Background: This study examined how FMR1 genetic testing impacts the reproductive decision-making of women with diminished ovarian reserve (DOR).

Methods: 120 women clinically diagnosed with DOR (elevated FSH and/or low AMH and/or low at trial follicle count, with regular menses), and without a family history of fragile X syndrome, received fragile X genetic testing (FMR1) and completed pretest questionnaires. A subset (n=7) were interviewed pretest. Surveys and interviews were analyzed separately and then integrated using sequential explanatory mixed methods.

Results: Approximately50% regarded carrying the FMR1 pre mutation as a serious condition, while 37.5% had a neutral position. Women were significantly more likely to be upset about being a carrier if they perceived the FMR1 pre mutation to be a serious condition (p< 0.01).Interviews reflect several inheritance concerns (immediate next generation, future generations, and extended family members) and the impact the test results might have on their future reproductive decisions.

Discussion: These qualitative/quantitative responses indicated that FMR1 screening (1) informed DOR patients’ view of an infertility diagnosis (2) prepared them for potential health consequences in future offspring and (3) impacted their future reproductive decisions.

•    Diminished ovarian reserve
•    FMR1
•    Female infertility
•    Mixed methods
•    Pre mutation
•    Reproductive decision-making
•    Qualitative study

Walker ER, Clark ML, Stelling J, Timko MP, Pastore LM (2017) The Impact of Genetic Carrier Testing in Reproductive Decision-Making: FMR1 Testing in Women with Diminished Ovarian Reserve. J Endocrinol Diabetes Obes 5(1): 1098.

FMR1: Fragile X Mental Retardation 1; FXS: Fragile X Syndrome; DOR: Diminished Ovarian Reserve; POF: Premature Ovarian Failure; POI: Primary Ovarian Insufficiency

Infertility affects approximately 9% of couples worldwide, or approximately 72 million women aged 20-44 years [1]. In the US, 12.1% of females aged 15-44 years have 12-month infertility or impaired fecundity, totaling 6.7 million women [2]. Forty-one percent of women with infertility/sub fertility seek assistance from fertility clinics [3]. Ten percent of women in an infertility clinic, totaling 275,000 women in the US, are diagnosed with DOR [4,5]. Reductions in oocyte quantity and quality with advanced age (typically themid-40s ages) are a normal physiologic occurrence termed diminished ovarian reserve (DOR)[6]. Some women experience DOR much earlier and become prematurely infertile (pathologic DOR).

Specific tri nucleotide repeat lengths in the fragile X mental retardation 1 (FMR1) gene (HGNC: 3775) are associated with ovarian dysfunction. Women with pre mutation level repeats in this gene (55-199 CGG) are at increased risk for premature ovarian failure (POF) [7-9], alternatively termed primary ovarian insufficiency (POI). The association of the FMR1 gene with other forms of ovarian dysfunction such as pathologic diminished ovarian reserve (DOR) is less clear, as reviewed in 2014 [10]. These clinical diagnoses differ DOR is diagnosed by abnormal ovarian hormone levels and regular periods [11-13], while POF is diagnosed by postmenopausal levels of ovarian hormones plus 4 or more months of secondary amenorrhea before the age of 40 [14].

Some reports have suggested that women with “low normal” repeat lengths [15-17], or “high normal” and/or intermediate repeat lengths [18,19], including the pre mutation range [20,21], may be associated with DOR or infertility. Others have reported no association between FMR1 repeat lengths and DOR [22] or infertility in general [23].

The American College of Medical Genetics testing guidelines recommend testing for pre mutation level FMR1 genes in “women with reproductive or fertility problems associated with elevated follicle stimulating hormone levels, especially if there is a family history of POF, fragile X syndrome, or undiagnosed mental retardation” [24], which encompasses the definition of DOR. The National Society of Genetic Counselors and the Genetics Committee of the American College of Obstetrics and Gynecology support this recommendation [25,26]. Recommendations call for FMR1 genetic carrier testing and pre-test genetic counseling for women with DOR [27].

Given these recommendations, more DOR patients will be undergoing genetic carrier testing who have no family history of fragile X syndrome (FXS), and are unfamiliar with its potential implications. A mother who carries a pre mutation level FMR1 repeat length has a 50% chance of passing the fully mutated gene (expansion to over 200 CGG repeats) to each of her children. Her children will either be pre mutation carriers or they will have the full mutation. The risk of passing on a full mutation is highest when at least one of the mother’s FMR1 alleles has over 100 CGG repeats [28]. Pre mutation carrier men will pass the pre mutation to all their daughters but none of their sons because men do not pass on their x chromosome. FXS symptoms include intellectual disabilities, attention deficit and hyperactivity, behavioral and learning challenges, sensory integration problems, and speech delays. Males are more frequently affected by FXS symptoms than females, and their symptoms are often more severe. Pre mutation carriers do not have FXS symptoms.

