Identification of barriers for good adherence to a guideline on recurrent miscarriage.

OBJECTIVE: Guidelines on recurrent miscarriage are poorly implemented in daily clinical practice. To ensure proper implementation, we identified existing barriers and facilitators for guideline adherence according to professionals and patients. DESIGN: Qualitative research. SETTING: Two different regions in the Netherlands. POPULATION: Forty-two professionals: gynecologists, residents in obstetrics and gynecology, fertility doctors and clinical geneticists. Ten patients with recurrent miscarriage. METHODS: Focus group interviews were performed with professionals and individual in-depth interviews with patients. Reports from the interviews were analyzed and barriers were identified. MAIN OUTCOME MEASURES: Identified barriers, categorized in four domains, including characteristics of: (I) the guideline, (II) professionals, (III) patients, (IV) organization. RESULTS: Ninety-six barriers, at all four domains, were identified among professionals. The most frequently mentioned barriers were: guideline being too complicated in the consultancy room and finding it difficult to refuse demands of insistent patients. Patients mentioned 40 barriers, of which lack of up-to-date patient information and lack of detailed knowledge about family history were most frequently mentioned. Potential facilitators, such as an electronic decision tool and patient questionnaires prior to their first visit, were mentioned by both professionals and patients. All participants agreed that complete adherence to the guideline was theoretically achievable. CONCLUSIONS: Both professionals and patients experienced barriers and facilitators for guideline adherence in recurrent miscarriage. Guideline implementation strategies should take these identified barriers into account.

Perspectives of couples with high risk of transmitting genetic disorders.

OBJECTIVE: To investigate the preference for preimplantation genetic diagnosis (PGD) as an alternative to prenatal diagnosis (PND) in a large group of couples representing a wide array of genetic disorders. We also investigated the couple's familiarity with PGD and presented time trade-off scenarios for PGD versus PND, as PGD treatment is regularly accompanied by waiting lists. DESIGN: Questionnaire study. SETTING: Patient organizations representing genetic disorders. PATIENT(S): A total of 210 couples carrying genetic disorders. MAIN OUTCOME MEASURE(S): Preference for PGD or PND and familiarity with PGD in carrier couples. RESULT(S): Fifteen organizations representing 38 genetic disorders agreed to participate. Nine hundred eighty-three couples responded. In total 210 couples were in their reproductive years (women 18-40 years) and had a desire to conceive. Ninety couples (42%) had never heard of PGD. After they were informed, 127 couples (60%) wanted to have diagnostic testing (PND or PGD) performed. Ninety-four (74%) of these couples preferred testing with PGD. When no waiting list was used 102 couples (80%) preferred PGD. With a 2-year waiting list for PGD, 58 couples (46%) would opt for PGD. CONCLUSION(S): Many carrier couples are unaware of the existence of PGD. When informed, most couples prefer PGD more than PND. The preference for PGD decreases with longer waiting lists.

Low uptake of prenatal diagnosis after established carrier status of a balanced structural chromosome abnormality in couples with recurrent miscarriage.

OBJECTIVE: To evaluate to what extent couples carrying a balanced structural chromosome abnormality follow up the advice to opt for invasive prenatal diagnosis (PND) in subsequent pregnancies. DESIGN: Index-control study. SETTING: Six centers for Clinical Genetics in The Netherlands. PATIENT(S): Couples referred for chromosome analysis after recurrent miscarriage between 1992 and 2001 and with at least one pregnancy after disclosure; 239 carrier couples and 389 noncarrier couples. INTERVENTION(S): Questionnaire, medical record checking. MAIN OUTCOME MEASURE(S): Uptake of invasive PND. RESULT(S): Only 53 of 239 (22%) carrier couples underwent a PND procedure (CVS or amniocentesis) in all subsequent pregnancies. A relatively high number, 105 (44%) carrier couples, refrained from PND in all subsequent pregnancies. More carrier couples with maternal age >or=36 years (20/33 = 61%) refrained from PND, compared with carrier couples with maternal age <36 years (85/206 = 41%). In women >or=36 years, an equal proportion of carrier and noncarrier couples refrained from PND (61% vs. 54%). CONCLUSION(S): The advice to opt for invasive PND in carrier couples is poorly followed, especially in carrier couples with maternal age >or=36 years. The motivations of carrier couples to opt for or refrain from invasive PND procedures should be the topic for further research to optimize clinical care and informative decision making.

Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells.

The Beckwith-Wiedemann syndrome (BWS) is a growth disorder for which an increased frequency of monozygotic (MZ) twinning has been reported. With few exceptions, these twins are discordant for BWS and for females. Here, we describe the molecular and phenotypic analysis of 12 BWS twins and a triplet; seven twins are MZ, monochorionic and diamniotic, three twins are MZ, dichorionic and diamniotic and three twins are dizygotic. Twelve twins are female. In the majority of the twin pairs (11 of 13), the defect on chromosome 11p15 was hypomethylation of the paternal allele of DMR2. In 5 of 10 twins, there was additional hypomethylation of imprinted loci; in most cases, the loci affected were maternally methylated, but in two cases, hypomethylation of the paternally methylated DLK1 and H19 DMRs was detected, a novel finding in BWS. In buccal swabs of the MZ twins who share a placenta, the defect was present only in the affected twin; comparable hypomethylation in lymphocytes was detected in both the twins. The level of hypomethylation reached levels below 25%. The exchange of blood cells through vascular connections cannot fully explain the degree of hypomethylation found in the blood cell of the non-affected twin. We propose an additional mechanism through which sharing of aberrant methylation patterns in discordant twins, limited to blood cells, might occur. In a BWS-discordant MZ triplet, an intermediate level of demethylation was found in one of the non-affected sibs; this child showed mild signs of BWS. This finding supports the theory that a methylation error proceeds and possibly triggers the twinning process.

Reproductive outcome after chromosome analysis in couples with two or more miscarriages: index [corrected]-control study.

OBJECTIVE: To compare reproductive outcomes in couples carrying a structural chromosome abnormality and non-carrier couples referred for chromosome analysis after two or more miscarriages. DESIGN: Index [corrected]-control study. SETTING: Six centres for clinical genetics in the Netherlands. PARTICIPANTS: 278 carrier couples and 427 non-carrier couples referred for chromosome analysis between 1992 and 2000 after two or more miscarriages before 20 weeks of gestation. Couples were followed up for at least 24 months after chromosome analysis. MAIN OUTCOME MEASURES: The birth of at least one healthy child, at least one more miscarriage, and viable offspring with unbalanced chromosomal abnormalities after parental chromosome analysis. RESULTS: Mean follow-up after chromosome analysis was 5.8 years. 120 of 247 (49%) carrier couples had one or more miscarriage after chromosome analysis compared with 122 of 409 (30%) non-carrier couples (difference 19%, 95% confidence interval 11% to 26%; P < 0.01). The percentage of couples with at least one healthy child was not significantly different in carrier couples (83%) and non-carrier couples (84%) (difference -1%, - 7% to 5%). Among 550 pregnancies in carrier couples, two viable unbalanced chromosome abnormalities were detected at prenatal diagnosis (0.4%) and the fetuses aborted and two children with an unbalanced karyotype were born (0.4%). CONCLUSIONS: Couples whose carrier status was ascertained after two or more miscarriages have a low risk of viable offspring with unbalanced chromosomal abnormalities. Their chances of having a healthy child are as high as non-carrier couples, despite a higher risk of miscarriage.

Counselor-counselee interaction in reproductive genetic counseling: Does a pregnancy in the counselee make a difference?

