijnenborg R.
      • Brosens I.A.
      A role for menstruation in preconditioning the uterus for successful pregnancy.
      ,
      • Finn C.A.
      Menstruation: a nonadaptive consequence of uterine evolution.
      This model is based on the observation that the degree of invasiveness of the placenta varies between species. This is even the case among species with so-called hemochorial placentation, that is, where the fetus is destroying not only the uterine luminal epithelium but also some of the uterine blood vessels so that the placenta is in direct contact with maternal blood. For instance, great apes have extravillous trophoblast cells, which invade the maternal blood vessels (spiral arterioles), the stroma, and even the muscular layer of the uterus (myometrium).
      • Pollheimer J.
      • Vondra S.
      • Baltayeva J.
      • Beristain A.G.
      • Knöfler M.
      Regulation of placental extravillous trophoblasts by the maternal uterine environment.
      Clinical observations have also revealed cases where a placenta embeds too deeply into the uterus, a condition called “placenta accreta” or “placenta percreta” depending on the depth of invasion. These conditions can threaten the life of the mother after birth because of massive uterine bleeding.
      • Booker W.
      • Moroz L.
      Abnormal placentation.
      Finally, one of the roles of the decidual cells is to both enable and limit the invasion of the placenta and thus regulate the depth of implantation even though the mechanisms are still unclear. Hence, it seems plausible that spontaneous decidualization is ensuring that a conceptus finds an environment that is prepared to allow and at the same time limit the degree of placental invasion. To our knowledge, no formal test of this model has been attempted. In particular, one would need a way to measure invasiveness of the conceptus in various animals, many of which are not laboratory models and thus hard to work with. Furthermore, we have no information whether and which close relatives of menstruating species are also menstruating to test for a correlation between menstruation and depth of placental invasion.
      The second model to explain the evolutionary origin of menstruation assumes that spontaneous decidualization is an adaptation to allow the female to “test” the viability of the conceptus before definite pregnancy ensues.
      • Teklenburg G.
      • Salker M.
      • Heijnen C.
      • Macklon N.S.
      • Brosens J.J.
      The molecular basis of recurrent pregnancy loss: impaired natural embryo selection.
      • Teklenburg G.
      • Salker M.
      • Molokhia M.
      • et al.
      Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation.
      • Brosens J.J.
      • Salker M.S.
      • Teklenburg G.
      • et al.
      Uterine selection of human embryos at implantation.
      • Macklon N.S.
      • Brosens J.J.
      The human endometrium as a sensor of embryo quality.
      This model is inspired by the observation that decidual cells have the ability to sense the vitality of the embryo and react with a stress reaction when the embryo is of inferior quality. The idea is that this ability of DSCs helps the mother avoid investing resources in an ultimately unsuccessful pregnancy and thus increases the reproductive fitness of the female by allowing her to achieve pregnancy sooner. This idea is supported by the fact that humans have a rate of pregnancy loss of 10% to 25%
      • Jarvis G.E.
      Early embryo mortality in natural human reproduction: what the data say.
      (higher estimates found in the literature seem to be spurious) and that spontaneous decidualization is primarily found in animals with a small litter size, that is, one or two neonates per pregnancy and thus with correspondingly higher investment into each offspring. The recently described, yet not fully evaluated, spiny mouse is somewhat an exception because its litter size is usually 2 or 3 but can be as high as 6.
      • Bellofiore N.
      • Ellery S.J.
      • Mamrot J.
      • Walker D.W.
      • Temple-Smith P.
      • Dickinson H.
      First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).
      Again, there is a dearth of comparative data to fully test this idea, given that we do not know the rate of pregnancy loss in most animals, and whether it is different between closely related species that differ in the presence or absence of spontaneous decidualization.

      III. An evolutionary argument for the validity of menstruation as a diagnostic tool

      In a later section, the utility of menstrual efflux as a diagnostic tool will be discussed in detail. Here, we review an evolutionary argument that supports the idea that menstruation may be predictive of pregnancy complications in the future.
      In the evolution of spontaneous decidualization, the decidualization process becomes independent of the actual initiation of pregnancy. Nevertheless, it is uncontroversial that the process of spontaneous decidualization is homologous to the process of embryo-induced decidualization as the former evolved from the latter.
      • Emera D.
      • Romero R.
      • Wagner G.
      The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.
      The only difference is the mode in which the decidualization is triggered, either by maternal hormones as in spontaneous decidualization (as in women) or by the embryo as in induced decidualization (as in the mice or rodents). This is the reason why experimental work on mice is a valid approach toward understanding human decidualization even though the mode of decidualization is different between these two species.
      At the end of the ovarian cycle, menstruation is caused by the withdrawal of the supportive function of progesterone for the decidua. As a consequence, menstruation has substantial mechanistic similarities with the processes that initiate labor.
      • Pavlicev M.
      • Norwitz E.R.
      Human parturition: nothing more than a delayed menstruation.
      Birth is also associated either with a systemic progesterone withdrawal through the degeneration of the corpus luteum (luteolysis) or by functional progesterone withdrawal caused by inhibition of progesterone signaling.
      • Csapo A.I.
      The ’see-saw’ theory of parturition.
      ,
      • Romero R.
      • Espinoza J.
      • Kusanovic J.P.
      • et al.
      The preterm parturition syndrome.
      Hence, it is likely that the mechanisms deployed in the uterus during menstruation are homologous to those during parturition.
      • Pavlicev M.
      • Norwitz E.R.
      Human parturition: nothing more than a delayed menstruation.
      If in fact menstruation and the uterine manifestations of parturition are homologous, it is likely that defects that affect the maintenance pregnancy or the initiation of parturition could also manifest themselves as aberrations in menstruation. Pavlicev and Norwitz
      • Pavlicev M.
      • Norwitz E.R.
      Human parturition: nothing more than a delayed menstruation.
      therefore suggest that substantial research effort should be dedicated toward testing whether biomarkers expressed during menstruation are associated with pregnancy complications that could be useful as preconception diagnosis of likely pregnancy complications.

      3. Menstruation in Humans

      3A. Menstruation and abnormal uterine bleeding

      Hilary O.D. Critchley, MD; Jacqueline A. Maybin, PhD

      I. The impact of menstrual bleeding complaints

      Understanding the mechanisms underpinning the pivotal human event of menstruation is critical to our understanding of AUB. AUB, which includes the symptom of HMB,
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      is a chronic complaint that affects the quality of life and well-being of 1 in 4 women of reproductive age (Figure 4).
      Royal College of Obstetricians and Gynaecologists
      National heavy menstrual bleeding audit. Final report. July 2014.
      Previously, women experienced menstruation approximately 40 times owing to pregnancy and lactation amenorrhea, whereas in developed economies today, women can expect up to 400 menses in their lifetime.
      • Short R.V.
      The evolution of human reproduction.
      Therefore, AUB is becoming more common and problematic for women and society. In contemporary society, women are delaying having children for a variety of reasons such as personal choice, prioritization of career, and other factors that impose a delay in childbearing. Therefore, these women wish to preserve their uterus alongside their fertility. As a consequence, surgical options are not always appropriate because these end fertility and may also involve higher risks than medical management alternatives. In a recent systematic review relevant to the United States, it was conservatively estimated that annual direct and indirect economic costs of menstrual bleeding complaints were in the order of $1 billion and $12 billion, respectively.
      • Cardozo E.R.
      • Clark A.D.
      • Banks N.K.
      • Henne M.B.
      • Stegmann B.J.
      • Segars J.H.
      The estimated annual cost of uterine leiomyomata in the United States.
      Leiomyoma (uterine fibroids) are common, present in 70% to 80% of women by the age of 50 years,
      • Baird D.D.
      • Dunson D.B.
      • Hill M.C.
      • Cousins D.
      • Schectman J.M.
      High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence.
      and associated with AUB or HMB. Among women in their 30s and 40s, leiomyomas are often the underlying cause of AUB, anemia, and iron deficiency anemia. When the presence of uterine fibroids is considered along with complaints of AUB, the annual estimated direct costs of this complaint in the United States, when surgery, hospital attendances, outpatient visits, and prescribed medications are taken into account, are as high as $4.1 billion to $9.4 billion. Furthermore, lost work hours resulted in costs ranging from $1.55 billion to $17.2 billion.
      • Cardozo E.R.
      • Clark A.D.
      • Banks N.K.
      • Henne M.B.
      • Stegmann B.J.
      • Segars J.H.
      The estimated annual cost of uterine leiomyomata in the United States.
      Figure thumbnail gr4
      Figure 4The modern effect of menstruation
      Previously, women experienced menstruation approximately 40 times in their lifetime, owing to pregnancy and lactational amenorrhea. Women may now expect to have more than 400 episodes of menstruation, mainly as a result of fertility management. Therefore, AUB is increasingly common. Women may experience significant anemia resulting in a poor physical quality of life. A negative financial effect occurs because of the cost of managing their blood loss and an inability to work outside the home. These costs, alongside a loss of caring ability, will have a negative effect on the wider family. The cost to society through loss of work days and healthcare costs is significant.
      AUB, abnormal uterine bleeding.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.

