Parental motivations for and adaptation to trio-exome sequencing in a prospective prenatal testing cohort: Beyond the diagnosis

Abstract

Purpose:

To understand motivations for and parental interpretation of results from trio-exome sequencing (ES) for fetal anomalies with a negative standard genetic diagnosis.

Methods:

Analysis of an ongoing, prospective prenatal trio-ES study of pregnancies with ultrasound-identified congenital anomalies and lack of a standard genetic diagnosis. After determination of pregnancy disposition, participants completed questionnaires and a semi-structured interview pre- and post-sequencing. Interviews were analyzed using a constructivist grounded theory methodology to identify themes. Associations between themes and ES result were also examined.

Results:

One hundred twenty-six trios have been sequenced. Of those, 45 (36%) resulted in fetal diagnosis. One hundred twenty-five women completed pre-sequencing surveys, and 91 women completed post-sequencing surveys. The main themes identified include (1) variable reasons to pursue ES, (2) limited expectations but high hopes from ES, (3) parental adaptation to uncertain results, (4) impact on personal health and reproduction, and (5) gratitude for the process.

Conclusion:

Participants pursued ES for various reasons, most often to identify a diagnosis and guide reproduction. Post-sequencing, most participants described the process, their interpretation of results, and the impact of receiving the results. Less frequently, but of most concern, participants expressed anxiety about testing and implications for themselves, relationships, and other family members, thus identifying an area of high need for additional support among patients undergoing prenatal ES.

1 |. INTRODUCTION

Congenital anomalies are a significant contributor to perinatal mortality, affecting 2%–5% of all pregnancies and accounting for 20% of all perinatal deaths.¹ Recent advances in prenatal diagnostic techniques, such as exome sequencing (ES), have sought to provide parents and maternal–fetal care teams with additional information. Such information is particularly useful when fetal anomalies cannot be explained by standard diagnostic test results.^2–6 Current data suggest that under such circumstances, diagnostic yield is 15%–19% in the setting of multisystem anomalies.^2,3 Given the recent adaptation of ES in the prenatal setting, implementation has primarily focused on resolving diagnostic yield and variant interpretation challenges; however, there are also significant ethical and counseling challenges that require further investigation.^7–9

Prior studies assessing the use of ES suggest that testing, particularly when results are available, helps patients reach closure and decide on next steps in diagnosis and care.¹⁰ However, data from our ongoing study show that such benefits may not extend to patients in the prenatal context; in fact, women who received results from ES after pregnancy completion had an increase in test-related anxiety and distress.¹¹ These results are consistent with findings from other studies on parental perceptions of prenatal ES, which found that the time to diagnosis, tension between uncertainty of results and desire for information, and perceived obligation to pursue testing despite potential limitations may increase anxiety and distress among parents.^12,13 Notably, current studies on parental perspectives, including our own, have been conducted with women who receive the results after determination of pregnancy disposition; thus, their perceptions are likely different from those receiving results in ongoing pregnancies.

The primary objective of this study is to describe parental motivations to pursue trio-ES in the context of fetal anomalies with a negative standard genetic diagnosis, and to show how parents interpret results. We hypothesize that parents have multiple reasons for pursuing ES beyond care of the immediate pregnancy, and that interpretation of and adaption to results occur in the context of their reproductive plans and social circumstances.

