Knowledge, Attitudes, Practices, and Associated Factors Toward Rh Incompatibility Among Pregnant Women in Addis Ababa, Ethiopia

Introduction: Rhesus (Rh) incompatibility occurs when an Rh-negative mother is exposed to Rh-positive fetal red blood cells, leading to maternal alloimmunization and production of anti-D antibodies. This condition may result in hemolytic disease of the fetus and newborn, hydrops fetalis, kernicterus, and significant perinatal morbidity and mortality. Despite the availability of effective preventive measures, awareness and utilization of anti-D prophylaxis remain limited in many developing countries, including Ethiopia. 
Methods: An institution-based cross-sectional study was conducted among 299 pregnant women attending antenatal care at SPHMMC from October 1 to November 30, 2020. Participants were selected using a systematic random sampling technique. Data were collected using a pretested interviewer-administered structured questionnaire adapted from previous studies. Data were entered and analyzed using Statistical Package for Social Sciences (SPSS) version 25. Descriptive statistics were used to summarize study variables. Binary and multivariable logistic regression analyses were performed to identify factors associated with knowledge, attitude, and practice regarding Rh incompatibility. Variables with p-values <0.05 at 95% confidence interval were considered statistically significant.
Results: Among the 299 pregnant women included in the study, 117 (39.1%) demonstrated good knowledge regarding Rh incompatibility, while 287 (96.0%) had a positive attitude and 262 (87.6%) had good practice. More than two-thirds of respondents (66.9%) did not know their husbands’ blood group and Rh status, and only 38.5% knew the appropriate preventive measures for Rh incompatibility. Educational level was significantly associated with knowledge and practice toward Rh incompatibility. Participants with a degree-level education were more likely to have good knowledge (AOR = 23.20, 95% CI: 4.67–115.25, p < 0.001) and good practice (AOR =8.46, 95% CI: 1.17–23.54, p = 0.043) compared with illiterate participants. In addition, para one mothers were more likely to have good knowledge than nulliparous mothers (AOR= 2.36, 95% CI: 1.22–4.61, p = 0.011). Urban residence was significantly associated with a positive attitude toward Rh incompatibility (COR = 7.94, 95% CI: 2.10–29.00, p = 0.002).
Conclusion: Most pregnant women attending antenatal care at SPHMMC had poor knowledge regarding Rh incompatibility despite demonstrating generally positive attitudes and good practices. Educational level was an important determinant of both knowledge and practice. Awareness regarding the causes, consequences, and prevention of Rh incompatibility remains inadequate.