      II. A classification system for abnormal uterine bleeding

      To provide diagnostic precision and specific treatment of AUB, classification of causes of uterine bleeding is crucial. The FIGO Menstrual Disorders Committee has led on the classification systems for causes of chronic AUB in the reproductive years.
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      ,
      • Munro M.G.
      • Critchley H.O.
      • Broder M.S.
      • Fraser I.S.
      FIGO Working Group on Menstrual Disorders
      FIGO classification system (PALM-COEIN) for causes of abnormal uterine bleeding in nongravid women of reproductive age.
      As already mentioned, there are 2 systems: the first system focuses on terminology with an encouragement for the removal of ill-defined terminologies such as “menorrhagia” and “dysfunctional uterine bleeding,” and the second system focuses on the underlying causes of AUB, using the acronym PALM-COEIN
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      ,
      • Munro M.G.
      • Critchley H.O.
      • Broder M.S.
      • Fraser I.S.
      FIGO Working Group on Menstrual Disorders
      FIGO classification system (PALM-COEIN) for causes of abnormal uterine bleeding in nongravid women of reproductive age.
      for structural and nonstructural causes, respectively (Figure 5). It is hoped that these 2 FIGO systems will be used globally to improve the management of women with AUB.
      Figure thumbnail gr5
      Figure 5The PALM-COEIN classification for abnormal uterine bleeding in the reproductive years illustrating the structural (PALM) and nonstructural causes (COEIN) and as described in Munro et al
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      ,
      • Munro M.G.
      • Critchley H.O.
      • Broder M.S.
      • Fraser I.S.
      FIGO Working Group on Menstrual Disorders
      FIGO classification system (PALM-COEIN) for causes of abnormal uterine bleeding in nongravid women of reproductive age.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.
      In the absence of any other features, for example, leiomyoma or a coagulopathy,
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      bleeding from the endometrium may represent a “primary endometrial disorder” (AUB-E). In the presence of structural features such as leiomyoma, polyp, and adenomyosis,
      • Munro M.G.
      • Critchley H.O.D.
      • Fraser I.S.
      Committee FIGO Menstrual Disorders Committee
      The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions.
      it is not known whether the presence of myometrial structural entities such as AUB-L (leiomyoma) or AUB-A (adenomyosis) actually result in a “secondary endometrial disorder” (Figure 6). There remains a true lack of knowledge about the phenotype of the endometrium when adenomyosis and leiomyomas are present.
      Figure thumbnail gr6
      Figure 6Potential mechanisms of “primary” and “secondary” endometrial AUB
      As the corpus luteum regresses in the absence of pregnancy, progesterone levels fall. This occurs irregularly in those with ovulatory or iatrogenic AUB. Progesterone withdrawal causes a local inflammatory response in the endometrium and may be increased in those with primary endometrial AUB. An increase in vasoactive factors results in intense vasoconstriction of spiral arterioles to limit blood loss; this may be decreased in primary endometrial AUB. Vasoconstriction may induce transient tissue hypoxia and stabilization of HIF-1, the master regulator of the cellular response to hypoxia, to coordinate endometrial repair. There is evidence that this is less intense in those with endometrial AUB. Efficient hemostasis limits menstrual blood loss at menstruation and this is defective in women with coagulopathy AUB. Structural and nonstructural pathologies have the potential to disrupt endometrial physiology at menstruation, leading to abnormal uterine bleeding; these mechanisms remain undefined.
      AUB, abnormal uterine bleeding; HIF-1, hypoxia-inducible factor 1.
      Critchley. Menstruation: science and society. Am J Obstet Gynecol 2020.
      This exciting area merits substantial research and many questions remain. What is the aberration in women with AUB-E? Do leiomyomas and adenomyosis contribute to the genesis of AUB or HMB? If so, is it because they directly affect the molecular mechanisms of endometrial hemostasis? Do leiomyomas actually need to be adjacent to the endometrium to cause AUB?
      • Sinclair D.C.
      • Mastroyannis A.
      • Taylor H.S.
      Leiomyoma simultaneously impair endometrial BMP-2-mediated decidualization and anticoagulant expression through secretion of TGF-β3.
      To answer these important questions, we need to fully understand endometrial physiology and pathology.

      III. Methods for the study of menstruation

      Identification of aberrations in endometrial function necessitates study of human endometrial tissue. Women must have a detailed clinical history and examination and undergo investigation to determine if structural disorders are present. For research purposes, women should have measurement of their menstrual blood loss to enable categorization as having heavy or normal menstrual bleeding. An objective measurement of blood loss may be obtained using the alkaline hematin method and total menstrual volume by using a menstrual cup.
      • Hallberg L.
      • Högdahl A.M.
      • Nilsson L.
      • Rybo G.
      Menstrual blood loss—a population study. Variation at different ages and attempts to define normality.
      ,
      • Donoso M.B.
      • Serra R.
      • Rice G.E.
      • et al.
      Normality ranges of menstrual fluid volume during reproductive life using direct quantification of menses with vaginal cups.
      Alternatively, a pictorial menstrual blood loss assessment chart has been validated to assess menstrual blood loss volume and duration.
      • Higham J.M.
      • O’Brien P.M.
      • Shaw R.W.
      Assessment of menstrual blood loss using a pictorial chart.
      In addition, tissue must be carefully classified to determine the correct stage of the menstrual cycle.
      Studies in women are often limited to generation of observational data. For more incisive functional studies, animal models of simulated menstruation have been developed.
      • Finn C.A.
      • Pope M.
      Vascular and cellular changes in the decidualized endometrium of the ovariectomized mouse following cessation of hormone treatment: a possible model for menstruation.
      ,
      • Brasted M.
      • White C.A.
      • Kennedy T.G.
      • Salamonsen L.A.
      Mimicking the events of menstruation in the murine uterus.
      • Menning A.
      • Walter A.
      • Rudolph M.
      • Gashaw I.
      • Fritzemeier K.H.
      • Roese L.
      Granulocytes and vascularization regulate uterine bleeding and tissue remodeling in a mouse menstruation model.
      • Cousins F.L.
      • Kirkwood P.M.
      • Saunders P.T.
      • Gibson D.A.
      Evidence for a dynamic role for mononuclear phagocytes during endometrial repair and remodelling.
      The nonhuman primate (rhesus macaque) has been studied extensively and provides an excellent model of the human menstrual cycle.
      • Brenner R.M.
      • Rudolph L.
      • Matrisian L.
      • Slayden O.D.
      Non-human primate models; artificial menstrual cycles, endometrial matrix metalloproteinases and s.c. endometrial grafts.
      ,
      • Brenner R.M.
      • Nayak N.R.
      • Slayden O.D.
      • Critchley H.O.
      • Kelly R.W.
      Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis.
      More recently, attention has focused on refinement of the mouse model of simulated menstruation.
      • Finn C.A.
      • Pope M.
      Vascular and cellular changes in the decidualized endometrium of the ovariectomized mouse following cessation of hormone treatment: a possible model for menstruation.
      ,
      • Cousins F.L.
      • Murray A.
      • Esnal A.
      • Gibson D.A.
      • Critchley H.O.
      • Saunders P.T.
      Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.
      ,
      • Brasted M.
      • White C.A.
      • Kennedy T.G.
      • Salamonsen L.A.
      Mimicking the events of menstruation in the murine uterus.
      ,
      • Cousins F.L.
      • Kirkwood P.M.
      • Saunders P.T.
      • Gibson D.A.
      Evidence for a dynamic role for mononuclear phagocytes during endometrial repair and remodelling.
      A detailed study of the cellular and histologic events occurring in the mouse endometrium during simulated menstruation has been reported to recapitulate several of the local events that occur in the human endometrium at the time of menstruation, these being apoptosis preceding cytokine and chemokine expression and extensive neutrophil influx into the endometrium.
      • Armstrong G.M.
      • Maybin J.A.
      • Murray A.A.
      • et al.
      Endometrial apoptosis and neutrophil infiltration during menstruation exhibits spatial and temporal dynamics that are recapitulated in a mouse model.
      There is an interesting recent discovery of a previously unrecognized menstruating rodent, the spiny mouse, which may provide another tool in the study of menstruation.
      • Bellofiore N.
      • Ellery S.J.
      • Mamrot J.
      • Walker D.W.
      • Temple-Smith P.
      • Dickinson H.
      First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).
      The combination of observational data generated from well-categorized human endometrial tissue and mechanistic studies in validated animal models will facilitate definitive experiments to determine menstrual physiology and pathology.