2 |. MATERIALS AND METHODS

This study was completed using an ongoing prenatal trio-ES cohort at University of North Carolina (UNC) at Chapel Hill, North Carolina. Trios (parents and fetus) with a pregnancy complicated by either isolated or multiple congenital anomalies and negative standard genetic testing (karyotype and microarray) were enrolled and underwent sequencing after pregnancy completion. Patients were identified from prenatal diagnosis clinics at UNC across the United States between July 2014 and November 2020.¹⁴ Providers from outside of the UNC healthcare system identified patients for referral based on knowledge of our ongoing study and its eligibility criteria. Such patients had already participated in reproductive genetic counseling with their institution and were continuing pregnancy management with their primary obstetric providers. Patients interested in study participation were then referred directly to our study coordinator. After ensuring that the patient met inclusion criteria, a certified prenatal genetic counselor at UNC performed pre-test counseling and consent procedures for the study. Trios were included in the study if the following inclusion criteria were met: (1) singleton gestation; (2) suspected genetic etiology of congenital anomalies identified on ultrasound (one or multiple anomalies not consistent with infectious, immune-mediated, or mechanical causes of structural change to the fetus); (3) lack of diagnosis after karyotype, microarray, and if indicated, gene-specific sequencing; and (4) presence of DNA from fetus, mother, and father. Parent–fetus trios were identified both prospectively and retrospectively and were enrolled at various gestational ages or after the pregnancy was completed. Trios enrolled prospectively were only approached for recruitment to the study after they had received standard clinical counseling and had made a decision about pregnancy management (e.g., continuation vs. termination of pregnancy) based on ultrasound findings and standard genetic diagnosis (karyotype and microarray) The study was not mentioned prior to this time to avoid impacting parental decision-making. A such, participant pre- and post-sequencing counseling and interviews were completed at varying time points during pregnancy, after termination, or after delivery depending on timing of referral to the study, timing of enrollment, and when sequencing occurred. Participants were also identified retrospectively through the UNC Perinatal Database, a repository of patients who received prenatal and delivery care at UNC (1996 to present). Women who previously indicated a desire to be re-contacted for additional fetal testing and who had fetal cells archived and available for DNA extraction were also approached for enrollment. The aforementioned individuals did not participate in the qualitative portion of the study that is presented here.

After enrollment, participants had pretest counseling by a certified prenatal genetic counselor regarding trio-ES and the possible results it can provide (genetic counseling was performed as previously described).¹⁴ This counseling specifically reviewed existing data regarding use of trio-ES in the prenatal setting, expected diagnostic yield from trio-ES in the setting of study inclusion criteria, and limitations to result interpretation in the setting of negative results. Additionally, given the novel nature of trio-ES, patients were clearly informed that testing was occurring in the context of study protocols. All participants agreed to learn findings that explained the fetal phenotype, medically actionable secondary findings in a parent, or carrier couple status for significant autosomal recessive conditions. After enrollment, we obtained maternal and paternal blood and extracted DNA in the Biospecimen Processing Facility, a core UNC-CH laboratory. Fetal DNA was extracted from stored specimens, such as amniocytes, chorionic villi, or umbilical cord blood as appropriate. For non-local cases, we directly received extracted fetal DNA from the outside institution. All genetic variants that were reported were confirmed by Sanger sequencing at the UNC Molecular Genetics Laboratory (MGL), a CLIA-certified and CAP-accredited facility, using a duplicate sample. After confirmation of results, parents were given the option to sign a separate consent form to have their own or their child’s variant placed in the medical record. Results were typically returned approximately 6–12 weeks to up to 6–8 months after enrollment, noting that enrollment could occur in a variety of circumstances as described above (e.g., after pregnancy termination, fetal demise, pregnancy continuation with live birth). As such, participants may have had differing time spans between the pregnancy event and interviews.

After enrollment, participants completed a demographics survey, which included self-reporting of race. Race categories were defined based on the U.S. Office of Management and Budget’s Revisions to the Standards for the Classification of Federal Data on Race and Ethnicity. As next-generation sequencing is not widely used at this time for prenatal diagnosis and racial and ethnic minority populations in the United States are consistently underrepresented in genetic and precision medicine research,¹⁵ race was tracked in order to appraise equity in enrollment and uptake, and to critically evaluate why certain groups may be underrepresented in our study in future research. Following demographic surveys, participants completed brief semi-structured interviews in which they were asked about their expectations of and reasons for pursuing ES. Pre-sequencing, participants were specifically asked:

In your own words, what is the main thing you expect to get out of genomic sequencing?

After return of results, participants were counseled about findings and completed a brief semi-structured interview. Specifically, participants were asked:

In your own words, what did you learn from your sequencing results?

This post-sequencing assessment was completed 2 weeks after sequencing results were delivered. Interviews and questionnaires were completed in person, online, by phone, or by web-based video platform dependent on the participant location. Interviews were limited to the aforementioned questions, with additional information often provided by participants unprompted.

