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International Journal of Fertility & Sterility logoLink to International Journal of Fertility & Sterility
. 2025 Jan 5;19(1):96–103. doi: 10.22074/ijfs.2024.2016523.1581

The Impact of Embryo Donation Technology on Child Psychological Adjustment and Parenting Styles: A Historical Cohort Study

Mahya Kaveh 1, Seyed Hamze Hosseini 2,*, Hamid Sharif-Nia 3, Sepideh Peivandi 4,*
PMCID: PMC11744203  PMID: 39827397

Abstract

Background:

Despite the remarkable advancements in the use of embryo donation, concerns have arisen regarding its potential effects on the psychological well-being of children conceived through this assisted reproductive technology and their parent-child relationships. The aim of the study is to evaluate children's psychological adjustment and parenting style in families with donor-conceived children and compare them with the normal population.

Materials and Methods:

A historical cohort study was conducted to assess the psychological adjustment of 31 children aged 3 to 7 years born via embryo donation and to compare the results with those of 30 age-matched children from families who conceived naturally using the Strengths and Difficulties Questionnaire. The sample size was determined using G power, and the samples were selected using a convenient sampling method. Parenting styles within these families were also evaluated using the Baumrind Parenting Styles Inventory through clinical interviews.

Results:

Although 8 out of 31 children born through embryo donation (25.8%) and 3 out of 30 children from families with natural conception exhibited psychological maladjustment, this difference was not statistically significant (P=0.249). Furthermore, there were no significant differences in parenting styles between the two groups (P values for permissive, authoritarian, and authoritative parenting styles were 0.424, 0.656, and 0.219, respectively).

Conclusion:

The lack of genetic parent-child relationships does not seem to be a dominant factor affecting the psychological adjustment of children or parenting styles.

Keywords: Adaptation, Child, Donation, Embryo, Psychology

Introduction

Infertility is a widespread issue globally, characterized by the inability to conceive after one year of unprotected sexual intercourse (1). In recent years, infertility has become increasingly prevalent, affecting approximately 15% of the global population (2). Assisted reproductive techniques, which began with in vitro fertilization in 1978, have made remarkable progress in treating infertility (3). Embryo donation is one such technique that has gained popularity in recent years. This method involves transferring an embryo from a different couple to the uterus of the infertile couple (4). In Iran, the prevalence of infertility varies according to different studies, ranging from 5 to 17% (5-7).

In recent years, the use of assisted reproductive techniques in Iran has seen significant growth, given the high prevalence of infertility and recent government policies aimed at boosting population growth (8). Embryo donation has been practised in Iran for nearly two decades, and its therapeutic use was approved by the Islamic Consultative Assembly in 2003 (9, 10). Embryo donation is a unique assisted reproductive technique because it does not involve a genetic connection between the child and the intended parents. As this method becomes more prevalent, concerns have arisen regarding the impact on family dynamics and the psychological well-being of children born from this type of pregnancy (11). It has been hypothesized that children born through ART may have lower cognitive, visualmotor, and language perception abilities compared to naturally conceived children (12). Weaknesses in these abilities can lead to weaker psychological adaptation in such children.

On the other hand, there is also the belief that parents who have no genetic connection with their children may become more hostile or distant, potentially leading to negative outcomes in the development and well-being of the children (13). Historically, concerns about nongenetic parents investing less in their offspring have been rooted in evolutionary psychology arguments (14). Similarly; in adoption studies where there is no genetic connection between the child and the parent, it is hypothesized that parents may not have sufficient confidence in their ability to love and express affection towards their offspring (15).

Additionally, there are reports indicating lower levels of mother-child interaction in families who conceive through egg donation compared to those who conceive naturally (16). Based on these findings, the dynamics of parent-child interaction and the child’s attachment style in such families are crucial. In such cases, attachment style is related to the quality of parenting, especially in terms of parental responsiveness and emotional warmth (17). Therefore, the absence of a genetic connection between parents and children may impact parental feelings towards the child and, as a result, the quality of parenting. This could potentially expose such children to harm (18).