Rhesus (Rh) incompatibility is a condition in which an Rh-negative mother develops anti-D antibodies after exposure to Rh-positive fetal red blood cells during pregnancy or delivery. Maternal sensitization occurs when fetal Rh-positive erythrocytes enter the maternal circulation, leading to alloimmunization and subsequent production of maternal anti-D antibodies. These antibodies can cross the placenta in subsequent pregnancies and cause hemolytic disease of the fetus and newborn (HDFN) [1]. Three conditions are required for Rh alloimmunization to occur: first, the mother must be Rh- negative and capable of producing antibodies against the Rh-D antigen; second, the fetus must be Rh-positive; and third, a sufficient amount of fetal Rh-positive red blood cells must enter the maternal circulation [1]. Rh incompatibility remains an important cause of fetal and neonatal morbidity and mortality worldwide. Hemolytic disease resulting from Rh alloimmune-zation may lead to fetal anemia, reticulocytosis, erythroblastosis, tissue hypoxia, hydrops fetalis, neonatal jaundice, and kernicterus. Hydrops fetalis, characterized by abnormal accumulation of fluid in fetal compartments, is associated with high perinatal mortality despite advances in prenatal diagnosis and treatment. In addition, kernicterus may result in severe long-term neurologic complications and death [1,2].
The introduction of postnatal anti-D immunoglobulin prophylaxis in the 1970s markedly reduced the incidence of maternal Rh alloimmunization from approximately 14% to 1–2%. Furthermore, the addition of routine antenatal anti-D prophylaxis reduced the incidence to nearly 0.1% in developed countries². Globally, the prevalence of alloimmunization among pregnant women has been reported to range from 0.4% to 2.7% [3]. In high-income countries, coordinated obstetric and neonatal care, routine blood group screening, and timely administration of anti-D immunoglobulin have substantially reduced the burden of Rh hemolytic disease [4]. However, Rh incompatibility continues to contribute significantly to perinatal morbidity and mortality in many low- and middle-income countries. Limited awareness, inadequate antenatal care utilization, lack of routine screening, and poor access to anti-D prophylaxis remain major challenges in these settings [5]. In Sub-Saharan Africa, prevention strategies for Rh alloimmunization following sensitizing events are still inadequate, and anti-D prophylaxis is inconsistently utilized³. In Ethiopia, evidence regarding awareness and preventive practices related to Rh incompatibility among pregnant women is limited, and there is a paucity of studies assessing knowledge, attitude, and practice regarding Rh incompatibility in antenatal care settings.
Perinatal Morbidity and Mortality
Maternal anti-D antibodies formed following Rh alloimmunization can cross the placenta and bind to Rh-positive fetal red blood cells, leading to hemolysis and fetal anemia. Progressive hemolysis may result in reticulocytosis, erythroblastosis, tissue hypoxia, elevated umbilical artery and venous lactate levels, and ultimately hydrops fetalis [6]. Hydrops fetalis is characterized by abnormal accumulation of fluid in two or more fetal compartments, including pleural effusion, ascites, pericardial effusion, and generalized skin edema. Despite advances in prenatal diagnostic and therapeutic interventions, hydrops fetalis remains associated with high perinatal mortality and frequently results in fetal or neonatal death before or shortly after delivery [7,8]. The neonatal manifestations of hemolytic disease of the fetus and newborn (HDFN) range from mild self-limited hemolysis with early neonatal hyperbilirubinemia to severe anemia and hydrops fetalis presenting at birth with shock and the need for emergency transfusion [9, 10]. Kernicterus, a severe neurologic complication of hyperbilirubinemia associated with Rh incompatibility, carries an estimated mortality rate of approximately 10% and morbidity rate of nearly 70%. Although kernicterus is irreversible once established, early detection and prompt treatment of neonatal hyperbilirubinemia can effectively prevent its occurrence [11,12].

Study Design and Period

An institution-based cross-sectional analytical study was conducted among pregnant women attending antenatal care ANC at St. Paul’s Hospital Millennium Medical College from October 1 to November 30, 2020.

Source Population

All pregnant women attending antenatal care (ANC) at St. Paul’s Hospital Millennium Medical College.

Study Population

All pregnant women attending ANC at St. Paul’s Hospital Millennium Medical College during the study period.

Inclusion Criteria

All pregnant women who attended ANC at St. Paul’s Hospital Millennium Medical College during the study period were included in the study.

Sample Size Determination

The sample size was determined using the single population proportion formula:

n = (Zα/2) ² × p (1 − p) / d²

Where:

n=required sample size; Zα/2 = standard normal value corresponding to a 95% confidence level (1.96); p = proportion of good knowledge regarding Rh incompatibility obtained from a previous study (23% or 0.23); d = margin of error (5% or 0.05)

Substituting the values into the formula:

n = (1.96) ² × 0.23 × (1 − 0.23) / (0.05) ²

n = 3.8416 × 0.23 × 0.77 / 0.0025

n = 0.6801 / 0.0025

n = 272

After adding a 10% non-response rate:

Final sample size = 272+ (272 × 0.10)

Final sample size = 272+ 27.2

Final sample size = 299.2 ≈ 299

Sampling Procedure

A systematic random sampling technique was employed. Based on hospital records, an average of 880 pregnant women attended antenatal care services monthly before the study period, resulting in an estimated 1,760ANC attendees during the two-month study period. The sampling interval ((k)) was determined by dividing the total study population by the sample size, yielding a value of approximately 6. Accordingly, every sixth pregnant woman attending ANC was selected for the study. The first participant was selected using the lottery method.