There is limited research on the cognitive and emotional reactions to and the impact on reproductive decision making from carrier testing for x-linked disorders. Carrier testing for autosomal and x-linked-recessive disorders has been associated with relief from fear and is useful in reproductive planning [29- 31]. Relief from fear was reported whether they already had children (afraid they had put their children at risk to pass on the disease to grandchildren) or not (had feared having a baby with the disorder) [30]. A study of population screening for FMR pre mutations [33,34] found that while women had active coping mechanisms, they also had concerns for the implications of their carrier status for their children or grandchildren, the results impacted reproductive decisions whether in hindsight or for their own future, and the women’s positive carrier status generally had minimal relevance unless it pertained to a current stage of life. Pre and post-test emotional reactions to FMR1 testing among 20 women with DOR reported that participants anticipated that their self-esteem would be unchanged if their results indicated they carried the pre mutation, and most projected that if they did have the pre mutation they would feel better knowing there was a medical explanation for their infertility [35]. A qualitative study of seven women with DOR (using the same cohort as in this report) plus their spouses, reported that their pregnancy-seeking journey was long and exhausting, the expense of fertility testing/ treatment was noteworthy, they understood the reproductive implications of carrying the FMR1 pre mutation, and they hoped for a negative test result [29]. A related study [36] found that n=92 women diagnosed with DOR viewed the FMR1 pre mutation as serious as women with a family history of fragile X syndrome [37]. For research related to the reaction to pre mutation screening among females with a positive family history of FXS, the reader is referred elsewhere [37-41].

This investigation began with the broad research question “how do women struggling with infertility approach genetic carrier testing in their reproductive decision-making?” Using a mixed methods analysis, the intent of this paper is to assess the impact of FMR1 carrier screening and the anticipated test results on reproductive decision-making among women diagnosed with DOR.

Overview and quantitative analysis

This is a post hoc analysis of data previously collected for the purpose of assessing the association between FMR1 repeat lengths and DOR. For this report, the study hypotheses were generated prior to the analysis. The focus of this investigation was how and to what extent the FMR1 genetic carrier testing (1) informed an individual’s view of her infertility; (2) prepared her for the potential health consequences for future biological children; and (3) impacted her reproductive decision-making.

Mixed methods study design

Using a version of Creswell’s sequential explanatory design for mixed methods research [42], collection and analysis of quantitative data was followed by the collection and analysis of qualitative data. The qualitative interview results were interpreted as a way to validate and expand upon the initial quantitative findings [42-44]. The source of the quantitative data was the FRAXELLE Study, which using a longitudinal multicenter cohort, investigated (1) the distribution of tri nucleotide repeats in the FMR1 gene among women diagnosed with DOR and (2) the emotions towards testing for and potentially carrying the fragile X pre mutation. For qualitative findings, longitudinal interviews were conducted among a subset of the FRAXELLE Study participants. The quantitative and qualitative results were analyzed separately and integrated at the point of data interpretation.

This investigation only included baseline survey data from participants (after consent and the blood sample were drawn, but prior to learning the results of the FMR1 carrier test). Similarly, while the FRAXELLE Study had conducted three longitudinal interviews, only the initial interview was included in this analysis (after consent and the baseline structured questionnaire was completed, but prior to learning the FMR1 test results).

Quantitative data collection -participants and tools

A total of 120 women clinically diagnosed with DOR, and without a family history of FXS, provided blood for FMR1 testing and completed baseline questionnaires. Women were excluded if there was a known cause of elevated follicle stimulating hormone (FSH) for one’s age unrelated to fragile X, or a family history of FXS. Study details have been previously published [18,36]. Participants were enrolled between March 2005 and December 2013 through academic Reproductive Endocrinology and Infertility clinics at University of Virginia (UVA, 15.00%), Stanford University (35.83%), and University of North Carolina at Chapel Hill (UNC, 15.83%), as well as private fertility practices in Virginia (29.17%) and North Carolina (2.50%). Two women (1.67%) self-referred into the study. This study was approved by the Institutional Review Board at all academic sites (#11448 UVA, #11-1535 UNC, #16182 Stanford University).

At baseline, participants completed two questionnaires. The Primary Questionnaire assessed demographics, and infertility/ reproductive history. The Emotions Questionnaire assessed the (1) perception of seriousness regarding fragile X pre mutations as a medical condition; (2) predicted level of impact a positive test result would have on reproductive decision-making; (3) level of upset at the potential for health consequences for future offspring; (4) importance of having a medical explanation for their infertility; and (5) Health Orientation Scale [45]. The 11-itemHealth Orientation Scale (HOS; “How would you describe your feelings at this moment when you consider that you are potentially a carrier of a fragile X pre mutation?”) contains semantic differential items anchored with a negative and a positive adjective (e.g., “bad” and “good”) corresponding to a score of 1 or 9, respectively, on a numbered scale [45]. See related reports for further details on the Emotions Questionnaire [32, 35, 36, 46].

Quantitative data analysis

All emotional reactions and opinions were measured using 9-point scales in which 1 = “not at all”, 5 = “neutral”, and 9 = “very much”. The Shapiro-Wilk W test indicated that the majority of the categories were not normally distributed. Therefore, Wilcox on signed rank nonparametric statistics were used to analyze these scaled questions with paired results [47]. All items tested for potential effect modification by parity (0 vs. 1+ live birth) used Wilcox on signed rank tests. All statistical analyses were based on two-sided tests with an alpha of 0.05 and 95% confidence intervals using SPSS software version 21.

