Women and girls with inherited bleeding disorders: Focus on haemophilia carriers and heavy menstrual bleeding.

Raising awareness and improving recognition, accurate classification, and enhanced access to new treatments represent current key challenges for carriers of haemophilia. Women and girls carrying genes for haemophilia often experience significant bleeding and/or low factor levels. The bleeding associated with female haemophilia is frequently overlooked, has a weak correlation with factor levels, and manifests differently than in males, with heavy menstrual bleeding being a predominant symptom. Recent changes in terminology now allow the diagnosis of haemophilia in females with low factor levels and differentiate between symptomatic and asymptomatic carriers of the gene. Observations from real-world experiences and limited clinical trial data have highlighted the positive impact of various new haemophilia treatments for women and girls with clotting factor deficiencies. There is an urgent need for initiatives that increase their access to these treatments and encourage well-designed clinical trials focusing on female-specific outcomes. In women with inherited bleeding disorders, early recognition and optimal management of heavy menstrual bleeding are crucial. However, treatment options and guidance from high-quality clinical trials are currently insufficient. Menstrual health assessment should be a regular part of monitoring women and girls with inherited bleeding disorders throughout their lives, emphasizing the importance of gathering data to improve future management.

Investigation of vascular endothelial growth factor (VEGF) polymorphism in patients with idiopathic heavy menstrual bleeding.

PURPOSE: To investigate whether there is a relationship between the VEGF polymorphisms and idiopathic heavy menstrual bleeding (HMB-E)Query. METHODS: Sixty-five patients diagnosed with HMB-E according to the FIGO classification system and 65 female healthy volunteers were included in the study. The polymorphic regions rs699947 (- 2578C > A), rs1570360 (- 1154G > A), rs2010963 (+ 405G > C), rs3025039 (+ 936C > T), rs25648 (c534C > T) in the VEGF were detected using Next Generation DNA Sequencing method. RESULTS: The - 2578C > A polymorphism CC genotype, CA + AA genotypes, and C allele, as well as the - 1154G > A polymorphism AA genotype, and A allele were associated with increased risk of HMB-E (p < 0.05 for all). However, no statistically significant difference was found between the patient group and the control group in terms of genotype and allele distributions in the 405G > C, + 936C > T, c534C > T polymorphic regions (p > 0.05 for all). While the - 2578/ - 1154/ + 405/c534 AGGC haplotype decreased the risk of HMB-E, the CAGC haplotype was found to increase the risk of HMB-E. CONCLUSION: VEGF - 2578C > A and - 1154G > A polymorphisms were significantly associated with the risk of HMB-E in the Turkish population.

Congenital factor XI deficiency caused by a novel F11 missense variant: a case report.

Hereditary factor XI (FXI) deficiency is a mild bleeding disorder, rare in the general population but relatively common among Ashkenazi Jews. The human F11 gene comprises 15 exons, spanning over 23 kb of the long arm of chromosome 4 (4q35). Homozygotes or compound heterozygotes typically show severe FXI deficiency, whereas heterozygotes show partial or mild deficiency. However, the genotype-phenotype relationship is difficult to establish, even among individuals within the same family. We report on a female patient with a heterozygous variant in F11 and FXI deficiency (49 IU/dL), who suffers from menorrhagia since menarche and easy bruising. She experienced excessive bleeding during thyroidectomy and after a cesarean section. Her younger sister, who carries the same heterozygous variant in F11 and has mild FXI deficiency (47 IU/dL), has menorrhagia without other bleeding difficulties although she has undergone several surgeries. Their father, who carries the same missense variant, has not experienced any bleeding difficulties (but he has not undergone any surgeries either). The family study revealed that the A428C mutation was inherited from the father. This variant has not previously been described in the literature and is the first F11 variant described in the Croatian population. Our study showed that even when family members have the same germline F11 variant, they still may experience phenotypic variability, making disease prognosis more complex.

Factor 11 single-nucleotide variants in women with heavy menstrual bleeding.