Study Variables

Dependent Variables

Knowledge

Attitude

Practice

Independent Variables

Age

Educational status

Residence

Parity

Religion

Marital status

Data Collection, Data Entry, and Analysis

Data were collected using an interviewer-administered structured questionnaire. Two trained midwives collected the data after receiving one day of training on the study objectives, data collection procedures, and ethical considerations. Written informed consent was obtained from each participant before the interview.
The questionnaire was adapted from previously published studies and modified to fit the local context and study objectives. It consisted of four sections: socio-demographic characteristics, knowledge regarding Rh incompatibility, attitude toward Rh incompatibility, and practices related to Rh incompatibility. Adaptation from previously validated instruments and modification by experts helped ensure the content validity of the questionnaire.
Prior to the actual data collection, a pretest was conducted on 5% of the total sample size among pregnant women attending antenatal care services outside the study setting. The pretest was used to assess the clarity, comprehensibility, and appropriateness of the questionnaire. Based on the findings of the pretest, necessary modifications were made to improve the quality of the data collection tool.
Data collection was conducted from October 1 to November 30, 2020. To maintain data quality, completed questionnaires were checked daily for completeness, consistency, and accuracy by the principal investigator and supervisors. Any identified errors or inconsistencies were corrected promptly before data entry. 
The collected data were coded and entered into the Statistical Package for Social Sciences (SPSS) version 25 for analysis. Prior to analysis, the data were cleaned and checked for completeness, consistency, missing values, and entry errors. Descriptive statistical analyses were performed, and the findings were summarized using frequencies, percentages, means, and standard deviations as appropriate. Knowledge, attitude, and practice scores were computed based on participants’ responses to the respective questionnaire items and categorized according to predefined scoring criteria.
Bivariable logistic regression analysis was performed to identify candidate variables associated with the outcome variables. Variables with a p-value <0.25 in the bivariable analysis were entered into a multivariable logistic regression model to identify independent predictors. Statistical significance was declared at a p-value <0.05 with a 95% confidence interval. The results were presented using tables, figures, and narrative summaries.

Operational Definitions

Good knowledge: Respondents who correctly answered ≥65% of the knowledge assessment questions.
Poor knowledge: Respondents who correctly answered <65>Positive attitude: Respondents who correctly answered ≥60% of the attitude assessment questions.
Negative attitude: Respondents who correctly answered <60% of the attitude assessment questions.
Good practice: Respondents who correctly answered ≥50% of the practice assessment questions.
Poor practice: Respondents who correctly answered <50>The cutoff values were adopted from previous studies [4,13].
Ethical Considerations
Ethical clearance was obtained from the Institutional Review Board (IRB) of St. Paul’s Hospital Millennium Medical College prior to data collection. A permission letter was obtained from the Research Directorate, and a letter of support was secured from the Department of Obstetrics and Gynecology. Participants were informed about the purpose, procedures, and benefits of the study before participation. Written informed consent was obtained from each participant. Confidentiality of the collected data was maintained throughout the study, and the information was used solely for research purposes.

Socio-demographic Characteristics of Respondents

A total of 299 pregnant women participated in the study. The majority of respondents, 120 (40.1%), were aged 25–29 years, while 179 (59.9%) were Orthodox Christians. Most participants, 283 (94.6%), were married, and 129 (43.1%) were nulliparous. Regarding occupation, more than half of the respondents, 155 (51.8%), were housewives. Concerning educational status, 109 (36.5%) had attended primary school, whereas only 50 (16.7%) had attained a degree or higher level of education. The majority of respondents, 278 (93.0%), were urban residents.