As consistent with previous research by Pastore et al. [36], three categories of response to “how serious do you think fragile X pre mutations are as a medical condition?” were created: “not serious” (response ≤ 3), “neutral” (response of 4, 5, or 6), and “serious” (response of ≥ 7). A priori analyses designed to stratify by the perception of FMR1 pre mutations being a serious medical condition (response ≥ 7) versus those who did not (response ≤ 6) used exact Mann-Whitney U tests for nonparametric independent samples.

Quantitative study

Sample description: Among the full sample that completed the questionnaires, the mean age at study participation was 38.0 years and the mean age at initial DOR diagnosis was 36.6 years (Table 1).

Table 1: Characteristics of the participants (n=120).

Factor	N (%)
Age at initial DOR diagnosis (years)	Mean 36.6 (sd 4.0) Median 37.8 Range 25-42
Age at study (years)	Mean 38.0 (sd = 4.3) Median 38.5 Range 26-49
Race: White Black Asian Mixed race/Other Hispanic Ethnicity	88 (73.3%) 4 (3.3%) 22 (18.3%) 6 (5.0%) 8 (6.7%)
Nulliparous (n=106) Nulligravid (n=111)	85 (80.2%) 42 (37.8%)
Smoking History Ever Smoked Regularly Current Smokers	17 (14.2%) 2 (1.7%)

Participants were primarily Caucasian (73.3%), with a sizeable minority of Asian women (18.3%). While 80% of participants had not given birth, almost two-thirds had been pregnant at least once.

Characteristics of the qualitative interviewees mirror the characteristics of the larger study population, with the exception that all interviewees were Caucasian. Six of the interviewees were married and 1 was single. Three of the interviewed women had never been pregnant, 2 had been pregnant once but had not given birth, 1 had been pregnant twice but had not given birth, and 1 had conceived once with fertility treatment resulting in a live birth. For further detail, the reader is referred elsewhere [48].

All of the women had tried multiple fertility treatments to achieve a pregnancy; of the 6 interviewed women who reported their treatment history in the structured questionnaire, all had used oral and/or inject able fertility medications, all had used intrauterine insemination, three had used IVF, and none had used a donor egg.

Quantitative results

As shown in Figure (1),

Figure 1 How serious do you think fragile X premutations are as a medical condition? Nulliparous (n=85). Parous (n=21). Parity missing for n=14.

61 participants (50.8%) regarded carrying the FMR1 pre mutation as a serious condition, while 45 participants (37.5%) had a neutral position, and only 14 participants (11.7%) did not consider this a serious condition. While no significant differences by parity (p=0.664) were found by the Exact Mann-Whitney U test, we noticed a potentially interesting skew in responses. Specifically, a higher percentage of nulliparous participants regarded the FMR1 pre mutation as a serious medical condition (response ≥ 7) than did the parous participants.

In response to the question “Without knowing your test result, how would you describe your feelings at this moment when you consider that you are potentially a carrier of a fragile X pre mutation?” participants reported feeling not guilty and un ashamed for the most part (Table 2).

Table 2: Health Orientation Scale (Baseline versus Imagine Positive).

Baseline (pretest)			Imagine Positive (pretest)		p-valueª
	Mean	Standard Deviation	Mean	Standard Deviation
Health Oriented Scale b
Good	4.5	1.953	3.16	1.754	<0.001
Unafraid	5.16	2.514	4.14	2.551	<0.001
Not Guilty	7.18	2.081	6.5	2.463	0.001
Unashamed	7.43	1.965	6.71	2.388	<0.001
Strong	5.99	2.056	5.25	2.377	<0.001
Relieved	4.89	1.939	4.18	2.624	0.002
Happy	4.01	1.994	2.98	1.808	<0.001
Able	5.72	2.235	5	2.48	<0.001
Pleased	4.73	1.566	4.02	1.815	<0.001
Healthy	6.36	1.974	5.38	2.334	<0.001
Unstigmatized	6.34	2.156	5.46	2.476	<0.001
a p-value calculated from paired sign rank statistics b 1=negative emotion, 9=positive emotion

They predicted they would still feel not guilty and unashamed if they were found to carry the pre mutation. The lowest mean scores (most negative emotions) were reported on the bad/good and sad/happy subscales for both baseline and imagine positive emotions. Mean scores on each component were significantly higher (more positive) for baseline emotions than for imagine positive emotions (p< 0.01). Mean scores between baseline emotions and imagine positive emotions had an average differential of 0.86. No significant differences were found by parity (data not shown). However, significantly more negative emotions were observed for 6 out of the 11 components at baseline items (bad/good, afraid/unafraid, relieved/shocked, happy/sad, able/unable, and pleased/ angry) when stratified by perceived seriousness of fragile X pre mutations (p< 0.05, data not shown).