Research on families with children conceived through embryo donation, particularly regarding the psychological adjustment of children and parenting styles, has been limited. Previous studies have primarily focused on families where at least one parent has a genetic connection to the child (19). Prominent differences in the function of these families in terms of parent-child relationships and psychological adjustment of children have not been found (11). For example, these authors discovered that the psychological adjustment of children conceived through egg and sperm donation did not differ significantly from that of other groups, including normally conceived children, at ages 3, 7, and 10 (20). In contrast, in 2009, Shelton reported that fathers of children born through egg donation reported higher levels of behavioral problems in their children compared to families using other assisted reproductive techniques and natural families (21). Many families choose to keep information confidential, which can complicate matters (22). The belief is that maintaining secrecy can be detrimental to the psychological well-being of these children (13), as secrecy can render communication patterns within the family less ideal and negatively affect intergenerational relationships (23). Moreover, studies have demonstrated that mothers who chose not to disclose information about their child’s genetic nature, specifically those conceived through sperm donation, exhibited less positive interactions with their offspring (22). In Iran, embryo donation and the role of families with donor-conceived children are significant issues due to the prevailing traditional context. A survey of infertile couples found that one-third of them oppose embryo donation as a treatment method. Additionally, 21% of women and 14% of men perceive this treatment method as inconsistent with religious standards (24). On the other hand, in a 2007 study conducted by MacCallum et al. (19) on 21 embryo donation families, 28 adoptive families, and 30 in vitro fertilization families in the field of psychological adjustment and parent-child relationships, no significant difference in psychological adjustment was observed among these children. However, the lack of a natural comparison group and the small number of embryo donation families may reduce confidence in the results. Therefore, due to the lack of published research that specifically deals with the issue of the function of embryo donation families and the increase in the use of embryo donation in Iran and the aforementioned reasons that raise concerns about the potential impact on the psychological adjustment of children born through this method (25-27), we decided to investigate the psychological adjustment of three to seven-year-old children and the parenting styles of families with donor-conceived children and compare them with the normal population during a historical cohort study.

Materials and Methods

Participants

Embryo donation families

In this historical cohort study, families with at least one child between the ages of 3 and 7 resulting from pregnancy through embryo donation were eligible for the embryo donation group. The families were recruited from the infertility treatment clinic of Imam Khomeini Educational Hospital in Sari, which is affiliated with Mazandaran University of Medical Sciences. A total of 39 families were invited to participate in this study via telephone contact, and 34 of them consented to take part. All of the children in this group lived with both of their parents. Out of these families, 30 were single-child families, and 4 were families with twins. In the twin families, information was randomly obtained for one of the children. The study process excluded families in which the child or parents had a congenital abnormality, chronic physical illness, or major psychiatric disorder. Some families chose not to participate in the study due to privacy concerns.

Natural families

Thirty-four families were randomly selected from those attending the outpatient clinic of the same aforementioned hospital. All of these families had at least one child aged 3 to 7 years who was conceived naturally. The families were similar to the embryo donation families, except for two cases of divorce and the father’s death; these children also lived with both of their biological parents. All of the children in the were singletons, and neither they nor their parents had any chronic physical or psychiatric illnesses or congenital abnormalities.

Recruitment

The effect size was calculated based on the mean and standard deviation of MacCallum et al.'s (19) study SDQ scores. Sample size estimation was performed using Gpower 3.1.9.6 Sample Size Estimation Software, taking into account the effect size, a significance level of less than 0.05, and a test power of 80% for two domains. Sampling was performed by a convenient sampling method. The coordinator of the infertility clinic, which is affiliated with Imam Khomeini Educational Hospital, initially contacted families via telephone to invite them to participate in the study. Families who expressed interest were provided with information about the study’s title, objectives, and the research team. After obtaining informed consent, demographic information was collected from all participants, who were mothers.