OBJECTIVE: To investigate the influence of a pregnancy and other counselee characteristics on several aspects of counselor-counselee interaction during the initial clinical genetic consultation. METHODS: The consultations, of a group of pregnant women (n = 82) and of a control group of non-pregnant women (n = 58), were compared specifically with regard to differences in global affective tone, extent of psychosocial exchange and women's participation in the decision-making process. Consultations were recorded, and subsequently coded from audiotape by 10 raters. RESULTS: Only two differences in outcome measures were found between the two study groups: the counselor was rated as slightly more nervous in consultations with pregnant women, and in consultations with non-pregnant women fewer decisions were taken. The length of the consultation, the contribution of a counselee's companion to the consultation and counselee characteristics (age, level of education, initiation of referral, affected person, degree of worry and preferred participation in decision-making) were more important in explaining the nature of the interaction. CONCLUSION: Our study yielded no important differences in counselor-counselee interaction during the initial clinical genetic consultation of pregnant versus non-pregnant women regarding the affective tone of the consultation, the degree to which psychosocial issues were discussed and the women's participation in the decision-making process. PRACTICE IMPLICATIONS: Our findings suggest that a negatively affected counselor-counselee interaction is not an important disadvantage in consultations with pregnant women. Given the limitations of our study, however, we advocate further studies on counselor-counselee interaction in reproductive genetic counseling, in order to improve the quality of reproductive genetic counseling.

Selective chromosome analysis in couples with two or more miscarriages: case-control study.

OBJECTIVE: To identify additional factors, such as maternal age or factors related to previous reproductive outcome or family history, and the corresponding probability of carrying a chromosome abnormality in couples with two or more miscarriages. DESIGN: Nested case-control study. SETTING: Six centres for clinical genetics in the Netherlands. PARTICIPANTS: Couples referred for chromosome analysis after two or more miscarriages in 1992-2000; 279 carrier couples were marked as cases, and 428 non-carrier couples served as controls. MAIN OUTCOME MEASURES: Independent factors influencing the probability of carrier status and the corresponding probability of carrier status. RESULTS: Four factors influencing the probability of carrier status could be identified: maternal age at second miscarriage, a history of three or more miscarriages, a history of two or more miscarriages in a brother or sister of either partner, and a history of two or more miscarriages in the parents of either partner. The calculated probability of carrier status in couples referred for chromosome analysis after two or more miscarriages varied between 0.5% and 10.2%. CONCLUSIONS: The probability of carrier status in couples with two or more miscarriages is modified by additional factors. Selective chromosome analysis would result in a more appropriate referral policy, could decrease the annual number of chromosome analyses, and could therefore lower the costs.

Mutations in the human BOULE gene are not a major cause of impaired spermatogenesis.

Mutation screening of the BOULE gene in 156 men with azoospermia or severe oligozoospermia revealed no relevant mutations; thus, mutations in BOULE can be eliminated as a major cause of impaired spermatogenesis.

Familial clustering of impaired spermatogenesis: no evidence for a common genetic inheritance pattern.

BACKGROUND: The aetiology of impaired spermatogenesis is unknown in the majority of cases. Evidence of a contribution of genetic factors is still scarce. Therefore, the aim of our study was to assess whether male factor subfertility due to impaired spermatogenesis has a familial component and to test different genetic models of inheritance. METHODS: Cases were all men with severe idiopathic impaired spermatogenesis attending our fertility clinic from January 1998 until December 2001. Controls were all men with normozoospermia attending our fertility clinic in the same period. Family data were collected from the medical records and by additional interviews of the probands. If subfertility of a first-degree relative was mentioned, permission was sought to contact the affected family member in order to obtain all medical information available, including the results of semen analyses. RESULTS: In total, 160 patients and 285 controls were included in the analysis. Family size and number of brothers and sisters were equally distributed in both groups. In the patient group, 1.63% of the brothers who had tried to father a child were mentioned to be subfertile compared to 5.8% in the control group [odds ratio 3.18 (95% confidence interval 1.59-6.37)]. The subfertility among the brothers in the patient group was more often due to reduced semen parameters compared to the control group. The data did not fit with frequent autosomal dominant or recessive segregation. CONCLUSION: Male factor subfertility due to impaired spermatogenesis appears to cluster in families. Our data suggests that heritable genetic factors play a role in a limited number of cases. Impaired spermatogenesis is not caused by a common genetic defect, but is most likely a complex disease in which several different factors play a role.