At baseline, an overwhelming majority (80.7%) of participants agreed (response of ≥ 7) with the statement “I am glad to know there may be a medical reason for my infertility”. Nulliparous participants were significantly more likely to agree with this statement than parous participants (p< 0.05). No significant differences were observed after stratification by perceived seriousness (p> 0.05).

In response to the question: “Genetic diseases are passed from parent to child. How upsetting would it be if you found out that fragile X runs in your family and that you could have a child or grandchild with fragile X?”, the majority (61.5%) of participants reported that they would be very upset (response of ≥ 7, Table 3).

Table 3: Relationship between how upsetting fragile X in their family would be (“if found out that fragile X runs in family and it could be passed on to future generations”) and pretest perceived seriousness of fragile X permutations.

Emotion	Percentage who reported this emotion among participants who consider premutations seriousª	Percentage who reported this emotion among all other participants b	p-valuec
Upset	73.8%	47.4%	0.005
Neutral/not upset	26.2%	52.6%
a n= 61 women in this category responded to this question b n= 57 women in this category responded to this question c Continuous variable used for statistical comparisons (exact Wilcoxon test)

Only 10.3% of participants said that this knowledge would not upset them (response ≤ 3), and 28.2% had a neutral position. Participants were significantly more likely to be upset if they perceived the FMR1 pre mutation to be a serious medical condition (p< 0.01).No significant differences were observed by parity (p> 0.05).

The following scaled question was only asked of nulliparous study participants (n=81 for this item): “If you were found to be a carrier, do you think this would influence how you might feel about potentially never having had a child?” Responses were nearly equal across the three categories (38.3% responded “makes me feel better”, 35.8% “neutral”, and 25.9% “makes me feel worse”). There was no significant difference by perceived seriousness (p> 0.05).

Through our mixed methods analysis, three key points became apparent about how these women diagnosed with diminished ovarian reserve approach genetic carrier testing. First, participants searching for the cause of their infertility welcomed an explanation for their condition. The participants indicated a desire for accurate information about their FMR1 results and their health and reproductive capacity even if the genetic test results held serious implications. Second, participants expressed strong concerns about potential health issues facing future biological children and the possibility of continuing a “negative” genetic legacy. Third, the process of having genetic carrier testing impacted the participant’s reproductive decision making, and they projected that the results of the test could be a factor in weighing their future reproduction options. If they were found to carry the pre mutation, participants anticipated being willing to use donor eggs or adopt or to stop altogether efforts to have a future child. These observations were supported by both the quantitative survey data and the qualitative interviews.

Currently in clinical practice, only pre mutation carriers are routinely informed that their FMR1 test results have relevance for reproductive decision-making. Some fertility clinics may also inform patients with intermediate repeat lengths ([45-54] CGG) of their risk of POI/POF (as reported in earlier reports [19,52,53] but not supported in more recent studies [54,55]) and/or the risk of expansion to a pre mutation in a subsequent generation (19% reported to either expand or contract in next generation [51]).

Research regarding the association between FMR1 tri nucleotide repeat lengths and DOR is ambiguous as previously discussed, thus genetic counseling may not even mention this potential association. Pre implantation genetic diagnosis testing can be used to identify embryos at risk for FXS; this testing is much more common now than when our sample participated in this study. The prevalence of the pre mutation is 1:151 in the general female population in the US [56] (1:250 among women without a family history of FXS [57]), and variation by ethnic heritage and country has been reported [58-60]. FMR1 repeat length variation by ethnic heritage and country has also been observed within the normal range [61]. Given that most women affected by the recommendations for FMR1 testing will receive negative test results, it is especially important to consider the emotional reactions to the testing process. This genetic test result may hold both welcome and unwelcome information for DOR patients: welcome in that an explanation for one’s DOR may provide comfort and closure for infertile women, and unwelcome because such a diagnosis carries clear implications for reproductive decision-making and the potential health of offspring.

Genetic carrier testing informs participants view of DOR diagnosis

The way in which a genetic test result serves to inform participants’ views of their diagnosis of DOR is contingent on individual factors, such as past fertility history, and the perceived seriousness of the genetic condition. Participants were significantly more likely to take comfort in a genetic explanation if they had never given birth. This observation is similar to that of a qualitative study of the general female population that found that the motivation for FMR1 genetic carrier testing and the need for information differ by family history of FXS and parental status [33]. Another qualitative study of the impact on future reproductive decision-making from having a FXS offspring reported that the majority of mothers chose not to have another biological child, while 20% either purposely became pregnant or continued an unplanned pregnancy after finding out they carried the FMR1 pre mutation [62]. In a commentary about population screening for genetic conditions including FXS, Archibald and Mc Claren [63], noted that initial judgments about the relevance of testing were centered on two key areas: reproductive state of life and health-related experiences. There is little prior research on the impact of previous parenthood with a healthy child on the neither emotional/psychological impact nor uptake of either prenatal or preconception genetic testing, thus this is an area ripe for future research.