Tools

Psychological adjustment of children

Strengths and Difficulties questionnaire

Goodman’s questionnaire, designed in 1997 in the United Kingdom, is based on ICD-10 diagnostic criteria for ages 3 to 16 years (28). Research conducted by Ghanizadeh et al. (29) in Iran found that the Persian version of this tool has a Cronbach’s alpha of 0.73, a sensitivity of 90%, and a specificity of 67%. The tool’s parent form of this tool, completed by parents, comprises 25 questions that measure four subscales of clinical symptoms, including emotional problems, conduct problems, hyperactivity-inattention problems, peer relationship problems, and a prosocial behavior subscale. A score is assigned to each subscale, of these subscales, and the total behavioral difficulty score is calculated by summing the scores from the four clinical symptom subscales. To evaluate each option, assign a score of 0, 1, or 2, indicating complete correctness, partial correctness, or incorrectness, respectively. After calculating scores for each clinical symptom subscale based on defined normal, borderline, and abnormal cutoff points, psychological compatibility in children is determined. A total score of 0 to 13 is considered normal, 14 to 16 is borderline, and 17 to 40 is abnormal. Psychological adjustment in children is also determined within each subscale. The scoring range for emotional symptoms is 0-3 for normal, 4 for borderline, and 5-10 for abnormal. Conduct symptoms have a normal range of 0-2, borderline at 3, and abnormal at 4-10. Hyperactivity-inattention symptoms have a normal range of 0-5, borderline at 6, and abnormal at 7-10. Peer relationship problems have a normal range of 0-2, borderline at 3, and abnormal at 4-10. Prosocial behaviors are considered normal when scores range from 6 to 10, borderline when the score is 5, and abnormal when the score is between 0 and 4.

Baumrind parenting styles inventory

Baumrind (1991) designed a tool consisting of 30 items, with each 10 items referring to one of the parenting styles: authoritative, authoritarian, and permissive (30). Respondents can express their opinion about each item by selecting from a 5-point scale (completely agree, somewhat agree, neutral, somewhat disagree, completely disagree), which is scored from 0 to 4.

Gunty and Bury (31) reported the reliability of this instrument. The permissive style was rated at 0.81 and 0.77 for mothers and fathers, respectively. The authoritarian style received ratings of 0.86 and 0.85, while the authoritative style received ratings of 0.78 and 0.88. The validity of the questionnaire was assessed through discriminant validity. The results showed that the authoritarian mother had an inverse relationship with the permissive mother (0.38) and the authoritative mother (0.48). Similarly, the authoritarian father had an inverse relationship with the permissive father (0.50) and the authoritative father (0.52).

Moradian et al. (32) research determined the reliability of the Persian-translated questionnaire as follows: 0.69 for permissive parenting style, 0.73 for authoritarian style, and 0.77 for authoritative style, using Cronbach’s alpha method.

Statistical analysis

Continuous variables were expressed as mean ± standard deviation (SD), while categorical variables were expressed as numbers (percentages). The study utilized three statistical methods for analysis: chi-square test, independent t test, and Pearson’s correlation coefficient. The chi-square test was used to determine the independence of two categorical variables, while the independent t test was used to compare the means of two groups. The Pearson correlation coefficient is utilized to assess if there is a significant correlation between two continuous variables. The significance level for all tests was set at less than a <0.05. The data's normal distribution was assessed by conducting the Shapiro-Wilk test. Data was analyzed using Statistical Package for Social Sciences version 26.0 software for Windows (IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp., USA).

Ethical considerations

After explaining the study objectives and assuring participants of the confidentiality of their information, we obtained their informed consent. The Ethics Committee of Mazandaran University of Medical Sciences approved this study under the code of ethics IR.MAZUMS. REC.1401.344, and it was conducted in an affiliated hospital of the university. Due to social restrictions imposed by the COVID-19 pandemic, clinical interviews were conducted by a psychiatrist via telephone.

Results

Sociodemographic data

Table 1 presents the sociodemographic information of the study sample based on the Chi-square and Fisher’s exact tests. Data from three families in the donation group and four families in the natural conception group were not included in the statistical analysis due to their withdrawal of consent.

The research sample consisted of 15 girls (48.4%) and 16 boys (51.6%) in the embryo donation children group, and 18 girls (60%) and 12 boys (40%) in the natural conception children group (P=0.257). The children's mean age in the donation group was 1.33 ± 5.04 years, and in the natural conception group, it was 0.96 ± 5.23 years (P=0.077). Table 1 shows that the parents' mean age did not differ significantly between the two groups.

In the donation group, 8 fathers (25.8%) had education levels lower than a diploma, 20 fathers (64.5%) had a diploma to bachelor's degree education, and 3 fathers (9.7%) had education levels higher than a bachelor’s degree. In contrast, in the natural conception group, 4 fathers (12.9%) had education levels lower than a diploma, 21 fathers (67.7%) had a diploma to bachelor’s degree education, and 6 fathers (19.4%) had education levels higher than a bachelor’s degree (P=0.427). The values for donor mothers were 5 (16.7%), 19 (63.3%), and 6 (20%), respectively. In comparison, the values for mothers in the natural group were 3 (10%), 22 (73.3%), and 5 (16.7%), respectively (P=0.887).