In a previous study it was shown that lower factor XI (FXI) levels in women with heavy menstrual bleeding (HMB). Our aim was to determine the single-nucleotide variants (SNVs) in the F11 gene in women with HMB. In addition, an extensive literature search was performed to determine the clinical significance of each SNV. Patients referred for HMB (PBAC-score >100) were included. With direct sequencing analysis of all 15 exons and flanking introns of the F11 gene, 29 different non-structural SNVs were detected in 49 patients with HMB. Interestingly, most of these SNVs have previously been associated with venous thrombosis instead of bleeding. These findings have not helped to elucidate the molecular basis of HMB. They also question the specificity of previously reported F11 variations in patients with thrombosis. More studies are needed to explain the lower FXI levels seen in patients with HMB. IMPACT STATEMENT Women with mild deficiencies of factor XI (FXI) (< 70%) are prone to excessive bleeding during menstruation. Bleeding manifestations are not well correlated with plasma FXI levels and bleeding episodes can vary widely among patients with similar low FXI levels. In a previous study we showed that women with heavy menstrual bleeding (HMB) had normal, but on average, lower levels of FXI than controls. In light of these findings, we performed F11 gene analysis to determine the single-nucleotide variants (SNVs) in women with HMB and performed an extensive literature search to determine the clinical significance of each SNV. By direct sequencing analysis of the F11 gene we found 29 different non-structural SNVs in 49 women with heavy menstrual bleeding. Remarkably, a number of these SNVs have previously been implicated in thrombosis. These findings have not helped to elucidate the molecular basis of lower FXI levels in HMB. They also question the specificity of previously reported F11 variations in patients with thrombosis. More studies are needed to explain the lower FXI levels seen in patients with HMB.

Factor XIII deficiency in Iran: a comprehensive review of the literature.

Factor XIII deficiency (FXIIID) is a rare bleeding disorder with an estimated prevalence of 1 in 2-million population worldwide. In Iran, a Middle Eastern country with a high rate of consanguineous marriages, there are approximately 473 patients afflicted with FXIIID. An approximately 12-fold higher prevalence of FXIIID is estimated in Iran in comparison with overall worldwide frequency. In this study, we have undertaken a comprehensive review on different aspects of FXIIID in the Iranian population. The distribution of this disease in different regions of Iran reveals that Sistan and Baluchestan Province has not only the highest number of patients with FXIIID in Iran but the highest global incidence of this condition. Among Iranian patients, umbilical cord bleeding, hematoma, and prolonged wound bleeding are the most frequent clinical manifestations. There are several disease causing mutations in Iranian patients with FXIIID, with Trp187Arg being the most common mutation in FXIIID in Iran. Traditionally, the management of FXIIID in Iran was only based on administration of fresh frozen plasma or cryoprecipitate, until 2009 when FXIII concentrate became available for patient management. Various studies have evaluated the efficacy and safety of prophylactic regimens in different situations with valuable findings. Although the focus of this study is on Iran, it offers considerable insight into FXIIID, which can be applied more extensively to improve the management and quality of life in all affected patients.

TMPRSS6 rs855791 polymorphism influences the susceptibility to iron deficiency anemia in women at reproductive age.

BACKGROUND: Genome-wide-association studies have identified the TMPRSS6 polymorphism rs855791 has the strongest association with red blood cell indices or iron parameters in general population. Whether this genetic variant influences the susceptibility of iron deficiency anemia (IDA) in women with menstruation has not been well studied. METHODS: In this case-control study, we enrolled 67 women with IDA and 107 healthy volunteers, and analyzed their complete blood counts, rs855791 genotypes, and menstrual amounts. Menstrual blood loss was evaluated with a pictorial blood-loss assessment chart. RESULTS: There were significantly fewer rs855791 C homozygotes in the IDA group than in the healthy group (11.9% vs. 25.2%, p = 0.03). The odds ratio (OR) of C homozygotes having IDA versus non-CC subjects having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). When the analysis was confined to study subjects with menorrhagia, this difference became more prominent (9.6% vs. 28.6%, p = 0.01; OR, 0.27, 95% CI, 0.09 - 0.77, p = 0.01). For women with non-CC genotypes, there was an inverse correlation between hemoglobin levels and menstrual loss (p < 0.001); however, this association was not found for those with genotypes CC (p = 0.15). CONCLUSIONS: Our study suggests homozygosity for TMPRSS6 rs855791 C genotype has a protective role against IDA in women at reproductive age, especially in those with menorrhagia.