Table 1: Shows socio-demographic characteristics of mothers (N=299)

Knowledge of Participants Toward Rh Incompatibility

Among the 299 respondents, 182 (60.9%) had poor knowledge, while 117 (39.1%) had good knowledge regarding Rh incompatibility based on the predefined operational definitions. More than half of the respondents, 165 (55.2%), did not know that differences in blood group and Rh status between partners could lead to miscarriage or stillbirth. In addition, 200 (66.9%) respondents did not know their husbands’ blood group and Rh status. Regarding precautions to be taken when a mother is Rh-negative, 184 (61.5%) respondents were unaware of the appropriate preventive measures.

Table 2: Frequency and percent distribution of mothers’ response for each knowledge question towards Rh incompatibility, (N=299)

 Attitude of Participants Toward Rh Incompatibility

Regarding attitudes toward Rh incompatibility, 287 (96.0%) of the 299 respondents had a positive attitude, while 12 (4.0%) had a negative attitude based on the predefined operational definitions. Only 20 (6.7%) respondents considered maternal–fetal incompatibility testing to be embarrassing. Similarly, 20 (6.7%) respondents reported fear of being Rh incompatible with their fetus.

Table 3: Frequency distributions of participants’ attitude towards Rh incompatibility among mothers attending their ANC at SPHMMC, Addis Ababa, Ethiopia (N=299)

Practice of Participants Toward Rh Incompatibility

Among the 299 pregnant women included in the study, 262 (87.6%) demonstrated good practice, while 37 (12.4%) had poor practice toward Rh incompatibility based on the predefined operational definitions. The majority of respondents, 285 (95.3%), had checked their blood group and Rh status. Of the 285 respondents who had ever checked their blood group and Rh status, 211 (74.0%) underwent testing during pregnancy, 39 (13.7%) before pregnancy, and 35 (12.3%) both before and during pregnancy. Regarding preventive measures, 242 (80.9%) respondents correctly stated that anti-D immunoglobulin should be administered to prevent Rh incompatibility. In addition, 235 (78.6%) respondents reported that they would seek medical care at a health facility if they experienced recurrent miscarriage or stillbirth related to Rh incompatibility. However, 29 (9.7%) respondents preferred seeking help from religious institutions, 27 (9.0%) from traditional healers, and 8 (2.7%) stated that they would not seek help because they believed the condition was determined by God.

Table 4: Frequency distributions of participants’ answer for each practice questions towards Rh incompatibility among mothers attending their ANC at SPHMMC, Addis Ababa, Ethiopia (N=299)

Factors Associated with Participants’ Knowledge Toward Rh Incompatibility

In the bivariate analysis, occupational status, educational level, parity, and residence were associated with knowledge toward Rh incompatibility at a p-value of <0.25 and were included in the multivariable logistic regression analysis.
In the multivariable logistic regression analysis, only educational level and parity remained significantly associated with knowledge toward Rh incompatibility. Participants with a degree-level education were 23.2 times more likely to have good knowledge regarding Rh incompatibility compared with illiterate participants (AOR = 23.195, 95% CI: 4.668–115.251, p < 0.001). Similarly, participants with diploma-level education showed a trend toward better knowledge compared with illiterate participants (AOR = 4.65, 95% CI: 0.959–22.64, p = 0.05). In addition, para one mothers were 2.36 times more likely to have good knowledge regarding Rh incompatibility compared with nulliparous mothers (AOR = 2.36, 95% CI: 1.215–4.610, p = 0.011). Occupational status and residence were not significantly associated with knowledge toward Rh incompatibility in the multivariable logistic regression analysis. 