2.1 |. Analysis

Participants’ demographic characteristics were analyzed using descriptive statistics. Questionnaire data was analyzed using descriptive statistics and with results previously published.¹¹ Interviews were reviewed, transcribed, and analyzed using grounded theory methods with a constructivist epistemology.¹⁶ Data were separated into pre- and post-sequencing responses and an iterative coding technique was used in accordance with grounded theory methodology. After initial review and memo-ing of the responses, open line-by-line coding was used. This included coding each line of participant response with appropriate summary codes. This was followed by focused coding, in which codes were grouped into categories and compared to one another, examined for overlap, and condensed. From this, a codebook was generated with primary codes and sub-codes determined from major categories that emerged in the data. Responses were then re-coded using this schema, constantly comparing occurrence of codes and ES results. Frequency and co-occurrence of codes were used to identify themes within the data.¹⁷

Interviews were coded by the primary author. Following this, participant responses and coding were reviewed by two additional members of the research team for agreement and internal validity. Any disagreements regarding codes, codebook, and emerging themes were resolved by debriefing with the research team. Themes that emerged were utilized to generate grounded theories of parental ES expectations and interpretations. Frequencies of applied codes and code-co-occurrences were also assessed using descriptive statistics. Analysis was completed using DeDoose software version 8.0.35 (SocioCultural Research Consultants, LLC www.dedoose.com) and STATA 15 statistical software.

3 |. RESULTS

3.1 |. Participant demographics

One hundred twenty-six trios have been sequenced to date and were asked to respond to the pre- and post-sequencing questionnaires and semi-structured interviews. Of those, 125 provided responses to the pre-sequencing questionnaires and 91 provided responses post-sequencing. Forty-five (36%) trios resulted in variants that provide a fetal diagnosis (pathogenic or likely pathogenic variants) and 6 (5%) resulted in medically actionable secondary findings in a parent (identified variants within this cohort are listed in Appendix 1). The majority of participants in our cohort identified as White women, were married, were employed, and had at least some college education. Additionally, 71 (58%) participants had genetic testing in a prior pregnancy before electing for ES (Table 1). There were no significant differences between those that completed the pre- and post-sequencing survey, and those that did not respond to the post-sequencing survey. Thirty-three (36%) of the participants that responded to the post-sequencing prompt received a result from ES: variant interpretations include 7—VUS, 10—likely pathogenic, and 16—pathogenic. Fifty-eight (64%) of individuals that responded post-sequencing had negative results.

TABLE 1.

Participant demographic characteristics

Characteristic	N = 126b
Maternal age (mean, SD)	31 (4.9)
Race
White	106 (84)
Hispanic	17 (13)
African American	9 (7)
Native American	2 (2)
Asian/Pacific Islander	8 (6)
College graduatea	96 (77)
Employed	80 (64)
Household income >$75,000	78 (62)
Married	106 (84)

^aCompleted 2-year college, 4-year college, or graduate degree.

^bShown as n (%) unless noted otherwise.

3.2 |. Pre- and post-sequencing interviews

Pre-sequencing themes included: (1) no expectations, (2) reasons for participation, (3) future decision-making, and (4) genetic anxieties. Post-sequencing themes included: (1) summarizing results, (2) adapting to negative results, (3) time to diagnosis, (4) gratitude for the process, and (5) impact on parental relationship (Table 2). It is noteworthy, and reflected by theme and theory construction, that many participants did not necessarily answer the questions as asked. Instead, participants used the interview prompts as an opportunity to reflect on the process and meaning they assigned to results. Below, exemplar quotes are provided in the context of major themes that emerged from synthesis of pre- and post-sequencing free responses.

TABLE 2.

Pre- and post-sequencing themes and frequency of themes

Pre-sequencing themes	N (%), N = 125	Post-sequencing themes	N (%), N = 91
No expectations of sequencing	17 (14)	Summarized results	59 (65)
Immediate reasons to participate	92 (74)
Altruism	21 (18)	Adapting to negative results	3 (36)
Identify the cause	77 (62)	Optimistic adaptation	9 (10)
Coping	9 (8)	Pessimistic adaptation	12 (13)
Future decisions	55 (45)	Time to diagnosis	6 (7)
Future pregnancy	39 (32)
Guidance for testing	17 (14)
Genetic anxieties	24 (20)	Gratitude for the process	6 (7)
‘Incompatibility’ with spouse	21 (18)
Fear of repeat experience	1 (1)
Last resort	3 (2)
Other family members	6 (5)
		Impact on parental relationship	4 (4)

Note: Themes are shown above subthemes. n (%) shown in columns 2 and 4. N = frequency the themes is identified in data, % = frequency of theme/all responses.

