The study population comprised mothers, sisters, daughters, cousins, and maternal aunts of persons with hemophilia A who were being monitored in various centers. They were identified as carriers after analyzing the family tree of each persons with hemophilia A under observation. Two types of carriers were distinguished: potential carriers and obligatory carriers. Obligatory carriers were defined as daughters of fathers with hemophilia, women who had multiple sons with hemophilia in separate births, or women who had a sick son and a confirmed hemophilic relative on their maternal side of the family. Potential carriers, on the other hand, were women with a hemophilic relative on the maternal side but had not yet given birth to a sick son or women who, despite the absence of a family history of hemophilia, had given birth to a son with hemophilia. Each pedigree was cross-verified with several family members to ensure its completeness. Excluded from the study were non-biological mothers of persons with hemophilia (PwH), sisters of PwH born to different fathers and mothers, women from families with acquired hemophilia or other constitutional bleeding disorders, women who use traditional reusable towels, carriers with jaundiced, hemolyzed, or lipemic plasma at the time of diagnosis, as well as those with a fibrinogen level between 0.6 g/L and 6 g/L The size of the study population was determined after systematic enrollment of all carriers of hemophilia A identified after review of the pedigrees of the 101 PwH followed in the hemophilia treatment center and after application of the study’s inclusion and non-inclu- sion criteria.

Between Avril 2021 and Mars 2022, we invited women identified as carriers of hemophilia A, to participate in this study conducted in three hospitals: in the South of Benin, at the University Clinic for Blood Disorders (CUMAS) located at the Hubert Koutoukou Maga National Hospital and University Center (CNHU- HKM) in Cotonou; in the Center, at the Zou-Collines Departmental Hospital Center (CHD) situated in the central region; and at the Borgou-Alibori Departmental University Hospital Center (CHUD) located in Parakou in the North of Benin. As for the laboratory tests, they were conducted at the hematology laboratory of CNHU-HKM in Cotonou. Candidates were recruited via pedigree of persons with hemophilia (PWHs) followed-up at the hospitals in Benin. A telephone directory was subsequently compiled, and the study participants were contacted via phone. Following the acquisition of their informed consent, these individuals were invited to visit one of the three aforementioned hospitals to take part in the study.

Data collected during interviews with the participants included socio-demo- graphic information, medical history, surgical history, obstetric history, and details regarding bleeding episodes. The Higham score were employed during data collection to assess the risk of menorrhagia. It is an objective measure for quantifying menstrual blood loss in reproductive-age women. It assigns a score based on an evaluation of menstrual losses, taking into account the extent of staining on sanitary products used by the women and the number of sanitary products used. When the Higham score is sensitivity and specificity exceed 100 it used to identify menorrhagia.

Following the completion of the data collection questionnaire, each carrier of hemophilia A was subjected to blood sampling. Samples collected into two tubes: one containing sodium citrate and the other containing ethylene diamine tetra acetic acid tripotassium. After double centrifugation of the citrated samples at 2500G for 15 minutes, with plasma decantation in between, the resulting platelet-poor plasmas were divided into cryotubes and frozen at −20˚C. These samples were then transported to the hematology laboratory of CNHU HKM in Cotonou using dry ice.

In hematology laboratory, the activated partial thromboplastin time (APTT) and the functional levels of factor VIII were determined using a chronometric method on a semi-automatic coagulometer with optical detection (CA 104 Sysmex). For each series of factor VIII assays, a calibration curve was constructed, and both normal and pathological control samples were processed under the same conditions as those applied to the carriers’ plasma. Moreover, the von Willebrand factor antigen level was determined using an immunological method on the Mini Vidas automated system from Biomérieux, and blood grouping within the ABO and RH1 antigens was conducted through microfiltration technique.

Internal quality controls results were systematically validated and all results obtained biological validation

All numerical data were reported as mean ± standard deviation along with the minimum and maximum values. Categorical data were expressed as percentages. Pearson’s Chi-2 test was employed to compare different groups in the study. A significance level of p < 0.05 was considered as the threshold for statistical significance, indicating that results were considered statistically significant when the p-value was less than 0.05. The statistical analysis was conducted using a computer based statistical program Stata version 15.

The study protocol received approval from the Local Ethics Committee for Biomedical Research at the University of Parakou (CLERB-UP) under the reference number N°464/CLERB-UP/P/SP/R/SA on August 9, 2021. The survey was conducted in accordance with the ethical guidelines outlined by this committee, including voluntary participation, the ability for participants to withdraw from the study at any point, ensuring anonymity, and maintaining data confidentiality. Also the carriers provided written informed consent for participating in the study. In the case of carriers who were minors, consent from one of their parents or legal guardians was obtained before their inclusion.