Table 5: Bivariate and multivariate logistic regression of factors associated with level of knowledge of participants towards Rh incompatibility

Key; ref= reference category, *P≤0.25, **P≤0.05; Abbreviation: COR=crude odds ratio, AOR= adjusted odds ratio, CI: confidence interval

Factors Associated with Participants' Attitude Toward Rh Incompatibility

In the bivariate logistic regression analysis, place of residence was significantly associated with attitude toward Rh incompatibility. Participants residing in urban areas had 7.94 times higher odds of having a positive attitude toward Rh incompatibility than those residing in rural areas (COR=7.94, 95%CI: 2.1–29.0, p=0.002).

Table 6: Logistic regression analysis of factors associated with attitude toward Rh incompatibility

Key; ref: reference category; Abbreviations; COR= crude odds ratio; AOR= adjusted odds ratio; CI = confidence interval.

Factors Associated with Participants' Practice Toward Rh Incompatibility

In the bivariate analysis, occupational status, educational level, and place of residence were associated with practice toward Rh incompatibility at a p-value of <0.25 and were included in the multivariable logistic regression analysis.
In the multivariable logistic regression analysis, only educational level remained significantly associated with practice toward Rh incompatibility. Participants with a degree-level education had 8.46 times higher odds of demonstrating good practice toward Rh incompatibility compared with illiterate participants (AOR=8.455, 95% CI: 1.17–23.54, p=0.043). Occupational status and place of residence were not significantly associated with practice toward Rh incompatibility in the multivariable logistic regression analysis.

Table 7: Bivariate and multivariate logistic regression of factors associated with level of practice of participants towards Rh incompatibility

Key; ref= reference category, *P≤0.25, **P≤0.05; Abbreviation: COR=crude odds ratio, AOR= adjusted odds ratio, CI: confidence interval