3.3 |. Theory construction and supporting quotations

3.3.1 |. Multiple reasons to seek one answer

Participants described several reasons for pursuing sequencing. The most common reason was to identify a genetic diagnosis that may explain the fetal findings (n = 77, 62%), altruistic reasons (n = 21, 18%), or to help them cope (n = 9, 8%) (Theme: “Immediate reasons to participate,” Table 2). Representative quotations are shown below, respectively:

“[We hope for] some explanation… just an answer toward why she was the way she was.”

—Participant # 8

“I’m really doing it for him [the baby] and to help others down the line. I hope to get an answer but I don’t have my hopes on it.”

—Participant #108

“I guess a reason why it occurred. I think that would give us some closure.”

—Participant #110

Further building on the coping theme, a few participants (n = 3, 2%) described ES as the “last step,” suggesting it was the final effort or even a necessary event having reached this point in the diagnostic process:

“This is the last step; a sense of relief. Knowing we completed this makes me feel better.”

—Participant #68

Finally, a significant proportion of patients chose to pursue sequencing to inform future reproductive decisions (n = 55, 45%), including pregnancy planning (n = 39, 32%) and guidance for future genetic testing (n = 17, 14%).

“Hoping to get some answers and hopefully be able to use that going forward to have more kids or testing with in vitro.”

—Participant #69

3.3.2 |. High hopes, limited expectations:

Seventeen (14%) participants stated that they had little or no expectations from ES. Despite this, some participants (n = 7, 42%) wanted to identify a specific cause of the anomalies noted on ultrasound (Theme: “Expectations of Sequencing,” Table 2). As stated by participant 6:

“I hope to understand her issues with genetics, but I don’t expect it. We are trying to put puzzle pieces together.”

—Participant #6

3.3.3 |. Adapting to uncertainty

Participants responded optimistically or pessimistically to ES results with pessimism driven by uncertainty of results. Participants had varied reactions to getting negative ES results (n = 33, 36%), with some expressing anxiety about the unknowns that may accompany a negative diagnostic result (pessimistic adaptation) (n = 12, 13%) and some expressing relief that a genetic diagnosis was not found (optimistic adaptation) (n = 9, 10%) (Theme: “Adapting to negative results,” Table 2). There were no significant differences in demographic characteristic among those who had optimistic or pessimistic responses to negative sequencing results. Examples of conflicting adaptations to results are cited below:

“They weren’t able to find anything specific, but it could be something we still need to worry about.”

— Participant #23

“We are grateful that no genetic issue was found in our baby.”

—Participant #50

As part of the post-sequencing questionnaire, subjects were also asked the following: “How certain are you that your results gave you accurate information about the cause of the findings of your baby/fetus/pregnancy?” Participants were able to respond “very uncertain,” “somewhat uncertain,” or “very certain.” Just over half (55/91, or 60%) of participants who completed the post-sequencing questionnaire answered this question. Thirty-six participants (40%) felt “very certain” about the ES results they received.

3.3.4 |. “Is it us?” Questions about genetics, personal health, and reproduction

A substantial number of participants (n = 21, 18%) expressed concern about their own health, their partners’ health, or genetic compatibility with a partner, and cited that as one of the reasons for pursuing trio-ES (Theme: “Genetic Anxieties,” Table 2). One participant summarized her reasons for pursuing sequencing as follows:

“To see if there is something wrong between us.”

—Participant #93

Four participants noted continued anxieties about genetic in-compatibility after trio-ES. Participant 93, mentioned above, stated the following after sequencing, demonstrating the burden that ES results placed on her relationship:

“[I learned]…that I can’t have any more babies, at least not with him. I don’t want to risk my life again so if he wants to have another baby it’s not with me. He still wants me to, but no.”

—Participant #93

Other participants noted anxieties about how recessive genes might impact their future children and other family members (n = 6, 5%):

“Definitely doing all that we can do for her health and seeing if this is something the rest of our children will have to face.”