      IV. Initiation of menstruation

      The human endometrium is a highly dynamic multicellular structure. Its physiological functions are preparation for implantation and, in the absence of pregnancy, menstruation. The regulation of normal menstruation is governed by sequential exposure to circulating sex steroids, estrogen, and then estrogen and progesterone followed by corpus luteum demise causing a fall in both circulating estrogen and progesterone. Progesterone withdrawal is the trigger for menstruation.
      • Maybin J.A.
      • Critchley H.O.
      Menstrual physiology: implications for endometrial pathology and beyond.
      ,
      • Critchley H.O.
      • Kelly R.W.
      • Brenner R.M.
      • Baird D.T.
      The endocrinology of menstruation—a role for the immune system.
      Menstruation involves a remarkable sequence of endometrial cell proliferation, differentiation, shedding, and regeneration that may occur as many as 400 times across the reproductive life course.
      • Short R.V.
      The evolution of human reproduction.
      The mechanisms underpinning menstruation still remain poorly understood. There are crucial interactions between the endocrine system and the immune system.
      • Maybin J.A.
      • Critchley H.O.
      Menstrual physiology: implications for endometrial pathology and beyond.
      ,
      • Critchley H.O.
      • Kelly R.W.
      • Brenner R.M.
      • Baird D.T.
      The endocrinology of menstruation—a role for the immune system.
      These cellular interactions, which are dependent on the menstrual cycle phase, involve epithelial and stromal cells along with an influx of innate immune cells and differentiation of the endometrial vasculature (spiral arterioles). The local endometrial events at the time of menses resemble those of an inflammatory event. There is an increase in endometrial blood vessel permeability and fragility, tissue breakdown, and an influx of innate immune cells into the endometrium, particularly neutrophils and macrophages.
      • Armstrong G.M.
      • Maybin J.A.
      • Murray A.A.
      • et al.
      Endometrial apoptosis and neutrophil infiltration during menstruation exhibits spatial and temporal dynamics that are recapitulated in a mouse model.
      ,
      • Thiruchelvam U.
      • Dransfield I.
      • Saunders P.T.
      • Critchley H.O.
      The importance of the macrophage within the human endometrium.
      ,
      • Salamonsen L.A.
      • Woolley D.E.
      Menstruation: induction by matrix metalloproteinases and inflammatory cells.

      V. Cessation of menstruation

      The cessation of menstrual bleeding and endometrial repair require 3 closely related events: these being vasoconstriction of the highly specialized spiral arterioles, local endometrial hemostasis, and reepithelialization of the injured endometrial mucosa (Figure 6). After menstruation, the restoration of the injured mucosal surface is a rapid event and the role of endometrial stem cells is addressed in the following contribution concerning endometrial regeneration. Endometrial repair of the denuded epithelial surface after menstruation has been described by Garry et al,
      • Garry R.
      • Hart R.
      • Karthigasu K.A.
      • Burke C.
      A re-appraisal of the morphological changes within the endometrium during menstruation: a hysteroscopic, histological and scanning electron microscopic study.
      using hysteroscopy, histology, and scanning electron microscopy. These imaging techniques detail the temporal repair of the epithelial surface, which occurs in a piecemeal fashion adjacent to actively menstruating tissue.
      • Garry R.
      • Hart R.
      • Karthigasu K.A.
      • Burke C.
      A re-appraisal of the morphological changes within the endometrium during menstruation: a hysteroscopic, histological and scanning electron microscopic study.
      The regulation of this endometrial repair process is not fully defined. There is a recent interesting interpretation of the link between human menstruation and separation of the placenta after delivery. Both are underpinned by progesterone withdrawal and critically involve uterine spiral arterial function.
      • Thomas V.G.
      The link between human menstruation and placental delivery: a novel evolutionary interpretation: menstruation and fetal placental detachment share common evolved physiological processes dependent on progesterone withdrawal.
      In women with complaints of HMB, impaired vascular differentiation caused by impaired spiral arteriole maturation has been described
      • Abberton K.M.
      • Healy D.L.
      • Rogers P.A.
      Smooth muscle alpha actin and myosin heavy chain expression in the vascular smooth muscle cells surrounding human endometrial arterioles.
      • Biswas Shivhare S.
      • Bulmer J.N.
      • Innes B.A.
      • Hapangama D.K.
      • Lash G.E.
      Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.
      • Biswas Shivhare S.
      • Bulmer J.N.
      • Innes B.A.
      • Hapangama D.K.
      • Lash G.E.
      Endometrial vascular development in heavy menstrual bleeding: altered spatio-temporal expression of endothelial cell markers and extracellular matrix components.
      along with exposure to an imbalance of locally generated vasoconstrictors and vasodilators.
      • Maybin J.A.
      • Critchley H.O.
      • Jabbour H.N.
      Inflammatory pathways in endometrial disorders.
      • Marsh M.M.
      • Malakooti N.
      • Taylor N.H.
      • Findlay J.K.
      • Salamonsen L.A.
      Endothelin and neutral endopeptidase in the endometrium of women with menorrhagia.
      • Smith O.P.
      • Jabbour H.N.
      • Critchley H.O.
      Cyclooxygenase enzyme expression and E series prostaglandin receptor signalling are enhanced in heavy menstruation.
      An increase in blood vessel radius will affect the resistance to blood flow (Poiseuille’s equation).
      • Maybin J.A.
      • Critchley H.O.
      • Jabbour H.N.
      Inflammatory pathways in endometrial disorders.
      The pivotal role for vasoconstriction after progesterone withdrawal was described nearly 80 years ago.
      • Markee J.E.
      Menstruation in intraocular transplants in the rhesus monkey.
      The study of autologous transplants of rhesus macaque endometrium into the anterior chamber of the eye and visualization of the events of menstruation through a slit-lamp ophthalmoscope revealed transient and intense vasoconstriction 4 to 24 hours before menstruation in response to steroid withdrawal. Authors proposed that this vasoconstriction was consistent with local tissue hypoxia. The presence and role of endometrial hypoxia in the process of menstruation have been debated. There is now experimental support for a pivotal role for transient physiological hypoxia because it has been reported to occur in the menstruating endometrium.
      • Maybin J.A.
      • Murray A.A.
      • Saunders P.T.K.
      • Hirani N.
      • Carmeliet P.
      • Critchley H.O.D.
      Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.
      The stabilization of hypoxia-inducible factor 1 (HIF-1; a marker for hypoxia) results in the generation of local repair factors to “heal” the injured mucosal surface (menstruating endometrium).
      • Maybin J.A.
      • Murray A.A.
      • Saunders P.T.K.
      • Hirani N.
      • Carmeliet P.
      • Critchley H.O.D.
      Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.
      Women with HMB have decreased endometrial HIF-1α at the time of menstruation, and these women also experience prolonged menstrual bleeding episodes. These observational data have been recapitulated in a mouse model of simulated menstruation in which physiological endometrial hypoxia is also reported to occur at the time of endometrial bleeding.
      • Maybin J.A.
      • Murray A.A.
      • Saunders P.T.K.
      • Hirani N.
      • Carmeliet P.
      • Critchley H.O.D.
      Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.
      Fibrinolysis is an important component of regulation of normal endometrial bleeding. The human endometrium contains tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA), along with plasminogen activator inhibitor (PAI) (inhibits fibrinolytic activity) and the u-PA receptor. Women complaining of HMB have raised levels of t-PA activity on the second day of bleeding when compared with those with normal menstrual blood loss, consistent with an overactive fibrinolytic system.
      • Gleeson N.
      • Devitt M.
      • Sheppard B.L.
      • Bonnar J.
      Endometrial fibrinolytic enzymes in women with normal menstruation and dysfunctional uterine bleeding.
      ,
      • Nordengren J.
      • Pilka R.
      • Noskova V.
      • et al.
      Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle.
      Tranexamic acid, a popular nonhormonal treatment for HMB in many countries, targets the overactivation of the fibrinolytic system with a reported 58% reduction in menstrual blood loss.
      • Gleeson N.C.
      • Buggy F.
      • Sheppard B.L.
      • Bonnar J.
      The effect of tranexamic acid on measured menstrual loss and endometrial fibrinolytic enzymes in dysfunctional uterine bleeding.