Chromosomal region 11p15 is associated with male factor subfertility.

The molecular aetiology of male factor subfertility, due to impaired spermatogenesis, is still unknown in the majority of cases. It is thought to be a complex disorder in which multiple genes are implicated. Cryptorchidism and reduced fecundity are symptoms in male Beckwith-Wiedemann patients and the ZNF214 and ZNF215 genes, localized on chromosomal region 11p15, are associated with this syndrome. We hypothesized that the ZNF214 and ZNF215 genes, which are predominantly expressed in the testis, could be involved in male factor subfertility in patients with idiopathic impaired spermatogenesis or in patients with impaired spermatogenesis due to cryptorchidism. Male partners of subfertile couples with idiopathic azoo- or severe oligozoospermia, male partners with azoo- or severe oligozoospermia and cryptorchidism in their medical history and men with normozoospermia were screened for nine single nucleotide polymorphisms in the ZNF214 and ZNF215 genes. An association study was performed based on allele and estimated haplotype frequencies. Statistically significant differences in allele frequencies and in estimated haplotype frequencies were found in both patient groups compared with controls. Thereafter, both genes were screened for mutations in all patients by PCR and single strand conformation polymorphism analysis. Aberrant patterns were confirmed by DNA sequencing. Mutation analysis in ZNF214 and ZNF215 revealed five new variants in the patients that were not present in the controls. At least three of these mutations were inherited from the mother. Our results suggest that chromosomal region 11p15 is associated with male factor subfertility due to impaired spermatogenesis with and without cryptorchidism.

Partial DAZ deletions in a family with five infertile brothers.

OBJECTIVE: To study the genetic cause of infertility in a family with five infertile brothers. DESIGN: Case report. SETTINGS: Center for reproductive medicine at a university medical center. PATIENT(S): Five brothers presenting with primary infertility due to severely impaired spermatogenesis; also, their parents and two other paternally related family members. INTERVENTION(S): Fluorescence in situ hybridization and sequence family variant analysis was performed in leukocyte DNA to determine the number of deleted in azoospermia (DAZ) genes. Linkage analysis was performed for X chromosome inheritance, and mitochondrial DNA (mtDNA) was screened for mutations. MAIN OUTCOME MEASURE(S): DAZ gene copy number, X chromosome linkage, and mtDNA sequence. RESULT(S): With conventional polymerase chain reaction (PCR) analysis, no deletions of the AZFc region were found, but with fluorescence in situ hybridization and sequence family variant analysis, only two DAZ genes instead of four were detected in all individuals tested. The five brothers did not share an identical X chromosomal locus, and no mutations were found in the mtDNA of the index patient. CONCLUSION(S): A reduced copy number of the DAZ genes is found in five infertile brothers with severely impaired spermatogenesis, as well as in their normospermic father and in two other fertile paternally related family members. This illustrates that the phenotype associated with a reduced copy number of the DAZ genes can be extremely variable.

Referral for genetic counselling during pregnancy: limited alertness and awareness about genetic risk factors among GPs.