The FMR1 pre mutation testing is important for women at risk of carrying the pre mutation and still considering conception with their own eggs. Provision of information regarding a woman’s chance of having a son or daughter with FXS or a daughter with the pre mutation is vital to her ability to make informed reproductive decisions. The quantitative data and qualitative interviews were consistent on the participants’ concerns regarding inheritance of FMR1 health effects and the legacy to future generations. Supporting our observation, others have reported that women with a family history of FXS more frequently reported a “concern for children” than a reproductive concern for themselves in structured interviews [37].

Genetic carrier testing impacts participants’ reproductive decision-making

Reproductive decision-making is a multi-factorial process for any woman. The inclusion of a potential inherited condition into the equation magnifies the complexity of this process. The women with DOR in this study projected that the results of the genetic carrier test would be a factor in weighing their future reproduction options. Consistent with previous studies, the study population generally perceived the FMR1 pre mutation as serious prior to testing, and recognized the medical importance of FMR1 testing for their future fertility planning [32,48]. These patient reactions mirror those of women with a family history of FXS [37,38,64].Of the interviewed participants that were still undergoing fertility treatments, most were waiting for the results of the test before moving forward, implying that their next reproductive decisions would be contingent on the results they received. Our findings are consistent with previous research on the general female population, where participants “expressed that positive [FMR1] pre mutation results could have led to their reconsidering life plans especially their decision to have children”[33].

Not surprisingly, the participants who considered fragile X pre mutations to be serious had more negative baseline “imagine positive” emotions regarding this genetic testing. This suggests that both the testing process and the imagining of a positive result may be distressing for this population. Dealing with infertility, the population is already in an emotionally distressing situation. Although no other studies of the Health Orientation Scale in a pretest situation were located outside of our own prior research [35-46], carrier testing for autosomal and x-linkedrecessive disorders has been associated with relief from fear and is useful in reproductive planning [30,31,48]. This relief from fear was reported whether they were parous (afraid they had put their children at risk to pass on the disease to grandchildren) or nulliparous (fear of having a child with the disorder) [30].

Strengths and limitations

Per the study protocol, participants had access to tailored educational materials and received genetic counseling prior to FMR1 testing, which may limit the generalize ability because typical fertility clinics provide limited pretest genetic counseling. This study-provided genetic counseling may account for the participants’ ability to understand the condition and the potential implications of a carrier result on future reproductive planning. This study is limited by its restriction to non-carriers, though our findings should be generalize able to women without a family history of FXS because the investigation analyzed pre-test emotions and both carriers and non-carriers enter the genetic testing arena without knowledge of their FMR1 status. The lack of carriers in the population only allows for analysis of participants’ predictions regarding the extent in which their reproductive decisions would be impacted if they were found to be a carrier; further investigation is needed to determine that the provision of a positive test result actually influences reproductive decision-making in carrier women without a family history of FXS. For literature on the impact on reproductive decision-making among women with a positive family history of FXS, see [62,64,65]. This study was limited to a patient population of women with DOR that sought input from a fertility clinic. It is further limited to women who chose to be tested for FMR1, and therefore may be more psychologically prepared or emotionally comfortable with undergoing genetic screening than the general population. As our study population was primarily white (with 18% Asian in the quantitative data and 0% minority races in the qualitative data), our results are limited regarding potential cultural differences in attitudes towards this genetic testing. Given the differences in the FMR1 CGG repeat distribution by race-ethnicity in the pre mutation [58] and normal ranges [61], it would be especially interesting for future research to explore attitudinal variation on FMR1 testing in OB/GYN clinics by heritage, similar to analogous research on prenatal genetic testing by others [66,67].

The primary study strength is the mixed methods approach, which considered both quantitative and qualitative findings. To our knowledge, this is the second mixed methods publication on reproductive decision-making related to FMR1 screening; the one prior report studied women with a family history of FXS [65]. As our interview and structured questionnaire data were consistent, it lends credibility to the findings. An additional strength is the clear clinical phenotype, thus the findings represent the experience of women with DOR without a family history of FXS

Implications for clinical practice and/or policy

As more women without a FXS family history undergo this genetic carrier testing, it is a critical responsibility of the physician and the genetic counselor to provide accurate and objective information about the implications, advantages, disadvantages and consequences of this genetic testing and its results. Additionally, reproductive decisions before and after genetic counseling requires mutual understanding and co-operation between the clinician and the patient.

In terms of practice implications of our findings, women undergoing this testing need to have easy access to tailored educational materials, genetic consultation, and supportive environments before and after testing in order to understand the information the test could provide them. Without the benefit of the pretest genetic counseling and education materials offered in this study, women may not fully understand the implications of carrying the pre mutation. Recommendations on the content of FMR1 pretest genetic counseling sessions have been published [25,68]. While specialists in reproductive medicine can provide a upportive environment to explain the meaning and implications of FMR1 testing [69], it is important that patients have access to tailored educational materials and consultation with genetic counselors before and after testing as needed. Additional research is needed to determine what supplemental educational materials would be best suited to substitute for pretest counseling where pretest genetic counseling is not available.