In the donation group, 93.5% of fathers and 41.9% of mothers were employed, while in the natural conception group, 90% of fathers and 60% of mothers were employed (P=0.668 for fathers and P=0.124 for mothers). In terms of income satisfaction, 45.2% of families who used donation and 26.7% of families who conceived naturally reported complete satisfaction. Additionally, 32.5% of donation families and 33.5% of natural conception families reported relative satisfaction. On the other hand, 22.6% of donation parents and 40% of natural conception parents reported dissatisfaction with their income levels (P=0.230).

Table 1.

Demographic information and its comparison in two groups based on Chi-square and Fisher’s tests


Groups/Variables Embryo donation Natural conception P value

Gender 0.257
Male 16 (51.6) 12 (40)
Female 15 (48.4) 18 (60)
Father’s education 0.427
Less than diploma 8 (25.8) 4 (12.9)
Diploma to bachelor 20 (64.5) 21 (67.7)
Master’s or higher 3 (9.7) 6 (19.4)
Mother’s education 0.887
Less than diploma 5 (16.7) 3 (10)
Diploma to bachelor 19 (63.3) 22 (73.3)
Master’s or higher 6 (20) 5 (16.7)
Marital status 0.344
Divorce 0 (0) 1 (3.3)
Married 31 (100) 28 (93.3)
Father’s death 0 (0) 1 (3.3)
Father’s job 0.668
Employed 29 (93.5) 27 (90)
Unemployed 2 (6.5) 2 (6.7)
Mother’s job 0.124
Employed 13 (41.9) 18 (60)
Unemployed 18 (58.1) 12 (40)
Income (million rials) 0.366
<50 9 (29) 4 (13.3)
50-100 8 (25.8) 7 (23.3)
100-200 5 (16.1) 9 (30)
>200 9 (29) 10 (13.3)
Addiction history 0.746
Father 1 (3.2) 1 (3.3)
Mother 0 (0) 0 (0)
Coefficient of dispersion
Child age (Y) 5.04 ± 1.33 5.23 ± 0.96 0.077
Mother age (Y) 38.06± 8.17 35.70 ± 4.98 0.089
Father age (Y) 40.65 ± 8.86 39.33 ± 6.60 0.521

Data are presented as mean ± SD or n (%).

There were no significant differences between the natural conception group and the donation group in terms of parental divorce or death (P=0.344). Addiction issues were present in one father from each group, but none of the mothers in either group had addiction issues (P=0.746).

Psychological adjustment of children

Table 2 presents a descriptive analysis of parental reports of psychological adjustment in children from two groups: embryo donation and natural conception, based on the SDQ questionnaire. The mean score of the SDQ questionnaire in the donation group (11.84 ± 5.41) was slightly higher than in the natural conception group (10.13 ± 5.07), although this difference was not statistically significant (P=0.412).

Overall, there were no significant differences in the SDQ questionnaire scores between children in the embryo donation group and those in the natural conception group. This applies to both the total score and all subcategories, including emotional problems, conduct problems, hyperactivity, peer relationship problems, and social behavior deficits.

As stated earlier, the psychological adjustment of each child and its subcategories, based on the SDQ questionnaire scores, is defined as normal, borderline, or abnormal. Table 3 reports that 8 children in the donation group and 3 children in the natural conception group had abnormal psychological adjustment. Additionally, 22 children in the donation group and 25 children in the natural conception group had normal psychological adjustment. There was no statistically significant difference in borderline psychological adjustment between the donation group and the natural conception group, with one child in the former and two children in the latter exhibiting such symptoms (P=0.249).

Additionally, there were no significant differences in the prevalence of emotional problems (P=0.221), conduct problems (P=0.490), hyperactivity (P=0.241), peer relationship problems (P=0.163), and social behavior deficits (P=0.322) between the two groups of children born from embryo donation and those conceived naturally.

Table 2.