BACKGROUND: In many countries, GPs play a key role in the referral to other medical specialists. Referral for reproductive genetic counselling during a pregnancy of women with a genetic risk factor already present before pregnancy has many disadvantages. Nevertheless, some 10-20% of the counsellees who attend a Department of Clinical Genetics for the first time are pregnant. OBJECTIVES: We aimed to explore the role of the GP in referring women for genetic counselling during, instead of before a pregnancy. METHOD: The GPs of 100 pregnant women who received genetic counselling were invited to participate in the study and asked to complete a questionnaire. The topics were: initiation and discussion of aspects of referral to the Department of Clinical Genetics; reasons for the referral during, instead of before a pregnancy; knowledge of genetic counselling; attitudes towards genetic counselling before a pregnancy; and attitudes towards abortion. RESULTS: To our surprise, 29% of the GPs indicated that they had not been involved in the referral to the Department of Clinical Genetics at all. Furthermore, the referral was initiated by the patient herself in most cases (40%) and by the GPs in 31% of the cases. Of the GPs who were involved in the referral, most of them (79%) talked to their patients to different extents about what to expect from their visit to the Department of Clinical Genetics; however, potential choices after an adverse outcome at prenatal diagnosis were discussed less often (60%). The main reason for referring the patient during, instead of before her pregnancy was because the GP was unaware of a potential risk factor before pregnancy (71%) and, consequently, never had a chance to talk about a referral before (71%). Other reasons for referral during pregnancy mentioned by the GPs were reassuring the patient about the health of her unborn child (32%) and the wish of the patient to be referred during pregnancy (31%). GPs considered their knowledge of clinical genetics to be limited (mean score 5, on a scale from 0 to 10). The majority of the GPs were in favour of genetic counselling taking place before, instead of during pregnancy, and they had no great objections to abortion. CONCLUSIONS: During pregnancy, the gatekeeper function of the GP in the referral for genetic counselling is undermined. Limited alertness and awareness among GPs about genetic risk factors in their patients played a major role in this undermined function and in the less appropriate timing of referral. Neither insufficient knowledge nor barriers to acceptance explained this lack of alertness and awareness. We advocate the implementation of routine family history taking in general practice.

The use of spermHALO-FISH to determine DAZ gene copy number.

The AZFc region of the human Y chromosome is frequently deleted in men with spermatogenic failure and contains many multicopy genes. The best-characterized gene family within this region is the Deleted in AZoospermia (DAZ) gene family, which is present in four nearly identical copies. Recent reports claim deletions of some but not all DAZ genes. The assays used in these studies, however, are unable to provide conclusive evidence on the number of DAZ genes. In this study we show that with the use of highly decondensed sperm nuclei with large DNA domains (spermHALO) it is possible to determine the number of DAZ genes accurately. Using this fluorescent in-situ hybridization (FISH) technique, which has both high resolution and high range, we show that in 10 normospermic men, in which PCR digest assays indicated a deletion of one or more DAZ genes, all four DAZ genes were present. Also we confirmed previous findings of a deletion of two DAZ genes in two men and identified a man with six DAZ genes. Our results indicate that spermHALO-FISH allows an accurate determination of DAZ gene copy number, while PCR digest assays do not. Therefore, confirmation of positive results from PCR digest assays with spermHALO-FISH is essential. Furthermore, the spermHALO-FISH technique should prove useful as a genetic mapping technique in other regions of the Y chromosome and similar repetitive regions throughout the genome.

Clinical relevance of partial AZFc deletions.

To determine the number of DAZ gene clusters in the Y-bearing spermatozoa of patients who underwent intracytoplasmic sperm injection (ICSI) and to compare the outcome with the number of clusters found in the spermatozoa of normospermic men. Prospective study. Academic hospital.Forty-seven patients with impaired spermatogenesis who were attending our clinic for ICSI and 56 semen donors. Peripheral blood was drawn to obtain somatic DNA for polymerase chain reaction (PCR) analysis and leukocytes for karyotyping and FISH analysis. Three-color FISH was performed on the spermatozoa remaining after ICSI and on the spermatozoa of semen donors to determine the presence of the X and Y chromosome as well as the number of DAZ gene clusters. Number of DAZ gene clusters in Y-bearing spermatozoa. Five patients had only one DAZ gene cluster, one patient had a complete AZFc deletion, and one patient had three clusters on average. One of the semen donors also showed three DAZ gene clusters in his Y-bearing spermatozoa. None of the semen donors had only one DAZ gene cluster. Besides complete AZFc deletions, partial deletions are also associated with impaired spermatogenesis. As a result, these partial deletions that are not recognized by routine PCR are reintroduced into the population by the ICSI technique.

Long-term follow-up of infants after transcervical chorionic villus sampling and after amniocentesis to compare congenital abnormalities and health status.