As women with DOR continue to undergo FMR1 testing in the future, it is important for clinicians and counselors to understand the reactions of these women with respect to genetic carrier testing.

We are grateful to all the participants of this study. We thank the study co-investigators who are not authors of this article: Dr. Lawrence Silverman and Dr. Ani Manichaikhul, University of Virginia; Dr. Steven Young, University of North Carolina at Chapel Hill; Dr. Valerie Baker, Stanford University; and Dr. Joel Finkelstein, Massachusetts General Hospital. We also thank the clinical research coordinators at all participating clinics: Parchayi Dalal, Hannah Spencer, Amy Brown, Amanda DeSmit, Angie Morey, Rebecca Briggs, and Janetta Phillips. This work was supported by the Eunice K. Shriver National Center for Child Health and Human Development at the National Institutes of Health (Grants R03HD052768, R21HD057485 and R01HD068440 to LMP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007; 22: 1506- 1512.

2. Chandra A, Copen C, Stephen E. Infertility and impaired fecundity in the United States, 1982-2010: Data from the National Survey of Family Growth. Natl Health Stat Report. 2013; 14:1-18.

3. Chandra A, Copen CE, Stephen EH. Infertility service use in the United States: data from the National Survey of Family Growth, 1982-2010. Natl Health Stat Report. 2014;1-21.

4. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT Jr. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril. 2001; 76: 666-669.

5. Scott RT Jr, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril.1995; 63: 1-11.

6. Sharara FI, Scott JRT, Seifer DB. The detection of diminished ovarian reserve in infertile women. Am J Obstet Gynecol. 1998; 179: 804-812.

7. Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D, Holden JJ, Yang KT, Lee C, et al. Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data. Am J Med Genet. 1999; 83: 322-325.

8. Uzielli ML, Guarducci S, Lapi E, Cecconi A, Ricci U, Ricotti G, et al. Premature ovarian failure (POF) and fragile X premutation females: from POF to to fragile X carrier identification, from fragile X carrier diagnosis to POF association data. Am J Med Genet. 1999; 84: 300-303.

9. Sullivan AK, Marcus M, Epstein MP, Allen EG, Anido AE, Paquin JJ, et al. Association of FMR1 repeat size with ovarian dysfunction. Hum. Reprod. 2005; 20: 402-412.

10. Pastore LM, Johnson J. The FMR1 Gene, Infertility and Reproductive Decision-Making: A Review. Front Genet. 2014; 5.

11. Ferraretti AP, Gianaroli L. The Bologna criteria for the definition of poor ovarian responders: is there a need for revision?. Hum Reprod. 2014; 29: 1842-1845.

12. Ferraretti AP, La Marca A, Fauser BCJM, Tarlatzis B, Nargund G, Gianaroli L, et al. ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011; 26: 1616-1624.

13. Sills ES, Alper MM, Walsh AP. Ovarian reserve screening in infertility: practical applications and theoretical directions for research. Eur J Obstet Gynecol Reprod Biol. 2009; 146: 30-36.

14. Coulam CB, Adamson SC, Annegars JF. Incidence of premature ovarian failure. Obstet Gynecol. 1986; 67: 604-606.

15. Gleicher N, Weghofer A, Barad D. Ovarian reserve determinations suggest new finction of FMR1 (fragile X gene) in regulating ovarian ageing. Reprod Biomed Online. 2010; 20: 768-775.

16. Gleicher N, Weghofer A, Oktay K, Barad D. Relevance of triple CGG repeats in the FMR1 gene to ovarian reserve. Reproductive Biomedicine Online. 2009; 19: 385-390.

17. Pastore LM, Young SL, Manichaikul A, Baker VL, Wang X, Finkelstein J. Distribution of the FMR1 Gene in Females by Race-Ethnicity: Women With Diminished Ovarian Reserve Versus Women With Normal Fertility (SWAN Study). Fertil Steril. 2017;107: 205-211.

18. Pastore LM, Young SL, Baker VM, Karns LB, Williams CD, Silverman LM. Elevated Prevalence of 35-44 FMR1 Trinucleotide Repeats in Women with Diminished Ovarian Reserve. Reprod Sci. 2012;19: 1226-1231.

19. Karimov CB, Moragianni VA, Cronister A, Srouji S, Petrozza J, Racowsky C, et al. Increased frequency of occult fragile X-associated primary ovarian insufficiency in infertile women with evidence of impaired ovarian function. Hum Reprod. 2011;26 : 2077-83.

20. Barasoain M, Barrenetxea G, Huerta I, Telez M, Carrillo A, Perez C, et al. Study of FMR1 gene association with ovarian dysfunction in a sample from the Basque Country. Gene. 2013; 521:145-149.

21. Streuli I, Fraisse T, Ibecheole V, Moix I, Morris MA, de Ziegler D. Intermediate and premutation FMR1 alleles in women with occult primary ovarian insufficiency. Fertil Steril. 2009; 92: 464-470.

22. Schufreider A, McQueen DB, Lee SM, Allon R, Uhler ML, Davie J, et al. Diminished ovarian reserve is not observed in infertility patients with high normal CGG repeats on the fragile X mental retardation 1 (FMR1) gene. Hum Reprod. 2015; 30: 2686-2692.