Descriptive analysis of SDQ scores and subscales based on t test in the donor and natural conception groups


Groups/Psychological adjustment Donor conception Natural conception P value Cohen’s d

SDQ scores 11.84 ± 5.41 10.13 ± 5.07 0.412 0.326
Emotional problems 2.39 ± 1.80 2.23 ± 1.75 0.539 0.090
Conduct problems 2.49 ± 2.04 2.60 ± 2.19 0.898 0.051
ADHD problems 4.19 ± 2.07 3.63 ± 2.04 0.755 0.272
Peer problems 2.39 ± 1.80 1.70 ± 1.51 0.217 0.415
Social behaviors 6.71 ± 2.47 7.30 ± 1.82 0.101 0.271

Data are presented as mean ± SD.

Table 3.

Relationship and frequency of psychological adjustment in emotional, conduct, hyperactivity, peer problems, and prosocial domains of children based on the Chi-Square test in donation and natural groups categorized as normal, borderline, and abnormal


Group/Psychological Adjustment Donor conception Natural conception P value

Psychological adjustment 0.249
Normal 22 (71) 25 (83.3)
Borderline 1 (3.2) 2 (6.7)
Abnormal 8 (25.8) 3 (10)
Emotional problems 0.221
Normal 21 (67.7) 23 (76.7)
Borderline 8 (25.8) 3 (10)
Abnormal 2 (6.5) 4 (13.3)
Conduct problems 0.490
Normal 15 (84.4) 19 (63.3)
Borderline 15 (16.1) 3 (10)
Abnormal 11 (35.5) 8 (26.7)
ADHD problems 0.241
Normal 22 (71) 25 (83.3)
Borderline 5 (16.1) 1 (3.3)
Abnormal 4 (12.9) 4 (13.4)
Peer problems 0.163
Normal 17 (54.8) 22 (73.3)
Borderline 15 (16.1) 5 (16.7)
Abnormal 9 (29.0) 3 (10)
Social behaviors 0.322
Normal 23 (74.2) 25 (83.3)
Borderline 2 (6.5) 3 (10)
Abnormal 6 (19.3) 2 (6.7)

Data are presented as n (%).

Parenting style

Table 4 compares the mean scores of various parenting styles in donation and natural conception families. The results indicate that although there were differences in the mean scores of parenting styles between the two groups, these differences were not statistically significant.

Table 4.

Investigation of the relationship and mean scores of different parenting styles based on t test in individuals participating in the two natural and donor groups


Pregnancy type/Parenting style Donor conception Natural conception P value

Permissive 16.42 ± 4.60 17.50 ± 5.82 0.424
Authoritarian 11.74 ± 7.33 10.97 ± 6.02 0.656
Authoritative 30.10 ± 7.09 31.93 ± 3.76 0.219

Data are presented as mean ± SD.

The Pearson correlation coefficients showed significant associations between the SDQ scores and parenting styles. Specifically, a positive and significant correlation was found between the SDQ score and the authoritarian parenting style (r=0.377, P<0.001), while a negative and significant correlation was found between the SDQ score and the authoritative parenting style (r=-0.402, P<0.001). However, no significant correlation was found between the SDQ score and the permissive parenting style (r=- 0.087, P=0.505).

Additionally, none of the 18 families in the embryo donation group had previously disclosed their children's biological origins. When asked about their plans to inform their children in the future, 5 families (27.7%) expressed a willingness to do so, 10 families (55.5%) intended not to inform their children, and 3 families (16.6%) had not yet made a decision.

These findings indicate that parents have different attitudes towards disclosing their children’s biological origins within the embryo donation group.

Discussion

According to our findings, this study is the first to compare the psychological adjustment of children and parenting styles in families who have become parents through embryo donation with natural conception families. The results did not reveal a significant difference in the psychological adjustment of children resulting from embryo donation and children born through natural conception. Thus, the hypothesis that children born through embryo donation have weaker psychological adjustment than naturally conceived children is unfounded. The sole other study conducted on families who received embryo donations found that these children did not have a higher risk of psychological maladjustment when compared to children who were adopted or conceived through in vitro fertilization (IVF). However, this study did not make a direct comparison with families who conceived naturally (19). Our findings are consistent with previous research (20-22, 33).

Additionally, our study found no association between parenting styles (permissive, authoritarian, and authoritative) and the type of family (embryo donation or natural conception). Previous research has also reported no significant differences in parenting styles among embryo donation families, adoptive families, or families who have undergone IVF (19, 33).