      VI. Endometrial pathology in structural abnormal uterine bleeding

      In the presence of structural pathologies (AUB-P/A/L), a secondary endometrial disorder has been proposed (Figure 6). The current literature presents lines of evidence to support the concept that there may be an element of resistance to normal progesterone-regulated events.
      • Benagiano G.
      • Brosens I.
      • Habiba M.
      Structural and molecular features of the endomyometrium in endometriosis and adenomyosis.
      ,
      • Patel B.G.
      • Rudnicki M.
      • Yu J.
      • Shu Y.
      • Taylor R.N.
      Progesterone resistance in endometriosis: origins, consequences and interventions.
      The latter is based on descriptive data and may certainly be implicated given the often reported poor response to many progestin-based therapies, for example, the levonorgestrel-releasing intrauterine system and oral, implant, or injectable progestins.
      Royal College of Obstetricians and Gynaecologists
      National heavy menstrual bleeding audit. Final report. July 2014.

      VII. Therapies targeting the progesterone receptor

      Progestins have long been used to modulate endometrial bleeding either through their action on ovarian function or abolition of ovulation along with a direct effect on the endometrium to reduce bleeding. All progestins, when delivered orally, systemically, or through an intrauterine route, improve menstrual experience in many women; however, there remains a consistent 20% who experience unscheduled endometrial bleeding and spotting. This is often a reason for the discontinuation of use of progestin therapies. The mechanisms underpinning this unscheduled bleeding still remain elusive despite studies focusing on many candidate pathways.
      • Guttinger A.
      • Critchley H.O.
      Endometrial effects of intrauterine levonorgestrel.
      • Weisberg E.
      • Hickey M.
      • Palmer D.
      • et al.
      A randomized controlled trial of treatment options for troublesome uterine bleeding in Implanon users.
      • Warner P.
      • Guttinger A.
      • Glasier A.F.
      • et al.
      Randomized placebo-controlled trial of CDB-2914 in new users of a levonorgestrel-releasing intrauterine system shows only short-lived amelioration of unscheduled bleeding.
      • Rogers P.A.
      • Martinez F.
      • Girling J.E.
      • et al.
      Influence of different hormonal regimens on endometrial microvascular density and VEGF expression in women suffering from breakthrough bleeding.
      • Jain J.K.
      • Nicosia A.F.
      • Nucatola D.L.
      • Lu J.J.
      • Kuo J.
      • Felix J.C.
      Mifepristone for the prevention of breakthrough bleeding in new starters of depo-medroxyprogesterone acetate.
      To date, there has been no reliable preventative intervention, albeit there are strategies to stop or reduce a heavy bleeding episode in users of progestin-only preparations.
      Faculty of Sexual and Reproductive Healthcare
      FSRH clinical guidance: problematic bleeding with hormonal contraception. 2015.
      Selective progesterone receptor modulators (SPRMs) reduced endometrial bleeding in women with uterine leiomyomas.
      • Donnez J.
      • Tatarchuk T.F.
      • Bouchard P.
      • et al.
      Ulipristal acetate versus placebo for fibroid treatment before surgery.
      ,
      • Donnez J.
      • Tomaszewski J.
      • Vázquez F.
      • et al.
      Ulipristal acetate versus leuprolide acetate for uterine fibroids.
      SPRMs inhibit ovulation in 90% of women and also affect the endometrium and many women experience amenorrhea.
      • Lakha F.
      • Ho P.C.
      • Van der Spuy Z.M.
      • et al.
      A novel estrogen-free oral contraceptive pill for women: multicentre, double-blind, randomized controlled trial of mifepristone and progestogen-only pill (levonorgestrel).
      The mechanisms of action of SPRMs on the endometrium in women with abnormal bleeding and uterine leiomyoma still remain poorly understood. SPRMs have an interesting antiproliferative effect, and the study of cell-to-cell interactions within the endometrium in women exposed to this class of drug is a current topic of investigation.
      • Whitaker L.H.
      • Murray A.A.
      • Matthews R.
      • et al.
      Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium.
      SPRM administration is associated with an unusual morphologic effect on the endometrium known as progesterone receptor–associated endometrial changes (PAECs). These morphologic features are associated with alterations in expression and localization of sex steroid receptors.
      • Whitaker L.H.
      • Murray A.A.
      • Matthews R.
      • et al.
      Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium.
      • Williams A.R.
      • Bergeron C.
      • Barlow D.H.
      • Ferenczy A.
      Endometrial morphology after treatment of uterine fibroids with the selective progesterone receptor modulator, ulipristal acetate.
      • Wagenfeld A.
      • Saunders P.T.
      • Whitaker L.
      • Critchley H.O.
      Selective progesterone receptor modulators (SPRMs): progesterone receptor action, mode of action on the endometrium and treatment options in gynecological therapies.
      • Mutter G.L.
      • Bergeron C.
      • Deligdisch L.
      • et al.
      The spectrum of endometrial pathology induced by progesterone receptor modulators.
      The fact that circulating estradiol levels remain consistently in midfollicular range has raised concerns among clinicians about the risks of hyperplasia and endometrial cancer. However, no studies to date that have explored in detail the endometrial impact have reported increases in either hyperplasia or endometrial cancer.
      • Stewart E.A.
      • Diamond M.P.
      • Williams A.R.W.
      • et al.
      Safety and efficacy of the selective progesterone receptor modulator asoprisnil for heavy menstrual bleeding with uterine fibroids: pooled analysis of two 12-month, placebo-controlled, randomized trials.
      ,
      • Kannan A.
      • Bhurke A.
      • Sitruk-Ware R.
      • et al.
      Characterization of molecular changes in endometrium associated with chronic use of progesterone receptor modulators: ulipristal acetate versus mifepristone.
      Moreover, a recent systematic review reporting the endometrial effects of SPRM (ulipristal acetate [UPA]) use in 10 studies involving 1450 women supports the current view that PAEC is essentially a benign endometrial morphology that is reversible on discontinuation of UPA use.
      • De Milliano I.
      • Van Hattum D.
      • Ket J.C.F.
      • Huirne J.A.F.
      • Hehenkamp W.J.K.
      Endometrial changes during ulipristal acetate use: a systematic review.

      VIII. Summary comment

      Understanding the pathology underlying AUB is essential to improve treatments for this common symptom that has a significant negative impact on women and society. Progesterone and progesterone receptor interactions play essential roles in uterine physiology and reproduction. Progesterone withdrawal remains the major trigger for the onset of endometrial bleeding. Menstruation itself involves repeated episodes of physiological “injury and repair” and a detailed knowledge of endometrial function is essential for understanding how disturbances in the endometrial function play a role in AUB. A particular gap is the understanding of endometrial function in women with myometrial structural features such as leiomyoma and adenomyosis and whether this represents a “primary or secondary endometrial disorder.” There is without doubt utility and validity of mouse models of simulated menstruation, particularly when used alongside human studies. Ligands for the progesterone receptor, that is, progestins and SPRMs, may reduce endometrial bleeding and modulate endometrial form and function. Identification of novel targets for the treatment of AUB is vital to address the significant personal and societal burden of this common disorder.

      3B. Regeneration after menstruation—the role of stem cells

      Hugh S. Taylor, MD

      I. Introduction

      In each monthly menstrual cycle, the endometrium is renewed from the basalis layer.
      • Du H.
      • Taylor H.S.
      Stem cells and female reproduction.
      • Gargett C.E.
      • Masuda H.
      Adult stem cells in the endometrium.
      • Santamaria X.
      • Mas A.
      • Cervelló I.
      • Taylor H.
      • Simon C.
      Uterine stem cells: from basic research to advanced cell therapies.
      This regenerative process recapitulates some features of development and includes production of all components of the endometrium, including glands, stroma, vasculature, and an influx of immune cells. The ability to rapidly and repetitively regenerate this tissue is fundamental to reproduction. Therefore, it is not surprising that there exists a population of cells that serve to replace and maintain the endometrium despite repetitive loss with menstruation.
      • Du H.
      • Taylor H.S.
      Stem cells and female reproduction.
      • Gargett C.E.
      • Masuda H.
      Adult stem cells in the endometrium.
      • Santamaria X.
      • Mas A.
      • Cervelló I.
      • Taylor H.
      • Simon C.
      Uterine stem cells: from basic research to advanced cell therapies.
      These stem cells maintain a reservoir of regenerative cells while simultaneously giving rise to more differentiated cells.