23. De Geyter C, M’Rabet N, De Geyter J, Zurcher S, Moffat R, Bosch N, et al. Similar prevalence of expanded CGG repeat lengths in the fragile X mental retardation I gene among infertile women and among women with proven fertility: a prospective study. Genet Med. 2014; 16: 374- 378.

24. Monaghan KG, Lyon E, Spector EB. ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics. Genet Med. 2013; 15: 575-86.

25. Finucane B, Abrams L, Cronister A, Archibald A, Bennett R, McConkie-Rosell A. Genetic counseling and testing for FMR1 gene mutations: Practice guidelines of the National Society of Genetic Counselors. J Genet Couns. 2012; 21: 752-760.

26. American College of Obstetricians and Gynecologists Committee on Genetics. ACOG Committee Opinion No. 469: Carrier screening for fragile X syndrome. Obstet Gynecol. 2010; 116: 1008-1010.

27. McConkie-Rosell A, Abrams L, Finucane B, Cronister A, Gane LW, Coffey SM, et al. Recommendations from multi-disciplinary focus groups on cascade testing and genetic counseling for fragile X-associated disorders. J Genet Couns. 2007; 16: 593-606.

28. Nolin SL, Glicksman A, Ding X, Ersalesi N, Brown WT, Sherman SL, et al. Fragile X analysis of 1112 prenatal samples from 1991 to 2010. Prenat Diagn. 2011; 31: 925-931.

29. Pastore LM, Karns LB, Ventura K, Clark ML, Steeves RH, Callanan NP. Longitudinal Interviews of Couples Diagnosed with Diminished Ovarian Reserve Undergoing Fragile X Premutation Testing. J Genet Couns. 2014; 23: 97-107.

30. Williams JK, Schutte DL. Benefits and burdens of genetic carrier identification. Western J Nurs Res. 1997; 19: 71-82.

31. Fanos JH, Johnson JP. Barriers to carrier testing for adult cystic fibrosis sibs: the importance of not knowing. Am J Med Genet. 1995; 59: 85-91.

32. Pastore LM, Karns LB, Pinkerton JV, Silverman LM, Williams CD, Camp TR. Acceptance of fragile X premutation genetic screening in women with ovarian dysfunction. Am J Obst Gyn. 2006; 194: 738-743.

33. Anido A, Carlson LM, Taft L, Sherman S. Women’s attitudes toward testing for fragile X carrier status: a qualitative analysis. J Genet Couns. 2005; 14: 295-306.

34. Anido A, Carlson LM, Sherman SL. Attitudes toward fragile X mutation carrier testing from women identified in a general population survey. J Genet Couns. 2007; 16: 97-104.

35. Pastore LM, Morris WL, Karns LB. Emotional reaction to fragile X premutation carrier tests among infertile women. J Genet Couns. 2008; 17: 84-91.

36. Pastore LM, Antero M, Ventura K, Penberthy JK, Thomas SA, Karns LB. Attitudes towards potentially carrying the FMR1 premutation: before vs after testing of non-carrier females with diminished ovarian reserve. J Genet Couns. 2014; 23: 968-75.

37. McConkie-Rosell A, Spiridigliozzi GA, Sullivan JA, Dawson DV, Lachiewicz AM. Longitudinal study of the carrier testing process for Fragile X Syndrome: perceptions and coping. Am J Med Genet. 2001; 98: 37-45.

38. McConkie-Rosell A, Spiridigliozzi GA, Iafolla T, Tarleton J, Lachiewicz AM. Carrier testing in the fragile X syndrome: attitudes and opinions of obligate carriers. Am J Med Genet. 1997; 68: 62-69.

39. McConkie-Rosell A, Spiridigliozzi GA, Sullivan JA, Dawson DV, Lachiewicz AM. Carrier testing in fragile X syndrome: effect on self-concept. Am J Med Genet. 2000; 92: 336-342.

40. Raspberry KA, Skinner D. Negotiating desires and options: how mothers who carry the fragile X gene experience reproductive decisions. Soc Sci Med. 2011; 72: 992-998.

41. Raspberry K, Skinner D. Enacting genetic responsibility: experiences of mothers who carry the fragile X gene. Sociol Health Illn. 201133: 420-433.

42. Creswell JW, Plano Clark VL. Designing and conducting mixed methods research. 2nd ed. Los Angeles: SAGE Publications. 2011.

43. Denzin NK, Lincoln YS. The SAGE handbook of qualitative research. 3rd ed. Thousand Oaks: Sage Publications. 2005.

44. Hesse-Biber SN. Mixed methods research : merging theory with practice. New York: Guilford Press. 2010.

45. Wooldridge EQ, Murray RFJ. The Health Orientation Scale: a measure of feelings about sickle cell trait. Social Biol. 1988; 35: 126-136.

46. Cizmeli C, Lobel M, Franasiak J, Pastore LM. Levels and associations among self-esteem, fertility distress, coping, and reaction to potentially being a genetic carrier in women with diminished ovarian reserve. Fertil Steril. 2013; 99: 2037-2044. 