—Participant #112

3.3.5 |. Gratitude for the process

Some participants expressed gratitude for the process, regardless of ES results. Six (7%) participants noted feeling grateful for the opportunity to participate in the study. Of those, four (66%) had negative sequencing results and two (33%) had results that explained the fetal findings (Theme “Gratitude for the Process,” Table 2). Respondents offered a range of reasons for gratitude, from self-discovery to closure:

“I learned that learning the answer doesn’t really change it. We are healed regardless of knowing the answer.”

—Participant #22

“ I feel like this was really important for me to be able to move on. Chances of this happening again are probably pretty slim…”

—Participant #45

4 |. DISCUSSION

Our study provides insight into the parental experience of using ES for fetal diagnosis in the setting of negative standard testing. Here, parents elected for advanced sequencing for several reasons beyond identifying a diagnosis, including desire to help other families, to inform future reproductive decisions, and to cope. A small fraction of participants indicated they pursued the testing because of its availability, feeling a necessity to “complete” the diagnostic process, a theme identified in prior studies.^12,13 However, our findings suggest a much broader range of factors beyond availability inform decisions to pursue ES.

Notably, some subjects reported continued uncertainty in the setting of both negative and positive ES results. Our prior assessment of this cohort suggests that participants who received ES results are more likely to have test-related anxiety and distress.¹¹ Our qualitative results extend those findings by indicating that uncertainty about results was common and depended to some extent on the nature of results returned. Prior literature has examined the risks and benefits of parents knowing uncertain results, and has noted possible longitudinal harm associated with “toxic knowledge.”^18–20 Moreover, a large proportion of those with negative results expressed concern about information that was not found. This is in contrast to the meaning patients and clinicians construct around negative ES results in areas of adult and pediatric medicine where ES has been adopted. Prior studies suggest that patients frame a negative result (the “nuanced negative”) with “nuanced optimism,” hoping that a genetic diagnosis may be found in the future or that there is no diagnosis to find.²¹ However, participants in our study expressed a continued apprehension about the “potentiality” of a negative result, which may be specific to the prenatal context in which the patient is interpreting results as implications for children instead of for herself.²²

In Wou et al.‘s assessment of parental perspectives of ES, 80% of participants desired to know ES results despite the level of uncertainty surrounding those results.¹³ However, clear guidelines for reporting results in the prenatal setting are still pending, with limited guidance for intensive pre- and post-test counseling and current focus on pathogenic and likely pathogenic variants.²³ As such, there is little information about how results, particularly uncertain or negative results, may impact families and relationship dynamics and what support should be offered in these situations, especially in the prenatal context in which parents are making partnership and reproductive decisions.

Our study, like others, demonstrates that genetic diagnosis is not only about the affected pregnancy, but also may be a referendum on genetic compatibility.²⁴ In our study, we identified specific examples of how results of ES proved challenging for parents regarding risks and benefits of attempting future pregnancy together. How that, in turn, impacts intimacy, bonding, and couples’ togetherness is unclear. In a prior study by Werner-Lin et al., the experience of prenatal microarray testing led to the pregnancy being experienced by couples as a “shared endeavor, but experienced in isolation.” Similar sentiments may have impacted our participants as they adapted to results, but more longitudinal follow-up is required to better characterize the impact on interpersonal relations.²⁴ Moreover, as partners often serve as support people for pregnant individuals, understanding the aforementioned impact on partnership is critical to providing informed counseling and appropriate support resources.

This study has several strengths. First, data presented here are from a large, prospective cohort of prenatal participants who provided responses immediately after pre- and post-sequencing counseling. As such, recall bias between counseling and interviews was minimized. However, participants enrolled in the study at various times after determining pregnancy disposition or after completion of pregnancy; thus, some of the participants may have been farther from that decision-making process than others. Additionally, this study extends upon our prior work, adding qualitative context to our quantitative findings. In our prior study, participants who received ES results that may explain the fetal findings were more likely to have test related anxiety and distress, and have more difficulty with psychological adaptation to results.¹¹ Qualitative findings from this cohort suggest that those who did not receive results that explain the fetal findings also vocalized some distress, likely related to both presentation and interpretation of negative results (e.g., the “nuanced negative”).²¹ As such, our study suggests that this group may require post-testing support, similar to those with positive findings, despite not having a diagnosis that requires follow-up and consultation.