      II. Endometrial stem cells

      Early research in stem cells centered on the hematopoietic system, because experimental transplants to repopulate bone marrow could be performed using tissue ablation.
      • Post Y.
      • Clevers H.
      Defining adult stem cell function at its simplest: the ability to replace lost cells through mitosis.
      These studies gave rise to the concept that stem cells divide asymmetrically, reproducing the stem cell and giving rise to a more differentiated cell, in contrast to the symmetrical division observed in somatic cells.
      • Post Y.
      • Clevers H.
      Defining adult stem cell function at its simplest: the ability to replace lost cells through mitosis.
      However, translation of this asymmetrical division concept to other tissues and organs has recently become controversial because tremendous plasticity in the fate of epithelial cells in the intestine, liver, and other organs is being uncovered.
      • Post Y.
      • Clevers H.
      Defining adult stem cell function at its simplest: the ability to replace lost cells through mitosis.
      Although specific mechanisms remain debated, stem cells throughout the body maintain the pool of regenerative stem cells for populating each tissue and organ. Most tissues contain a collection of stem and progenitor cells that replace adult cells lost to age or damage. In many organs, stem cells divide only under unusual conditions such as in response to injury. In other organs characterized by rapid turnover, such as the gastrointestinal tract, stem cells regularly divide to replace worn or damaged cells as part of normal tissue homeostasis.
      • Post Y.
      • Clevers H.
      Defining adult stem cell function at its simplest: the ability to replace lost cells through mitosis.
      In the endometrium, the vast majority of cells are lost every month, making the need for frequent stem cell division more acute and essential.
      Totipotent, pluripotent, and multipotent stem cells give rise to many different tissues, whereas tissue-specific stem and progenitor cells give rise to a limited set of differentiated cells in a local environment. Tissue-specific stem and progenitor cells may give rise to a single cell type or several types of cells that make up an individual organ. In the endometrium, multiple lines of evidence in mice and humans support the presence of a population of stem and progenitors that give rise to stromal fibroblasts and another population that gives rise to epithelia. Much of the current knowledge on endometrial stem cells comes from the studies in mice, where cell lineages can be traced using molecular tags and reporters, but understanding of the human endometrium is accelerating as more signatures of stem and progenitor cells in other organs are identified, investigated, and validated in the endometrium.
      • Du H.
      • Taylor H.S.
      Stem cells and female reproduction.
      • Gargett C.E.
      • Masuda H.
      Adult stem cells in the endometrium.
      • Santamaria X.
      • Mas A.
      • Cervelló I.
      • Taylor H.
      • Simon C.
      Uterine stem cells: from basic research to advanced cell therapies.
      ,
      • Gargett C.E.
      • Schwab K.E.
      • Deane J.A.
      Endometrial stem/progenitor cells: the first 10 years.
      ,
      • Tempest N.
      • Maclean A.
      • Hapangama D.K.
      Endometrial stem cell markers: current concepts and unresolved questions.
      Although early and even more recent studies in humans suggest that endometrial stem and progenitor cells are localized to the basalis layer,
      • Valentijn A.J.
      • Palial K.
      • Al-Lamee H.
      • et al.
      SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis.
      • Hapangama D.K.
      • Drury J.
      • Da Silva L.
      • et al.
      Abnormally located SSEA1+/SOX9+ endometrial epithelial cells with a basalis-like phenotype in the eutopic functionalis layer may play a role in the pathogenesis of endometriosis.
      • Gargett C.E.
      • Schwab K.E.
      • Zillwood R.M.
      • Nguyen H.P.
      • Wu D.
      Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.
      more recent evidence of stem and progenitor cell markers in the luminal region suggests a more complex picture of wider dispersal
      • Tempest N.
      • Baker A.M.
      • Wright N.A.
      • Hapangama D.K.
      Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?.
      because they are in nonmenstruating species such as the mouse.
      • Jin S.
      Bipotent stem cells support the cyclical regeneration of endometrial epithelium of the murine uterus.
      Moreover, recent studies using tracers in the mouse endometrium have identified stem and progenitor cells that give rise to both epithelial glandular and luminal epithelial cells,
      • Jin S.
      Bipotent stem cells support the cyclical regeneration of endometrial epithelium of the murine uterus.
      • Syed S.M.
      • Kumar M.
      • Ghosh A.
      • et al.
      Endometrial Axin2+ cells drive epithelial homeostasis, regeneration, and cancer following oncogenic transformation.
      • Yin M.
      • Zhou H.J.
      • Lin C.
      • et al.
      CD34+KLF4+ stromal stem cells contribute to endometrial regeneration and repair.
      whereas other tissue-resident stem and progenitor cells give rise to stromal cells in the mouse endometrium.
      • Yin M.
      • Zhou H.J.
      • Lin C.
      • et al.
      CD34+KLF4+ stromal stem cells contribute to endometrial regeneration and repair.
      It is possible that there is a common stem cell that gives rise first to stromal cells and can also differentiate into a distinct bipotential epithelial progenitor cell, as suggested by studies in mice showing gene expression evidence of mesenchymal-epithelial transitions (METs)
      • Cousins F.L.
      • Murray A.
      • Esnal A.
      • Gibson D.A.
      • Critchley H.O.
      • Saunders P.T.
      Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.
      and morphologic evidence of such METs in humans.
      • Garry R.
      • Hart R.
      • Karthigasu K.A.
      • Burke C.
      Structural changes in endometrial basal glands during menstruation.
      These tissue-resident stem and progenitor cells regenerate the endometrium after menstruation in each menstrual cycle.

      III. Bone marrow–derived stem cells

      There also exist multipotent stem cells in several tissues that can divide and differentiate into multiple types of cells and are found in many tissue types. Most notably, bone marrow hosts both hematopoietic stem cells, which give rise to circulating white blood cells, red blood cells, and platelets, and mesenchymal stem cells, which give rise to bone, cartilage, and fat.
      • Crane G.M.
      • Jeffery E.
      • Morrison S.J.
      Adult haematopoietic stem cell niches.
      Bone marrow hematopoietic and mesenchymal stem cells are found in the circulation, where they can be recruited to sites of injury and contribute to tissue repair in ways that are still incompletely understood in humans.
      • Zhang M.
      • Malik A.B.
      • Rehman J.
      Endothelial progenitor cells and vascular repair.
      • Fadini G.P.
      • Ciciliot S.
      • Albiero M.
      Concise review: perspectives and clinical implications of bone marrow and circulating stem cell defects in diabetes.
      • Myerson D.
      • Parkin R.K.
      Donor-derived hepatocytes in human hematopoietic cell transplant recipients: evidence of fusion.
      In human patients who received bone marrow transplants, allowing donor cells to be tracked through sex chromosomes or human leukocyte antigen type, early studies reporting that bone marrow cells differentiated into hepatocytes or other epithelial tissue types are now mostly attributed to cell fusion or artifactual protocols.
      • Myerson D.
      • Parkin R.K.
      Donor-derived hepatocytes in human hematopoietic cell transplant recipients: evidence of fusion.
      However, bone marrow fusion to endometrial stromal cells has been characterized in mice and is rare compared with bone marrow cells directly contributing to endometrial cell fates.
      • Tal A.
      • Tal R.
      • Shaikh S.
      • Gidicsin S.
      • Mamillapalli R.
      • Taylor H.S.
      Characterization of cell fusion in an experimental mouse model of endometriosis†.
      Although the ability of mesenchymal stem cells to transdifferentiate broadly into cells in other tissue types remains controversial,
      • Robey P.
      ”Mesenchymal stem cells”: fact or fiction, and implications in their therapeutic use.
      convincing evidence from human studies using single-cell sequencing indicates that bone marrow–derived donor cells differentiate into mature adipocytes—a known cell fate for mesenchymal stem cells.
      • Arner P.
      • Rydén M.
      The contribution of bone marrow-derived cells to the human adipocyte pool.
      Studies in mice and humans support the idea that bone marrow–derived stem and progenitor cells also contribute to the reproductive tract, supplementing the resident stem and progenitor cells. In both the mouse model and in humans, bone marrow–derived cells are incorporated into the endometrium where they differentiate into endometrial stromal cells, epithelial cells, and endothelial cells.
      • Taylor H.S.
      Endometrial cells derived from donor stem cells in bone marrow transplant recipients.
      ,
      • Du H.
      • Taylor H.S.
      Contribution of bone marrow-derived stem cells to endometrium and endometriosis.
      The vast majority of bone marrow–derived endometrial cells are stromal cells with epithelial cells differentiating slowly and in smaller numbers. Other groups have subsequently confirmed a bone marrow origin for endothelial cells in the human
      • Mints M.
      • Jansson M.
      • Sadeghi B.
      • et al.
      Endometrial endothelial cells are derived from donor stem cells in a bone marrow transplant recipient.
      and for stromal and epithelial cells in mouse,
      • Morelli S.S.
      • Rameshwar P.
      • Goldsmith L.T.
      Experimental evidence for bone marrow as a source of nonhematopoietic endometrial stromal and epithelial compartment cells in a murine model.
      ,
      • Gil-Sanchis C.
      • Cervelló I.
      • Khurana S.
      • Faus A.
      • Verfaillie C.
      • Simón C.
      Contribution of different bone marrow-derived cell types in endometrial regeneration using an irradiated murine model.
      establishing a potential role of bone marrow in endometrial repair in humans and prompting human clinical studies aimed at treating endometrial disorders.
      • Santamaria X.
      • Mas A.
      • Cervelló I.
      • Taylor H.
      • Simon C.
      Uterine stem cells: from basic research to advanced cell therapies.
      Perhaps because of the depletion after menstruation, exogenous stem cells may be even more essential in the uterus than in other organs. Furthermore, increased recruitment and engraftment of these cells to the uterus occur in response to injury such as hypoxia or inflammation to aid in repair and regeneration.
      • Du H.
      • Naqvi H.
      • Taylor H.S.
      Ischemia/reperfusion injury promotes and granulocyte-colony stimulating factor inhibits migration of bone marrow-derived stem cells to endometrium.