Genetic screening of PLA1/PLA2 polymorphous marker of integrin beta 3 (ITGB3) GP IIIA gene in adolescent girls with puberty menorrhagia.

Puberty menorrhagia is one of the urgent problems of modern reproductive medicine. The study aimed to investigate the relationship between polymorphism of the GP IIIa (PLA1/PLA2) gene and improve the diagnosis of puberty menorrhagia in girls with thyroid gland pathology. Ninety-seven girls at puberty age were divided into three groups: group 1 (main) - girls with puberty menorrhagia and thyroid gland pathology (30 individuals), group 2 (comparison) - 40 girls with puberty menorrhagia, group 3 (control) - 27 practically healthy girls. Polymorphism of the GP IIIa (PLA1/PLA2) gene was studied by isolating genomic DNA from peripheral blood leukocytes, followed by amplification with a polymerase chain reaction. Results showed that mutation in the 17th chromosome of q21.32 of the GP IIIa gene occurred in 8.6% of cases among adolescents with menorrhagia, in contrast to the control group, where it was not observed at all. The A1A1-genotype occurred by 11.7% (X2=4.01, p=0.041) more often in adolescents with menorrhagia than in girls with concomitant thyroid gland pathology and by 15.0% (X2=4.54, p=0.033) more often than in the control group. It was also found that the presence of the A1A2-genotype unreliably reduced the chances of uterine bleeding in adolescent girls by 1.45 times (OR=2.12) and was a protective factor in the puberty menorrhagia occurrence (OR=0.47). It may be concluded that the identification of a hereditary factor of the reproductive system diseases of adolescent girls fundamentally changes the point of view on the tactics of disease management and subsequent therapy.

Coexpression of factor VIII and factor von Willebrand variants in a woman with heavy menstrual bleeding.

Heavy menstrual bleeding is one of the most common causes of consultation in haematology. We present the clinical case of a 20-year-old woman referred by her gynaecologist due to heavy menstrual bleeding since menarche, complicated by iron deficiency anaemia. Haemostasis work-up was initially suggestive of a von Willebrand disease type 1. Genetic analyses by whole exome sequencing lead to a fortuitous discovery of haemophilia by identifying a heterozygous missense mutation in F8 , exon 8 c.1127T>G:p.Val376Gly, previously reported in a patient with mild haemophilia A. The bleeding phenotype worsened by concomitant low von Willebrand factor (VWF) due to VWF variants influencing VWF levels. Our case highlights how whole exome sequencing can help to correct an erroneous diagnosis and identify polymorphisms that eventually contribute to the overall haemostatic balance.

Whole-exome analysis of adolescents with low VWF and heavy menstrual bleeding identifies novel genetic associations.

Adolescents with low von Willebrand factor (VWF) levels and heavy menstrual bleeding (HMB) experience significant morbidity. There is a need to better characterize these patients genetically and improve our understanding of the pathophysiology of bleeding. We performed whole-exome sequencing on 86 postmenarchal patients diagnosed with low VWF levels (30-50 IU/dL) and HMB and compared them with 660 in-house controls. We compared the number of rare stop-gain/stop-loss and rare ClinVar "pathogenic" variants between cases and controls, as well as performed gene burden and gene-set burden analyses. We found an enrichment in cases of rare stop-gain/stop-loss variants in genes involved in bleeding disorders and an enrichment of rare ClinVar "pathogenic" variants in genes involved in anemias. The 2 most significant genes in the gene burden analysis, CFB and DNASE2, are associated with atypical hemolytic uremia and severe anemia, respectively. VWF also surpassed exome-wide significance in the gene burden analysis (P = 7.31 × 10-6). Gene-set burden analysis revealed an enrichment of rare nonsynonymous variants in cases in several hematologically relevant pathways. Further, common variants in FERMT2, a gene involved in the regulation of hemostasis and angiogenesis, surpassed genome-wide significance. We demonstrate that adolescents with HMB and low VWF have an excess of rare nonsynonymous and pathogenic variants in genes involved in bleeding disorders and anemia. Variants of variable penetrance in these genes may contribute to the spectrum of phenotypes observed in patients with HMB and could partially explain the bleeding phenotype. By identifying patients with HMB who possess these variants, we may be able to improve risk stratification and patient outcomes.