Furthermore, the increased difficulty with psychological adaptation among those that received results may be related to impact on the parental relationship, worry about future pregnancies, or worry about personal health. The myriad of anxieties that impact parents seeking genetic diagnosis likely contribute to the challenge of adapting to such genetic information, but more importantly, may have longitudinal repercussions on the family unit. As such, our findings confirm and expand upon the limited existing literature about parental perspectives of ES in the prenatal period and elucidate the need for additional data to inform best practices for counseling.

Our study is not without limitations. First, our participants received ES results after pregnancy completion. Thus, responses produced by our cohort may not be representative of women who receive ES results during pregnancy. Importantly, clinical applicability of this data is limited, as prenatal diagnosis, particularly in the setting of uncertain fetal prognosis, is often used by parents as a tool to assist with decision making regarding pregnancy management. Given this, our results may not truly capture the psyche of parents who are using ES for pregnancy-specific decisions. Moreover, our study does not address the logistical issues that may arise with incorporating ES into intra-pregnancy decision-making, including timing and access to termination of pregnancy, turnaround time on test results and how that impacts maternal and fetal care, and access to prenatal diagnostic services in a variety of care settings. Future studies should explore how ES results influence maternal decision-making regarding ongoing pregnancy and impact on pregnancy experience, delivery, and neonatal care. Future studies should also assess how to equitably and systematically incorporate ES into the diagnostic algorithm, given the variety of factors that may determine clinical usability of the data derived from the test.

Secondly, our patient population primarily consisted of White, educated, high-earning cis-gendered women. Thus, our population and their perspectives are unlikely to be representative of the diverse range of individuals who may elect or benefit from ES. Although we attempted to recruit the diverse patient population that our institution serves (approximately 50% White people, 25% Black people, 20% Hispanic people, and 5% Asian people according to hospital census data 2019), the individuals that most often elected to participate were of the aforementioned demographics. Barriers to recruitment of other ethnic and racial groups for our particular study are unclear, however, may relate to individual and community perceptions about genetic and genomic medicine, plans for pregnancy management, or religiosity. Additionally, individuals may have declined participation if they anticipated difficulty with follow-up, including limited access to digital communication tools (e.g., Zoom, smartphone); this may have specifically prevented lower income people from participating in this study. This limitation of our study is an example of a larger issue in genomic medicine of poor representation and lack of inclusive results.¹⁵ Future studies at our institution are planned to assess community perspectives of diagnostic testing and advanced sequencing in the setting of fetal anomalies. We hope this will inform how to generate inclusive and equitable practices when considering how to best incorporate ES.

Finally, our qualitative results are limited to brief, free responses; thus we did not capture the full narrative of the participants’ diagnostic odyssey during our study. Finally, as our study was completed in real time with counseling, we were unable to assess longitudinal adaptation and future reproductive decisions after prenatal ES.

As advanced sequencing strategies for prenatal diagnosis become more common, it remains unclear which patients benefit most and from what information. Our study underscores the importance of a precise, patient-centered counseling approach, detailing whether or how knowledge revelation should be based on patient’s desire for information, support system, and need for future support.²⁵ It is well known that the interaction with providers throughout the diagnostic process influences parents’ ultimate genetic decision-making.²⁶ As such, future studies should focus on best practices for this process, detailing how to best disclose variants of uncertain significance and secondary findings, maintaining patient autonomy while providing accurate support for knowledge that could have “toxic” ramifications for self and family.^27,28 Finally, our study, like others, suggests that sequencing results may impact parental relationships and ultimate reproductive decisions.²⁹ Future studies detailing the longitudinal adaptation of families to reproductive genetic information are critical to understanding and developing the support that individuals and families may need after a prenatal testing and genetic diagnosis.

Key points

What’s already known about this topic?

• Exome sequencing (ES) for prenatal diagnosis increases diagnostic yield in the setting of fetal anomalies and negative standard genetic diagnosis (chromosomal microarray).

• Existing studies have centered on operationalizing this testing strategy, with focus on improving diagnosis and naming previously undetected variants.

• Little is known about parental motivations for pursuing this diagnostic strategy and post-sequencing interpretation of results, particularly when results are uncertain.

What does this study add?

• This study provides some patient perspectives of electing for this diagnostic strategy and their post-sequencing interpretations of ES results.

• In particular, this study illustrates how parents conceptualize results to inform their relationship, future reproductive decisions, and achieving closure after the affected pregnancy.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.