Our results are consistent with previous studies, indicating that children from families with authoritarian parenting styles exhibit weaker psychological adjustment (34). Conversely, children from families with authoritative parenting styles demonstrate better psychological adjustment.

In conclusion, these findings suggest that the lack of a genetic parent-child relationship does not significantly impact the psychological adjustment of children. This aligns with Golombok et al'. (20) suggestion that the maternal-child relationship during pregnancy can be just as influential on children’s psychological well-being, and the absence of a genetic link is not detrimental. However, it is important to consider some notable points in this study. For example, although not statistically significant, children in the embryo donation group had higher average psychological adjustment scores than those in the natural conception group. Furthermore, 25.8% of children born through embryo donation and 10% of natural conception families exhibited abnormal psychological adjustment. Despite the noticeable difference, it was not statistically significant. However, It is important to recognize that societal stigma surrounding embryo donation as a fertility treatment may lead to less than truthful parental responses (24). MacCallum et al. (19) discovered that families who received embryos through donation displayed more defensive responses than families who had children through adoption and IVF. Like our study, they also used the SDQ questionnaire to assess children’s psychological adjustment. Therefore, future studies that use observational assessment tools may produce more reliable results.

One notable finding from the recent research is that none of the 18 donor families who were surveyed had disclosed the genetic origin of their children. Only 5 out of the 18 families (27.7%) were willing to share this information in the future. MacCallum et al.' (19) research revealed that only 2 out of 21 families (9%) who donated embryos had informed their 2-5-year-old children about their genetic origin. Additionally, 5 cases (24%) were willing to disclose in the future. This rate was significantly lower compared to IVF and adoptive families. Given that the rate of disclosure is higher in families with sperm or egg donation (35), it is worth considering whether the attitudes of embryo recipient parents, who have no genetic relationship with their child, differ from those of sperm or egg recipient parents who have a genetic connection with one of the parents. Moreover, it prompts us to explore whether the absence of a genetic relationship can be perceived as a greater threat to the parent-child relationship.

One limitation of this research is the use of questionnaires. Future studies should consider employing observational tools and assessing parenting styles and children’s psychological adjustment in their living environment. Additionally, the stigma surrounding embryo donation may lead to defensive responses from families, which can be mitigated by using observational assessment methods. To increase the reliability of results, it is recommended to use a larger sample size in future studies. This is due to the limitations of our current study, which had a small sample size.

Another important point to consider for future research is how children’s attitudes develop as they age after learning about the conception process. It is unclear whether this factor will have an impact on their psychological well-being.

Conclusion

This study suggests that the psychological adjustment of donor-conceived children is not significantly impacted by the absence of a genetic connection between parents and children. The study found no significant differences in parenting styles across different family types. It emphasizes the importance of enhancing communication and transparency in embryo donation. Furthermore, future studies could benefit from using observational methods to evaluate parenting styles and child adjustment, which would help reduce potential biases.

The strength of our study titled lies in several aspects: Randomized sample: Although not explicitly stated that it's a randomized controlled trial, the use of a sample may provide insights into potential trends and patterns associated with embryo donation technology.

Standardized assessment tools: Utilizing validated instruments such as the Strengths and Difficulties Questionnaire allows for objective measurement of child psychological adjustment and comparison across different studies.

Comparative data: Providing comparisons between embryo donation and natural conception groups helps identify possible effects of embryo donation on family dynamics and child development.

However, as noted above, some limitations also exist: Sample size: While the specific number of participants is not provided in the summary, smaller sample sizes can limit the ability to detect subtle differences and make robust conclusions.

Self-report measures: Relying solely on self-reports by parents could introduce bias due to subjectivity and memory errors.

Single-center study: Results might not be fully representative of broader populations if the study only took place at a single center.

Acknowledgements

The authors are grateful to the clients who trusted us and participated in this research project. The authors declare no conflict of interest and state that the project was funded by Mazandaran University of Medical Sciences.

Authors’ Contributions

M.K.; Designed the project, Conducted participant interviews, Collected study data, and Wrote the manuscript. S.H.H.; Designed and Supervised the project. S.P.; Contributed to study design and Sample preparation. H.Sh.- N.; Contributed to study design, Statistical analysis, and Manuscript review. All authors read and approved the final manuscript.

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