The endometrial response to modulation of ligand-progesterone receptor pathways is reversible.

OBJECTIVE: To study the impact of the progesterone receptor modulator (PRM), ulipristal acetate (UPA), on endometrial morphology and function. DESIGN: Cross-sectional. SETTING: University Research Institute. PATIENT(S): Endometrial biopsies from 16 patients with heavy menstrual bleeding with a structurally normal uterus or in association with structural abnormalities identified on radiological imaging (fibroids, adenomyosis or a combination of fibroids and adenomyosis). INTERVENTION(S): Participants received UPA (5 mg once daily) for three 12-week courses, each separated by 4 weeks without treatment. MAIN OUTCOME MEASURE(S): Gene expression by real-time quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and digital image analysis were analyzed to investigate the endometrial impact of modulation of progesterone receptor pathways upon expression of steroid receptors, steroid metabolizing enzymes, cell proliferation, and progesterone-regulated genes in the same patients at 3 time points: before, during, and after discontinuation of PRM treatment. RESULT(S): Ulipristal acetate treatment resulted in increased messenger ribonucleic acid (mRNA) levels of steroid receptors compared with pretreatment secretory endometrium; decreased mRNA levels of 17- and 11-beta-hydroxysteroid dehydrogenases compared with pretreatment proliferative endometrium and pretreatment secretory endometrium; reduced cell proliferation compared with pretreatment proliferative endometrium; and altered mRNA levels of progesterone-regulated genes. A strong consistency between immunohistochemistry-digital image analysis and real-time quantitative reverse transcription polymerase chain reaction results was evident. Alterations in the mRNA levels and endometrial morphology returned to a pretreatment phenotype after the cessation of PRM exposure. CONCLUSION(S): The endometrial impact of the modulation of progesterone receptor pathways with PRM (UPA) treatment is reversible. CLINICAL TRIAL REGISTRATION NUMBER: Ulipristal acetate versus conventional management of heavy menstrual bleeding (UCON) trial (EudraCT 2014-003408-65; REC14/LO/1602).

Prolonged use of finasteride-induced gonadal sex steroids alterations, DNA damage and menstrual bleeding in women.

The aim of the present study was to examine the effect of prolonged use of finasteride on serum levels of dihydrotestosterone (DHT), estradiol (E2), progesterone, testosterone and androstenedione in women during the menstrual period. Further, to screen and compare the 5α-reductase activities through the expression of SRD5A1, SRD5A2 and AR gene and to determine the level of VEGF, VKOR and SAA gene expression and DNA damage. A total of 30 Saudi women aged between 25 and 35 years were enrolled in the study. The selected women were divided into two groups. The first group (n = 15) received 5 mg finasteride/day for prolonged period of one year and second group (n = 15) was taken as a healthy control. ELISA technique was used for measuring the serum levels of the targeted hormones, and Comet assay was used for checking the DNA integrity. Our findings revealed significant decrement of DHT, E2, progesterone and androstenedione levels and elevated levels of testosterone in group treated with daily oral doses of 5 mg finasteride/day compared with the control subjects. mRNA expression suggested that finasteride has concrete effects on the gene expression of the selected genes from the treated group in comparison with the control group. In addition, finasteride induced DNA damage, and heavy menstrual bleeding was noted in women treated with finasteride. In conclusion, the present findings revealed that finasteride has adverse health effects in women associated with gonadal sex steroids alterations, DNA damage and heavy menstrual bleeding with no consensus in the treatment of androgenetic alopecia in women.

Factor VII Deficiency.

The complex formed between the procoagulant serine protease activated factor VII (FVII) and the membrane protein tissue factor, exposed on the vascular lumen upon injury, triggers the initiation of blood clotting. This review describes the clinical picture of FVII deficiency and provides information on diagnosis and management of the disease. FVII deficiency, the most common among the rare congenital coagulation disorders, is transmitted with autosomal recessive inheritance. Clinical phenotypes range from asymptomatic condition, even in homozygotes, to severe disease characterized by life-threatening and disabling symptoms (central nervous system and gastrointestinal bleeding and hemarthrosis), with early age of presentation and the need for prophylaxis. In females, menorrhagia is prevalent and affects two thirds of the patients of fertile age. Although FVII gene mutations are extremely heterogeneous, several recurrent mutations have been reported, a few of them relatively frequent. The study of genotype-phenotype relationships indicates that modifier (environmental and/or inherited) components modulate expressivity of FVII deficiency, as reflected by patients with identical FVII mutations and discordant clinical phenotypes. Several treatment options are available for FVII deficiency: the most effective are plasma-derived FVII concentrates and recombinant activated FVII (rFVIIa). Treatment-related side effects are rare.