OBJECTIVES: Next to procedure-related fetal loss, other adverse effects of invasive prenatal diagnosis have been reported: limb defects after chorionic villus sampling (CVS) or early amniocentesis and respiratory distress after amniocentesis (AC). Because minor abnormalities may be overlooked in routine follow-up, we obtained long-term follow-up data after CVS and AC. METHODS: 1509 women with a singleton pregnancy who had transcervical CVS were matched by age and season of conception with 1509 women with singleton pregnancies who had AC. All procedures were performed during 1985-1991 for advanced maternal age >35 years. Data regarding congenital malformations (classified according Eurocat), neonatal and paediatric morbidity and complications of motor development, speech, hearing and visual function were obtained by questionnaire in 1993-1995. RESULTS: Short-term outcome was known in all but ten infants. Questionnaires with a structured design were mailed to all women with a surviving infant (n=2810); 86.7% responded. No difference was detected between infants after CVS compared with infants after AC regarding congenital malformations (7.2% versus 6.3%), neonatal morbidity (15.1% versus 15.9%), paediatric morbidity with clinical treatment (7.7% versus 6.3%) or outpatient treatment only (43.9% versus 40.3%) and evident function disturbance (2.0% versus 2.0%) or doubtful function disturbance (6.3% versus 6.8%). The number of infants with physical growth <10th centile for Dutch infants was equal (10.1%). Sub-analysis for limb abnormalities or respiratory complications did not demonstrate differences between infants after CVS and AC. Only 10% of all congenital malformations were already known through routine post-partum follow-up. CONCLUSIONS: An extensive long-term survey could not demonstrate differences of health status between infants after prenatal diagnosis by transcervical CVS and AC.

Trisomy 13 or 18 (mosaicism) in first trimester cytotrophoblast cells: false-positive results in 11 out of 51 cases.

OBJECTIVE: The finding of full or mosaic trisomy 13 or 18 in first trimester chorionic villus sampling (CVS) may be a false-positive result. This report provides incidence and outcome information that may be helpful in counselling individual patients and in choosing adequate follow-up. STUDY DESIGN: From a series of 6820 CVS cases, we retrospectively collected data on all patients (n=51) with full (n=30) or mosaic (n=5) trisomy 18, and full (n=13) or mosaic (n=3) trisomy 13 in cytotrophoblast cells. RESULTS: Five false-positives were seen in patients with full trisomy 18 and three in the mosaic cases. One false-positive result was observed in full trisomy 13 and two false-positives in cases of mosaicism. No false-negative results were reported. CONCLUSION: The diagnosis of trisomy 13 or 18 in cytotrophoblasts should be confirmed in other tissues, unless fetal abnormalities are seen at ultrasound. In case of mosaicism, follow-up amniocentesis is advised.

Reduced copy number of DAZ genes in subfertile and infertile men.

OBJECTIVE(S): To determine the copy number and identity of the DAZ genes on the Y chromosomes of infertile patients. DESIGN: Prospective study. SETTING: University medical center. PATIENT(S): One hundred and thirty-nine patients with male factor infertility. INTERVENTION(S): The separate genes were detected by polymerase chain reaction (PCR) digestion assays of sequence family variants in leukocyte DNA and by fluorescence in situ hybridization of interphase nuclei and chromatin fibers. MAIN OUTCOME MEASURE(S): Number of DAZ genes present. RESULT(S): One hundred twenty-nine patients had four genes, 6 patients had two genes, and 4 patients had none. Three patients had a deletion of the two proximal DAZ genes, and three were missing both distal genes. Semen analysis showed a less severe phenotype in patients with only two DAZ genes compared with patients missing all four genes. CONCLUSION(S): In six patients, two different partial deletions were found that were not detected by PCR with conventional markers. One patient with an AZFb deletion appeared to also have a partial AZFc deletion that was not detected by routine PCR. Phenotypic differences between patients with different deletions suggest a dose effect of the DAZ genes.