      IV. Consequences of stem and progenitor cell loss

      Infection and iatrogenic trauma can lead to endometrial destruction and loss of progenitor cells, causing failure to regenerate lost tissue and resulting in permanent damage. Multipotent stem cells circulate to the endometrium and engraft, contributing to the regeneration of damaged endometrium and mitigating endometrial atrophy, thin endometrium, and Asherman’s syndrome.
      • Alawadhi F.
      • Du H.
      • Cakmak H.
      • Taylor H.S.
      Bone marrow-derived stem cell (BMDSC) transplantation improves fertility in a murine model of Asherman’s syndrome.
      ,
      • Yi K.W.
      • Mamillapalli R.
      • Sahin C.
      • Song J.
      • Tal R.
      • Taylor H.S.
      Bone marrow-derived cells or C–X-C motif chemokine 12 (CXCL12) treatment improve thin endometrium in a mouse model.
      However, these circulating bone marrow–derived stem cells are found in only very limited numbers in the circulation. In the setting of severe injury, the number of stem cells may prove insufficient to repair the damage. We have shown that augmented numbers of bone marrow cells in the circulation can prevent injury to damaged tissue including the endometrium. Transfer of bone marrow cells to mice after endometrial injury led to subsequently normal fertility, whereas those receiving placebo had severe infertility because of Asherman’s syndrome. Several case reports and nonrandomized trials have explored delivery of endometrial stem cells to women with inadequate endometrial development or Asherman’s syndrome with promising results for this potential novel therapy.
      • Santamaria X.
      • Cabanillas S.
      • Cervelló I.
      • et al.
      Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman’s syndrome and endometrial atrophy: a pilot cohort study.
      ,
      • Singh N.
      • Mohanty S.
      • Seth T.
      • Shankar M.
      • Bhaskaran S.
      • Dharmendra S.
      Autologous stem cell transplantation in refractory Asherman’s syndrome: a novel cell based therapy.
      Understanding normal menstruation and endometrial repair may provide insight into several endometrial pathologies.
      We also found that the chemokine CXCL12 attracts bone marrow–derived mesenchymal stem cells to the endometrium.
      • Wang X.
      • Mamillapalli R.
      • Mutlu L.
      • Du H.
      • Taylor H.S.
      Chemoattraction of bone marrow-derived stem cells towards human endometrial stromal cells is mediated by estradiol regulated CXCL12 and CXCR4 expression.
      In a mouse model, we found that the administration of CXCL12 to the damaged uterus can mobilize and recruit stem cells from the bone marrow to the uterus. In a mouse model of Asherman’s syndrome, intrauterine administration of CXCL12 led to restoration of normal fertility.
      • Sahin Ersoy G.
      • Zolbin M.M.
      • Cosar E.
      • Moridi I.
      • Mamillapalli R.
      • Taylor H.S.
      CXCL12 promotes stem cell recruitment and uterine repair after injury in Asherman’s syndrome.
      ,
      • Moridi I.
      • Mamillapalli R.
      • Cosar E.
      • Ersoy G.S.
      • Taylor H.S.
      Bone marrow stem cell chemotactic activity is induced by elevated CXCL12 in endometriosis.
      Similarly, in a mouse model of thin endometrium, treatment with either bone marrow supplementation or CXCL12 administration restores normal endometrial architecture and fertility.
      • Yi K.W.
      • Mamillapalli R.
      • Sahin C.
      • Song J.
      • Tal R.
      • Taylor H.S.
      Bone marrow-derived cells or C–X-C motif chemokine 12 (CXCL12) treatment improve thin endometrium in a mouse model.
      Future therapy for Asherman’s syndrome may make use of chemokines that mobilize and attract bone marrow cells without the need for bone marrow stem cell transplantation.

      V. Menstruation and potential role of endometrial stem cells in endometriosis

      Although rapid endometrial regeneration is essential for reproduction in menstruating species, one of the adverse consequences of menstruation and a rapidly regenerating endometrium is endometriosis. Menstruation allows for retrograde menstruation and the possibility of ectopic implantation of endometrial tissue. Continued menstrual flow regularly feeds the endometriosis and allows for lesion expansion. Retrograde menstruation of stem cells in particular contributes to the lesions.
      • Cousins F.L.
      • O D.F.
      • Gargett C.E.
      Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis.
      Furthermore, bone marrow stem cells contribute to the continued growth of endometriosis lesions.
      • Du H.
      • Taylor H.S.
      Contribution of bone marrow-derived stem cells to endometrium and endometriosis.
      ,
      • Figueira P.G.
      • Abrão M.S.
      • Krikun G.
      • Taylor H.S.
      Stem cells in endometrium and their role in the pathogenesis of endometriosis.
      Bone marrow–derived stem cells may be responsible for those rare endometriosis cases outside of the peritoneal cavity such as endometriosis occasionally seen in the lungs or brain. The very processes designed to regenerate and repair the endometrium after menstruation can lead to disease. Here, the circulating stem cells can even lead to endometriosis in areas where endometrial cells cannot reach even through retrograde menstruation.
      Although retrograde menstruation is a well-established cause of endometriosis, in reality endometrial cell trafficking is common; we have previously shown that stem cells from endometriosis can be found in the circulation in a mouse endometriosis model.
      • Li F.
      • Alderman 3rd, M.H.
      • Tal A.
      • et al.
      Hematogenous dissemination of mesenchymal stem cells from endometriosis.
      Similarly, we have shown that endometrial cells can be identified in very small numbers in multiple organs not typically associated with endometriosis including the brain, lung, spleen, and liver.
      • Samani E.N.
      • Mamillapalli R.
      • Li F.
      • et al.
      Micrometastasis of endometriosis to distant organs in a murine model.
      This vast cell migration may explain many of the systemic effects of endometriosis. Women with this disease are more likely to have depression, anxiety, autoimmune disease, and a lower average body mass index.
      • Alderman 3rd, M.H.
      • Yoder N.
      • Taylor H.S.
      The systemic effects of endometriosis.
      The regenerative ability of endometrium and use of circulating stem cells may allow for regeneration after menstruation and enhance fertility; however, it may predispose menstruating animals to endometriosis and associated disease. Endometriosis can be considered a systemic disease in which widespread cell trafficking contributes to the pathophysiology.
      • Alderman 3rd, M.H.
      • Yoder N.
      • Taylor H.S.
      The systemic effects of endometriosis.

      VI. Endometrium, stem cells, and pregnancy

      Finally, endometrium has an essential role in the establishment of pregnancy. Indeed, many complications throughout pregnancy have their origin at the time of implantation.
      • Vannuccini S.
      • Clifton V.L.
      • Fraser I.S.
      • et al.
      Infertility and reproductive disorders: impact of hormonal and inflammatory mechanisms on pregnancy outcome.
      It is not surprising that stem cells are an important part of endometrial and decidual function in pregnancy. We recently reported that there is a major flux of bone marrow–derived stem cells to the uterus in pregnancy.
      • Tal R.
      • Shaikh S.
      • Pallavi P.
      • et al.
      Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy.
      These cells differentiate into endothelial cells and decidual cells that have a functional role in pregnancy. In a mouse model of infertility based on an endometrial receptivity defect, administration of normal bone marrow can restore fertility and successful pregnancy in otherwise infertile animals. This leads to the fascinating conclusion that some instances of infertility or pregnancy loss may be caused by inadequate bone marrow rather than defects in reproductive organs or gametes. Indeed, one can now include the bone marrow as a key reproductive organ!