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation.

The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene (Xq27.1-27.2). A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1-70 years); median bleeding score: 1 (range 0-8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score >/=3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 +/- 10.3% (95% CI 36.4-47.7) while a score >3 had involvement of /=3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.

Turner syndrome and 45,X/47,XXX mosaicism.

The occurrence of double aneuploidy is a relatively rare phenomenon. We report on a 17-year-old woman with short stature, minimal pubic and axillar hair and short hands. In cultured lymphocyte a double aneuploidy mosaicism was detected, consisting of a cell line with trisomy for X chromosome and a cell line with monosomy for the X-chromosome and no cell line with a normal karyotype. To our knowledge, this is the first case of mosaic 45,X/47,XXX in Turkey.

A case of a severe factor XI deficiency in a Chinese woman with heavy menorrhagia.

: The current study was to elucidate the molecular defect in a 32-year-old Chinese woman with heavy menorrhagia and delayed wound healing. The F11 gene was amplified by PCR and screened for mutations. Then identified mutations were analyzed by in-silico programs and molecular modeling analysis. This woman was found to have severely low levels of factor XI (FXI) (FXI:C: 2.0%; FXI:Ag: 5.4%) by surgical screening. Further DNA sequencing of F11 reveled a novel mutation (p.Ser295Ile) in the Ap4 domain and an already known mutation (p.Trp228stop) in the Ap3 domain. Pedigree analysis showed that the new mutation was inherited from her father (FXI:C: 41%), whereas the other was inherited from her mother (FXI:C: 62%). Modeling analysis indicated Ser295Ile mutation probably determining important structural changes in the protein folding. Both of the heterozygous mutation contribute to the severe FXI deficiency by interfering with correct assembly of the region.

[Expression profile of aquaporin 1 in patients with menorrhagia].

OBJECTIVE: To investigate the expression of aquaporin 1 (AQP1) in endometrium of patients with menorrhagia. METHODS: RT-PCR and immunohistochemistry were carried out in twenty women with normal menstrual cycle to confirm the expression of AQP1 in endometrium and locate it. Then 51 women with menorrhagia and 40 women with normal menstrual cycle were included in the study. RT-PCR and immunohistochemistry were used to examine the expression of AQP1. RESULT: AQP1 mRNA was expressed in the human endometrium throughout menstruation cycle, which was mainly located in the endothelia of the capillaries and small blood vessels. Quantification of the immunostaining revealed higher density during secretary phase than that in proliferative phase (P<0.01). The staining intensity and density of AQP1-positive microvessel decreased significantly in simple hyperplasia group (P<0.01) and then gradually increased in complex hyperplasia and atypical hyperplasia groups (P<0.001). CONCLUSION: Decreased expression of AQP1 may lead to disturbed endometrial vascular remodeling and may be involved in the occurrence of menorrhagia.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

Context: Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. Objective: To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Patients/Setting: Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. Design: CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. Results: CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. Conclusions: These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.

Gene expression profiling of mid to late secretory phase endometrial biopsies from women with menstrual complaint.

OBJECTIVE: The purpose of this study was to test whether a quantitative high-throughput molecular screen can be used to probe human endometrium and initiate the development of molecular diagnostic tools with potential for identification of therapeutic targets in women with menstrual complaints. STUDY DESIGN: Endometrium was collected from 10 patients with complaint of heavy bleeding, classified into mid or late secretory phase of the menstrual cycle by histologic dating and serum progesterone concentration. Total RNA was extracted and gene activity assessed using high-density oligonucleotide arrays. RESULTS: Statistical testing identified 83 'signature' genes whose expression levels differentiated the mid and late secretory phases of the menstrual cycle. CONCLUSION: The results show that the endometrium, a complex heterogeneous tissue, is amenable to high-throughput molecular analyses and this work provides further support for the future application of molecular profiling to clinical diagnosis.