All the carriers included in the study were aged between 1 and 73 years, with a median age of 23 years and a mean age of 23.2 ± 15 years. Approximately 63 carriers (88.7%) had some level of education, with 4.8% having attended nursery school, 34.9% having completed primary education, 44.4% having secondary education and 15.9% universary education. Regarding socio-professional categories, 8.5% were in the preschool age group, followed by students (39.4%), shopkeepers (21.1%), and employee (14.1%). It is worth noting that 11.3% of the carriers were artisans and 5.6% were homemakers. The distribution of carriers by ethnic groups revealed that 68.4% of the included women were Fon, followed by Adja (15.2%), Yoruba (10.1%) and Bariba (2.5%). The Dendi, Ottamari, and YoaLokpa ethnic groups were the least represented (1.3% each). Concerning the departments of Benin in which the carriers resided, 38% of 71 included carriers lived in the Littoral region of Benin. Other highly represented departments included Atlantique (18.3%) and Borgou (16.9%), while the department with the fewest recruited carriers in the study was Ouémé (12.7%), Zou (12.7%) and Collines (1.4%). Table 1 illustrates the socio-demographic characteristics of the study population.

During the study, family interviews and pedigree analysis allowed for the identification of 41 obligatory carriers and 30 potential carriers. Regarding the severity of hemophilia, 36 carriers (50.7%) had a family history of moderate hemophilia A, and 35 had a family history of severe hemophilia A (49.3%). No carriers of mild hemophilia were identified during the study.

Among the 71 carriers studied, 36 carriers (50.7%) reported having one or more brothers with hemophilia. The median number of brothers with hemophilia per carrier was 1, ranging from 1 and 3 brothers. Among these 36 carriers, 25 of them (69.4%) had a single brother with hemophilia, 10 carriers (27.8%) had two, and 1 carrier (2.8%) had 3 hemophilic brothers. Furthermore other than brothers, 21 of 71 carriers included (29.6%) had another family member with hemophilia, including cousins (76.2%), uncles (28.6%), nephews (47.6%), and fathers (33.3%). One carrier had grandfather with hemophilia, and another one

had grandchild with hemophilia. Among the 71 carriers included, 30 had children with hemophilia (42.3%). The mean number of children with hemophilia per carrier was 1.8 ± 1.00 children with extremes between 1 and 4.

In the study population, 16 carriers (22.5%) had medical histories that included epigastric pain (31.3%), asthma (31.3%), and high blood pressure (31.3%). Hemorrhoidal disease (12.5%) and migraine (12.5%) were the least frequently reported medical histories among carriers. Long-term medication use was found in 6 carriers (8.5%), including three on hormonal contraception, two on antihypertensive medication, and one on bronchodilators.

In the study population, 31 carriers (43.7%) reported having abnormal bleeding symptoms. The most frequently reported symptoms were menorrhagia (71%), postpartum hemorrhage (45.2%), gastrointestinal bleeding (19.4%), post-operative bleeding (12.9%) and gingivorragy (9.7%). The least common bleeding symptoms included epistaxis (3.2%), bleeding from wounds after minor trauma (3.2%), and hemarthrosis (3.2%). Hematuria, hematomas, and bleeding from the nervous system weren’t observed in the carriers during this study. Among the 71 hemophilia A carriers, 45 were of reproductive age. Of whom 22 (48.8%) had a Higham score exceeding 100, indicating menorrhagia. The average Higham score in this study was 144.4 ± 143.3, with a median of 78.00 and a range from 2 to 603.

During the study, the activated partial thromboplastin time (APTT) was prolonged in 7 carriers, representing 9,9% of the study population. The average factor VIII:C level was 68.8 ± 34.9 IU/dL with ranging from 5.5 to 150.00 IU/dL. The average FVIII:C level in obligatory carriers was 56.9%, with extremes of 5.5% and 127.9%. Among potential carriers, the average FVIII:C level was higher at 80.4% with extremes of 11.5% and 132%. It is worth noting that out of the 71 carriers of hemophilia A, 12 had a factor VIII level < 40%, indicating a frequency of 16.9% for female hemophilia. Of these 12 female with hemophilia A, eight were obligatory carriers and four were potential carriers. The distribution of these female according to the severity of hemophilia revealed 11 cases of mild hemophilia and one case of moderate hemophilia.

Furthermore, the FVIII/FvWAg ratio was less than 0.7 in 52 carriers, accounting for a frequency of 73.2% of the study population. They include 32 obligatory carriers and 20 potential carriers. Regarding the distribution of carriers by ABO blood group, those with blood group O were the most common (53.5%), followed by blood group A (29.6%), B (14.1%), and AB (2.8%).

The study of factors favoring the occurrence of abnormal bleeding in carriers, regardless of the type, revealed that the status of being an obligatory carrier (p = 0.00003) and a FVIII/FvWAg ratio < 0.7 (p = 0.003) were associated with the occurrence of abnormal bleeding. Of the 19 womens with a FVIII:C/FvWAg ratio < 0.7, eight had abnormal bleeding symptoms without statistically significant relationship. However, prolonged APTT, a family history of hemophilia, the presence of a family history of severe hemophilia and blood group O were not statistically associated with the occurrence of abnormal bleeding in carriers. Furthermore, in this study, blood group O (p = 0.000) and a FVIII/FvWAg ratio < 0.7 (p = 0.0016) were associated with a higher risk of menorrhagia. Table 2 and Table 3 show the factors associated respectively with development of abnormal bleeding symptoms in carriers of haemophilia A in Benin and with hypermenorrhoea in these carriers.