      VII. Beyond the uterus: menstrual blood–derived stem cells in the context of regenerative medicine

      The fast, scarless regenerative power of the endometrium, along with the relatively easy access to endometrial stem cells from menstrual effluent (see section 4), has spurred efforts to use menstrual blood–derived endometrial stem and progenitor cells therapeutically for a range of regenerative medicine applications beyond those in the uterus mentioned previously.
      • Chen L.
      • Qu J.
      • Cheng T.
      • Chen X.
      • Xiang C.
      Menstrual blood-derived stem cells: toward therapeutic mechanisms, novel strategies, and future perspectives in the treatment of diseases.
      ,
      • Emmerson S.
      • Mukherjee S.
      • Melendez-Munoz J.
      • et al.
      Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.
      Endometrial mesenchymal stem and progenitor cells (MSCs)
      • Gargett C.E.
      • Schwab K.E.
      • Deane J.A.
      Endometrial stem/progenitor cells: the first 10 years.
      ,
      • Gargett C.E.
      • Schwab K.E.
      • Zillwood R.M.
      • Nguyen H.P.
      • Wu D.
      Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.
      share many properties with mesenchymal stem cells derived from the bone marrow, adipose tissue, and other sources.
      • Alcayaga-Miranda F.
      • Cuenca J.
      • Luz-Crawford P.
      • et al.
      Characterization of menstrual stem cells: angiogenic effect, migration and hematopoietic stem cell support in comparison with bone marrow mesenchymal stem cells.
      Similar to mesenchymal stem cells from these other sources, they can be readily expanded in culture, show features of differentiation into the canonical mesenchymal stem cell connective tissues (bone, cartilage, and fat), and produce a range of immunomodulating cytokines, chemokines, and growth factors.
      • Chen L.
      • Qu J.
      • Cheng T.
      • Chen X.
      • Xiang C.
      Menstrual blood-derived stem cells: toward therapeutic mechanisms, novel strategies, and future perspectives in the treatment of diseases.
      ,
      • Alcayaga-Miranda F.
      • Cuenca J.
      • Luz-Crawford P.
      • et al.
      Characterization of menstrual stem cells: angiogenic effect, migration and hematopoietic stem cell support in comparison with bone marrow mesenchymal stem cells.
      Reports that mesenchymal stem cells from endometrium and other sources can transdifferentiate into a variety of nonconnective tissues, including liver, pancreatic beta cells, and hepatocytes, both in vitro and in animal models or in studies of human bone marrow transplant patients, have been attributed to experimental artifacts, as described previously and in section 4.
      More than 1000 clinical trials involving human mesenchymal stem cells or their products are currently listed on ClinicalTrials.gov as of May, 2020, with many in advanced phase III stages of testing. Of these trials, about 400 are listed as involving autologous cells and about 300 as involving allogeneic cells (others do not specify in a searchable term; they may involve cell products such as matrix or exosomes). Clinical applications listed on ClinicalTrials.gov may broadly be divided into direct, permanent regeneration of connective tissues (bone, cartilage, fat, or related tissues), where autologous cells are required, and immunomodulatory applications that tilt healing toward regeneration rather than fibrotic repair through transient action of the therapeutic cells. For example, a phase III clinical trial is underway at the Cleveland Clinic to treat Crohn’s disease fistulas with allogeneic bone marrow–derived mesenchymal stem cells after a successful phase III trial outside the United States using adipose-derived mesenchymal stem cells.
      • Panés J.
      • García-Olmo D.
      • Van Assche G.
      • et al.
      Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn’s disease: a phase 3 randomised, double-blind controlled trial.
      Similarly, advanced clinical trials using allogeneic or autologous bone marrow–derived mesenchymal stem cells to modulate inflammation are underway for aplastic anemia,
      • Pang Y.
      • Xiao H.W.
      • Zhang H.
      • et al.
      Allogeneic bone marrow-derived mesenchymal stromal cells expanded in vitro for treatment of aplastic anemia: a multicenter phase II trial.
      ,
      • Fouillard L.
      • Bensidhoum M.
      • Bories D.
      • et al.
      Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma.
      liver,
      • Forbes S.J.
      • Newsome P.N.
      New horizons for stem cell therapy in liver disease.
      lung,
      • Behnke J.
      • Kremer S.
      • Shahzad T.
      • et al.
      MSC based therapies-new perspectives for the injured lung.
      ,
      • Wilson J.G.
      • Liu K.D.
      • Zhuo H.
      • et al.
      Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial.
      and many other acute or chronic inflammation pathologies.
      Of the mesenchymal stem cell clinical trials listed on ClinicalTrials.gov, only 2 use menstrual blood–derived cells, both taking place at Zhejiang University in Hangzhou, China: 1 for chronic liver disease
      • Cen P.P.
      • Fan L.X.
      • Wang J.
      • Chen J.J.
      • Li L.J.
      Therapeutic potential of menstrual blood stem cells in treating acute liver failure.
      and 1 for type 1 diabetes. However, because menstrual blood–derived mesenchymal stem cells are an attractive source for autologous transplant in regeneration of connective tissues in women,
      • Chen L.
      • Qu J.
      • Cheng T.
      • Chen X.
      • Xiang C.
      Menstrual blood-derived stem cells: toward therapeutic mechanisms, novel strategies, and future perspectives in the treatment of diseases.
      especially considering that connective tissue cells exhibit strong sex-based phenotypic differences, several regenerative applications are advancing through large animal studies. Particularly promising is the potential for endometrial mesenchymal stem cells to repair pelvic organ prolapse by seeding cells onto degradable scaffolds.
      • Emmerson S.
      • Mukherjee S.
      • Melendez-Munoz J.
      • et al.
      Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.
      These and other connective tissue applications are moving toward human trials. Whether the ease of collection of human menstrual effluent–derived stem cells, or performance factors of these cells, will overcome the established infrastructure that relies on bone marrow, adipose tissue, and other sources for regenerative medicine or other purposes is difficult to predict, but they are in the running.

      VIII. Conclusion

      In summary, menstruation in humans requires rapid regeneration of endometrium that is facilitated by stem cells. Stem and progenitor cells in the basalis layer are the major source of new endometrium each cycle. Bone marrow stem cells are engaged to repair endometrium after damage and are now known to be functionally important for pregnancy in mice.
      • Tal R.
      • Shaikh S.
      • Pallavi P.
      • et al.
      Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy.
      Stem cells play a crucial role in reproduction. With this stem cells flux comes the possibility of disease related to aberrant endometrial growth, namely, endometriosis. Endometriosis is a systemic disease of inappropriate stem cell differentiation. Menstruation is far more complex than a simple loss of endometrium and regrowth—it requires contributions from stem cells both within the uterus and bone marrow. Menstruation also predisposes to endometriosis, which also involves far more than just the immediate surroundings of the uterus where most endometriosis settles.

      3C. What does fibroid (leiomyoma) research teach us about endometrial function?