This study assessed the knowledge, attitude, and practice of pregnant women attending antenatal care regarding Rh incompatibility. The study found that only 39.1% of participants had good knowledge, while 96.0% demonstrated a positive attitude and 87.6% exhibited good practice toward Rh incompatibility. Educational status and parity were significantly associated with knowledge, whereas urban residence was associated with a positive attitude toward Rh incompatibility.
The proportion of participants with good knowledge (39.1%) was higher than reports from Kampala International University Teaching Hospital (17.5%) and Ambo University (23.58%), and was similar to findings from Nigeria (39%). Awareness of Rh incompatibility and Rh factor (39.8%) was also comparable to that reported in Kampala (37.5%). However, knowledge regarding preventive measures was lower than that observed among Ambo University students (61.6%). Similarly, only 5.7% of participants correctly identified Rh incompatibility as occurring when a Rh-negative mother carries an Rh-positive fetus, which was lower than the 12.5% reported in Kampala. More than half of respondents (55.2%) were unaware that Rh incompatibility could result in miscarriage or stillbirth, and only one-third (33.3%) knew their husbands’ blood group and Rh status, indicating important gaps in awareness. The prevalence of positive attitude in the present study (96.0%) was substantially higher than that reported by Ambo University (13.29%). Practice-related findings were generally comparable with those from Kampala, where approximately 75% of participants reported seeking medical care for complications related to Rh incompatibility.
The association between higher educational status and better knowledge may reflect greater exposure to health information and improved understanding of antenatal counseling messages. Similarly, para one mothers may have acquired knowledge through previous interactions with healthcare providers during earlier pregnancies. The significant association between higher educational status and better practice may reflect greater access to health information, improved understanding of preventive measures, and increased utilization of maternal healthcare services among educated women. The high level of positive attitude despite relatively low knowledge may suggest that pregnant women generally trust healthcare recommendations even when their understanding of Rh incompatibility is limited. Furthermore, the study was conducted at a tertiary referral hospital where counseling and health education services may contribute to favorable attitudes toward prevention and treatment. Urban residents were more likely to have a positive attitude than rural residents, possibly reflecting better access to healthcare services and health information. The wide confidence intervals observed for some variables may indicate limited precision resulting from the relatively small sample size and the low number of highly educated participants.
The findings highlight the need for strengthened health education on Rh incompatibility during antenatal care visits. ANC providers should routinely educate pregnant women about Rh blood group status, the consequences of Rh incompatibility, and the importance of anti-D immunoglobulin prophylaxis. Hospitals should ensure consistent counseling and encourage partner blood group and Rh status testing. Health authorities should develop community-based awareness programs targeting rural and less educated women, who may have lower access to health information. Furthermore, approximately one-fifth of respondents reported that they would seek help from religious institutions, traditional healers, or would not seek medical care if faced with complications related to Rh incompatibility. This underscores the need for community education and improved linkage to formal healthcare services. Improving knowledge of Rh incompatibility could contribute to earlier identification of at-risk pregnancies and reduce preventable adverse maternal and neonatal outcomes. The findings should be interpreted cautiously because the majority of participants were urban residents and may not be representative of pregnant women living in rural Ethiopia.
Recommendations
1. Healthcare providers working in antenatal care clinics, including midwives, interns, residents, and obstetricians, should provide regular counseling and health education regarding Rh incompatibility, its causes, complications, preventive measures, and the importance of timely anti-D immunoglobulin administration when indicated. 
2. St. Paul’s Hospital Millennium Medical College should strengthen awareness creation and health education programs on Rh incompatibility among pregnant women attending antenatal care services.
3. Government health authorities, media organizations, and other stakeholders should promote community-based awareness regarding blood groups, Rh status, and the prevention of Rh incompatibility-related complications, particularly among rural populations.
4. Further multicenter studies involving diverse populations should be conducted to improve the generalizability of findings regarding knowledge, attitude, and practice toward Rh incompatibility in Ethiopia.
Limitation of the study
This study has several limitations that should be considered when interpreting the findings. First, the study was conducted at a single tertiary hospital, St. Paul’s Hospital Millennium Medical College, which may limit the generalizability of the findings to pregnant women attending other health facilities in Ethiopia. Second, the majority of participants were urban residents, which may have introduced urban bias and limited the applicability of the findings to rural populations, where access to health information and maternal healthcare services may differ substantially.
Third, knowledge, attitude, and practice data were collected through self-reported responses, making the study susceptible to recall bias and social desirability bias. This may have resulted in overestimation of positive attitudes and good practices regarding Rh incompatibility. Fourth, the cross-sectional nature of the study limits the ability to establish temporal relationships or causal inferences between the identified associated factors and the outcome variables. 
In addition, some categories of explanatory variables contained relatively small numbers of participants, which may have reduced the precision of the estimates and contributed to wider confidence intervals. Furthermore, although the questionnaire was adapted from previously published studies and pretested before data collection, formal psychometric evaluation was not performed.
Finally, some potentially important factors related to knowledge, attitude, and practice toward Rh incompatibility may not have been assessed or controlled for in the analysis.
Therefore, residual confounding cannot be excluded.

Most pregnant women attending antenatal care at St. Paul’s Hospital Millennium Medical College had poor knowledge regarding Rh incompatibility. Only a small proportion correctly identified that Rh incompatibility occurs when an Rh-negative mother carries an Rh-positive fetus. Educational status and parity were significantly associated with knowledge. Urban residence was significantly associated with attitude, while educational status was significantly associated with practice toward Rh incompatibility. Despite the low level of knowledge, most respondents demonstrated positive attitudes and good practices toward Rh incompatibility. These findings highlight important gaps in awareness and underscore the need for strengthened health education and counseling during antenatal care visits, particularly among less educated and rural women, to improve awareness and help prevent Rh incompatibility-related adverse pregnancy outcomes.

Conflict of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding
This study was financially supported by St. Paul Millenium Medical College.

Acknowledgments

We would like to acknowledge St. Paul's Hospital Millennium Medical College for providing institutional support for this research.
We also thank the administration and staff of St. Paul's Hospital Millennium Medical College for their cooperation during the study period. Our appreciation extends to the data collectors for their commitment and diligence. 
Finally, we are grateful to all study participants for their willingness to take part in this.