47. Wuensch KL. Nonparametric statistics. In. 2004.

48. Pastore LM, Karns LB, Ventura K, Clark ML, Steeves RH, Callanan NP. Longitudinal Interviews of Couples Diagnosed with Diminished Ovarian Reserve Undergoing Fragile X Premutation Testing. J Genet Couns. 2014; 23: 97-107.

49. Sandelowski M. Sample size in qualitative research. Res Nurs Health. 1995; 18: 179-183.

50. Kockelmans JJ. Toward an Interpretative or Hermeneutic Social Science. Graduate Facul Philos J. 1975; 5: 73-96.

51. Nolin SL, Brown WT, Glicksman A, Houck GE Jr., Gargano AD, Sullivan A, et al. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles. Am J Hum Genet. 2003; 72: 454- 464.

52. Bretherick KL, Fluker MR, Robinson WP. FMR1 repeat sizes in the gray zone and high end of the normal range are associated with premature ovarian failure. Hum Genet. 2005; 117: 376-382.

53. Bodega B, Bione S, Dalpra L, Toniolo D, Ornaghi F, Vegetti W, et al. Influence of intermediate and uninterrupted FMR1 CGG expansions in premature ovarian failure manifestation. Hum Reprod. 2006; 21: 952-957.

54. Voorhuis M, Onland-Moret NC, Janse F, Ploos van Amstel HK, Goverde AJ, Lambalk CB, et al. The significance of fragile X mental retardation gene 1 CGG repeat sizes in the normal and intermediate range in women with primary ovarian insufficiency. Hum Reprod. 2014; 29: 1585-1593.

55. Murray A, Schoemaker MJ, Bennett CE, Ennis S, Macpherson JN, Jones M, et al. Population-based estimates of the prevalence of FMR1 expansion mutations in women with early menopause and primary ovarian insufficiency. Genet Med. 2014; 16: 19-24.

56. Seltzer MM, Baker MW, Hong J, Maenner M, Greenberg J, Mandel D. Prevalence of CGG expansions of the FMR1 gene in a US populationbased sample. Am J Med Genet B Neuropsychiatr Genet. 2012; 159: 589-597.

57. Cronister A, Teicher J, Rohlfs EM, Donnenfeld A, Hallam S. Prevalence and instability of fragile X alleles: implications for offering fragile X prenatal diagnosis. Obstet Gynecol. 2008; 111: 596-601.

58. Genereux DP, Laird CD. Why do fragile X carrier frequencies differ between Asian and non-Asian populations? Genes Genet Syst. 2013; 88: 211-224.

59. Crawford DC, Meadows KL, Newman JL, Taft LF, Scott E, Leslie M, et al. Prevalence of the fragile X syndrome in African-Americans. Am J Med Genet. 2002; 110: 226-233.

60. Weiss K, Orr-Urtreger A, Kaplan Ber I, Naiman T, Shomrat R, Bardugu E, et al. Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women. Genet Med. 2014; 16: 940-944.

61. Pastore L, Manichaikhul A, Wang X, Finkelstein J. FMR1 CGG Repeats: Reference Levels and Race-Ethnic Variation in Women With Normal Fertility (Study of Women’s Health Across the Nation). Reprod Sci. 2016; 9: 1225-1233.

62. Raspberry K, Skinner D. Enacting genetic responsibility: experiences of mothers who carry the fragile X gene. Sociol Health Illn. 2011; 33: 420-433.

63. Archibald AD, McClaren BJ. Perceived relevance of genetic carrier screening: observations of the role of health-related life experiences and stage of life in decision making. J Community Genet. 2012; 3: 47- 54.

64. McConkie-Rosell A, Heise EM, Spiridigliozzi GA. Influence of genetic risk information on parental role identity in adolescent girls and young women from families with fragile X syndrome. J Genet Couns. 2012; 21: 59-71.

65. Raspberry KA, Skinner D. Negotiating desires and options: how mothers who carry the fragile X gene experience reproductive decisions. Soc Sci Med. 2011; 72: 992-998.

66. Chen L-S, Zhao M, Zhou Q, Xu L. Chinese Americans’ views of prenatal genetic testing in the genomic era: a qualitative study. Clin Genet. 2012; 82: 22-27.

67. Thompson S, Noblin SJ, Lemons J, Peterson SK, Carreno C, Harbison A. Perceptions of Latinas on the Traditional Prenatal Genetic Counseling Model. J Genet Couns. 2015; 24: 675-82.

68. McConkie-Rosell A, Finucane B, Cronister A, Abrams L, Bennett RL, Pettersen BJ. Genetic counseling for fragile x syndrome: updated recommendations of the national society of genetic counselors. J Genet Couns. 2005;14: 249-270.

69. Wittenberger MD, Hagerman RJ, Sherman SL, McConkie-Rosell A, Welt CK, Rebar RW, et al. The FMR1 premutation and reproduction. Fertil Steril. 2007; 87: 456-465.