      Elnur Babayev, MD; Serdar E. Bulun, MD

      I. Pathophysiology of uterine fibroid (leiomyoma) formation and growth

      Uterine fibroids are extremely common. More than half of women will develop uterine fibroids by the age of 50 years.
      • Baird D.D.
      • Dunson D.B.
      • Hill M.C.
      • Cousins D.
      • Schectman J.M.
      High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence.
      Patients present with AUB or pressure symptoms such as pelvic discomfort and pain, constipation, or changes in urinary habits. Submucosal fibroids are also associated with infertility and early pregnancy loss.
      • Bulun S.E.
      Uterine fibroids.
      • Ikhena D.E.
      • Bulun S.E.
      Literature review on the role of uterine fibroids in endometrial function.
      • Hapangama D.K.
      • Bulmer J.N.
      Pathophysiology of heavy menstrual bleeding.
      Fibroids are benign uterine tumors characterized by disordered monoclonal proliferation of uterine smooth muscle cells embedded in an abundant extracellular matrix. One proposed mechanism of fibroid formation involves genetic and epigenetic changes in multipotent stem cells in the myometrium that lead to abnormal proliferation and differentiation. Physiological fluctuations in sex steroid levels with subsequent growth and involution of myometrial cells during the menstrual cycle make these stem cells vulnerable to mutations or epigenetic changes and fibroid formation. The genetic, epigenetic, molecular, and paracrine mechanisms underlying fibroid pathophysiology are highly diverse, which explains the observed variations in individual tumors’ clinical behavior (growing, stable, or regressing) and response to medications.
      • Bulun S.E.
      Uterine fibroids.
      ,
      • Ikhena D.E.
      • Bulun S.E.
      Literature review on the role of uterine fibroids in endometrial function.
      Fibroid growth is hormone dependent, and the sex steroids estrogen and progesterone are important regulators of fibroid growth. Most fibroids decrease in size after menopause, whereas pregnancy can lead to an increase in the size of fibroids. Both systemic and local estrogen can stimulate fibroid growth, but local estrogen production through aromatase activity in fibroid tissue seems to play an important role in fibroid pathophysiology.
      • Imir A.G.
      • Lin Z.
      • Yin P.
      • et al.
      Aromatase expression in uterine leiomyomata is regulated primarily by proximal promoters I.3/II.
      • Ishikawa H.
      • Reierstad S.
      • Demura M.
      • et al.
      High aromatase expression in uterine leiomyoma tissues of African-American women.
      • Sumitani H.
      • Shozu M.
      • Segawa T.
      • et al.
      In situ estrogen synthesized by aromatase P450 in uterine leiomyoma cells promotes cell growth probably via an autocrine/intracrine mechanism.
      Estrogen induces progesterone receptor expression and progesterone responsiveness of the tumor. Progesterone has been shown to be essential for fibroid growth in animal studies.
      • Ishikawa H.
      • Ishi K.
      • Serna V.A.
      • Kakazu R.
      • Bulun S.E.
      • Kurita T.
      Progesterone is essential for maintenance and growth of uterine leiomyoma.
      Progesterone may regulate fibroid growth indirectly through its action on differentiated smooth muscle cells, which in turn secrete paracrine molecules that stimulate proliferation of multipotent stem cells.
      • Ono M.
      • Qiang W.
      • Serna V.A.
      • et al.
      Role of stem cells in human uterine leiomyoma growth.
      ,
      • Mas A.
      • Cervelló I.
      • Gil-Sanchis C.
      • et al.
      Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells.

      II. The role of vasoactive substances and inflammatory molecules in the pathogenesis of abnormal uterine bleeding secondary to fibroids

      Fibroids may interfere with normal endometrial function. In fact, heavy menstrual or irregular bleeding is the most common clinical presentation of fibroids and can affect the physical, social, and emotional well-being of women. The degree of endometrial dysfunction seems to be related to the size and location of the fibroids. Submucosal fibroids located immediately beneath the endometrium are more likely to disrupt endometrial integrity and cause AUB. Subserosal fibroids are less likely to do so. Intramural fibroids represent an intermediate pathology, although large intramural fibroids that distort the endometrial cavity will likely lead to abnormal menstruation.
      • Ikhena D.E.
      • Bulun S.E.
      Literature review on the role of uterine fibroids in endometrial function.
      ,
      • Hapangama D.K.
      • Bulmer J.N.
      Pathophysiology of heavy menstrual bleeding.
      Dissecting the mechanisms of interaction between fibroids and endometrium can help us understand menstrual biology and may lead to the development of novel therapeutic modalities for AUB.
      Fibroids are space-occupying lesions that, depending on their size, can place significant mechanical stress on uterine architecture. Fibroids can increase the amount of bleeding by simply increasing the surface area of the endometrium. In addition, changes in cell shape and stretch can affect gene expression in the myometrium and endometrium.
      • Rogers R.
      • Norian J.
      • Malik M.
      • et al.
      Mechanical homeostasis is altered in uterine leiomyoma.
      • Payson M.
      • Malik M.
      • Siti-Nur Morris S.
      • Segars J.H.
      • Chason R.
      • Catherino W.H.
      Activating transcription factor 3 gene expression suggests that tissue stress plays a role in leiomyoma development.
      • Norian J.M.
      • Owen C.M.
      • Taboas J.
      • et al.
      Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma.
      Large intramural and submucosal fibroids may interfere with the myometrial contractions that occur during menstruation. These contractions help to evacuate menstruation material from the uterus and decrease blood loss from endometrial vessels under physiological conditions; thus, even a small submucosal fibroid can lead to a significant blood loss in these patients.
      • Bulun S.E.
      Uterine fibroids.
      Endothelin 1 (ET-1) is a vasoconstrictor that affects spiral arterioles in the endometrium
      • Maybin J.A.
      • Critchley H.O.
      Menstrual physiology: implications for endometrial pathology and beyond.
      and plays an important role in myometrial contractility.
      • Masaki T.
      Endothelins: homeostatic and compensatory actions in the circulatory and endocrine systems.
      Altered expression levels of ET-1 and endothelin receptors (ETA-R and ETB-R) in uterine fibroids may interfere with the normal physiological function of the myometrium during menstruation. Fibroids have higher levels of ETA-R and lower levels of ETB-R than normal myometrium.
      • Pekonen F.
      • Nyman T.
      • Rutanen E.M.
      Differential expression of mRNAs for endothelin-related proteins in human endometrium, myometrium and leiomyoma.
      ,
      • Farrer-Brown G.
      • Beilby J.O.
      • Tarbit M.H.
      Venous changes in the endometrium of myomatous uteri.
      Thus, it may be envisioned that altered endothelin biology induced by a uterine fibroid may affect the vascular function of the adjacent endometrium, giving rise to its irregular development or shedding.
      Menstruating endometrium is rich in cytokines and prostaglandins. How this inflammatory milieu affects fibroids and vice versa is an active area of investigation. The composition of inflammatory cells is different in areas of the endometrium that overlay fibroids compared with distant sites. Perifibroid endometrium has increased numbers of macrophages in all phases of menstrual cycle; however, the number of uterine natural killer (uNK) cells is decreased in the secretory phase.
      • Kitaya K.
      • Yasuo T.
      Leukocyte density and composition in human cycling endometrium with uterine fibroids.
      Prostaglandin F2α levels are increased in fibroid uteri, which may explain the disordered contractility and increased blood loss observed in these patients. Moreover, prostaglandin E2 (PGE2), which is produced in the normal menstruating uterus, affects leiomyoma (fibroid) cells. Normal myometrial cells do not show any changes in gene expression in response to PGE2, whereas leiomyoma (fibroid) cells demonstrate increased expression of antiapoptotic microRNAs.
      • Kim H.G.
      • Jung G.Y.
      • Park S.B.
      • Cho Y.J.
      • Han M.
      Assessment of the effects of prostaglandins on myometrial and leiomyoma cells in vitro through microRNA profiling.
      Celecoxib, a cyclooxygenase 2 inhibitor, reduces the proliferation rate of leiomyomas (fibroids) through a nuclear factor κB–mediated decrease in expression of cytokines and growth factors.
      • Park S.B.
      • Jee B.C.
      • Kim S.H.
      • Cho Y.J.
      • Han M.
      Cyclooxygenase-2 inhibitor, celecoxib, inhibits leiomyoma cell proliferation through the nuclear factor κB pathway.
      Our understanding of the role of prostaglandins in fibroid pathogenesis may lead to new therapeutic approaches. Gene expression analysis of endometrial biopsies from women with HMB has also indicated differential expression of antigen processing pathway genes in women with and without fibroids.
      • Girling J.E.
      • Lockhart M.G.
      • Olshansky M.
      • et al.
      Differential gene expression in menstrual endometrium from women with self-reported heavy menstrual bleeding.
      Thus, specific molecular pathways might be responsible for abnormal bleeding associated with fibroids.

      III. Growth factors as primary mediators of endometrial dysfunction in fibroid uteri

      Fibroid uteri demonstrate rich vascularity and increased venous plexus.
      • Ikhena D.E.
      • Bulun S.E.
      Literature review on the role of uterine fibroids in endometrial function.
      ,
      • Hapangama D.K.
      • Bulmer J.N.
      Pathophysiology of heavy menstrual bleeding.
      There also seems to be defective vasoconstriction as evidenced by dilated venous spaces and vasocongestion. Increased angiogenesis is also apparent in patients with fibroids,
      • Farrer-Brown G.
      • Beilby J.O.
      • Tarbit M.H.
      Venous changes in the endometrium of myomatous uteri.
      ,
      • Anania C.A.
      • Stewart E.A.