Preimplantation genetic testing for more than one genetic condition: clinical and ethical considerations and dilemmas.

STUDY QUESTION: Which clinical and ethical aspects of preimplantation genetic testing for monogenic disorders or structural rearrangements (PGT-M, PGT-SR) should be considered when accepting requests and counselling couples for PGT when applied for more than one condition (combination-PGT; cPGT-M/SR)? SUMMARY ANSWER: cPGT is a feasible extension of the practice of PGT-M/SR that may require adapting the criteria many countries have in place with regard to indications-setting for PGT-M/SR, while leading to complex choices that require timely counselling and information. WHAT IS KNOWN ALREADY: Although PGT-M/SR is usually performed to prevent transmission of one disorder, requests for PGT-M/SR for more than one condition (cPGT-M/SR) are becoming less exceptional. However, knowledge about implications for a responsible application of such treatments is lacking. STUDY DESIGN, SIZE, DURATION: Retrospective review of all (40) PGT-M/SR applications concerning more than one genetic condition over the period 1995-2018 in the files of the Dutch national PGT centre. This comprises all relevant national data since the start of PGT in the Netherlands. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Data regarding cPGT-M/SR cases were collected by means of reviewing medical files of couples applying for cPGT-M/SR. Ethical challenges arising with cPGT-M/SR were explored against the background of PGT-M/SR regulations in several European countries, as well as of relevant ESHRE-guidance regarding both indications-setting and transfer-decisions. MAIN RESULTS AND THE ROLE OF CHANCE: We report 40 couples applying for cPGT-M/SR of which 16 couples started their IVF treatment. Together they underwent 39 IVF cycles leading to the birth of five healthy children. Of the couples applying for cPGT, 45% differentiated between a primary and secondary condition in terms of perceived severity. In the light of an altered balance of benefits and drawbacks, we argue the 'high risk of a serious condition' standard that many countries uphold as governing indications-setting, should be lowered for secondary conditions in couples who already have an indication for PGT-M/SR. As a consequence of cPGT, professionals will more often be confronted with requests for transferring embryos known to be affected with a condition that they were tested for. In line with ESHRE guidance, such transfers may well be acceptable, on the condition of avoiding a high risk of a child with a seriously diminished quality of life. LIMITATIONS, REASONS FOR CAUTION: We are the first to give an overview of cPGT-M/SR treatments. Retrospective analysis was performed using national data, possibly not reflecting current trends worldwide. WIDER IMPLICATIONS OF THE FINDINGS: Our observations have led to recommendations for cPGT-M/SR that may add to centre policy making and to the formulation of professional guidelines. Given that the introduction of generic methods for genomic analysis in PGT will regularly yield incidental findings leading to transfer requests with these same challenges, the importance of our discussion exceeds the present discussion of cPGT. STUDY FUNDING/COMPETING INTEREST(S): The research for this publication was funded by the Dutch Organization for Health Research and Development (ZonMw), project number: 141111002 (Long term safety, quality and ethics of Preimplantation Genetic Diagnosis). None of the authors has any competing interests to declare.

BRCA1 mutation carriers have a lower number of mature oocytes after ovarian stimulation for IVF/PGD.

PURPOSE: The aim of this study was to determine whether BRCA1/2 mutation carriers produce fewer mature oocytes after ovarian stimulation for in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD), in comparison to a PGD control group. METHODS: A retrospective, international, multicenter cohort study was performed on data of first PGD cycles performed between January 2006 and September 2015. Data were extracted from medical files. The study was performed in one PGD center and three affiliated IVF centers in the Netherlands and one PGD center in Belgium. Exposed couples underwent PGD because of a pathogenic BRCA1/2 mutation, controls for other monogenic conditions. Only couples treated in a long gonadotropin-releasing hormone (GnRH) agonist-suppressive protocol, stimulated with at least 150 IU follicle stimulating hormone (FSH), were included. Women suspected to have a diminished ovarian reserve status due to chemotherapy, auto-immune disorders, or genetic conditions (other than BRCA1/2 mutations) were excluded. A total of 106 BRCA1/2 mutation carriers underwent PGD in this period, of which 43 (20 BRCA1 and 23 BRCA2 mutation carriers) met the inclusion criteria. They were compared to 174 controls selected by frequency matching. RESULTS: Thirty-eight BRCA1/2 mutation carriers (18 BRCA1 and 20 BRCA2 mutation carriers) and 154 controls proceeded to oocyte pickup. The median number of mature oocytes was 7.0 (interquartile range (IQR) 4.0-9.0) in the BRCA group as a whole, 6.5 (IQR 4.0-8.0) in BRCA1 mutation carriers, 7.5 (IQR 5.5-9.0) in BRCA2 mutation carriers, and 8.0 (IQR 6.0-11.0) in controls. Multiple linear regression analysis with the number of mature oocytes as a dependent variable and adjustment for treatment center, female age, female body mass index (BMI), type of gonadotropin used, and the total dose of gonadotropins administered revealed a significantly lower yield of mature oocytes in the BRCA group as compared to controls (p = 0.04). This finding could be fully accounted for by the BRCA1 subgroup (BRCA1 mutation carriers versus controls p = 0.02, BRCA2 mutation carriers versus controls p = 0.50). CONCLUSIONS: Ovarian response to stimulation, expressed as the number of mature oocytes, was reduced in BRCA1 but not in BRCA2 mutation carriers. Although oocyte yield was in correspondence to a normal response in all subgroups, this finding points to a possible negative influence of the BRCA1 gene on ovarian reserve.

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice.

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection.

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.

[Results from 10 years of preimplantation-genetic diagnostics in The Netherlands]. / Resultaten van 10 jaar preimplantatiegenetische diagnostiek in Nederland.

OBJECTIVE: To report the data from couples who were referred for preimplantation-genetic diagnostics (PGD) and treatment due to a significantly increased risk of offspring with a serious genetic disorder. DESIGN: Descriptive, prospective. METHOD: Data were collected from couples that underwent PGD in the period 1993/'03 at Maastricht University Hospital. Embryos produced by means of in-vitro fertilisation (IVF) were subjected to genetic tests several days after fertilisation. Subsequently 1 or 2 unaffected embryos were transferred to the uterus. Where there was an increased risk of a male with an X-linked genetic disorder, the gender was determined using fluorescence in-situ hybridisation (FISH). This method was also used to detect structural chromosomal abnormalities. The polymerase chain reaction (PCR) method was used for mutation detection and/or marker analysis of monogenetic disorders. RESULTS: A total of 691 couples were referred for PGD. The most frequent indications were X-linked disorders (30%), in particular Fragile-X syndrome, Duchenne/Becker muscular dystrophy and haemophilia A/B. This was followed by autosomal dominant disorders (26%), such as Huntington's disease and myotonic dystrophy, and then structural chromosomal abnormalities (24%). A total of 120 women underwent 260 PGD cycles. An embryo transfer was possible in 158 of the cycles and this resulted in 45 successful pregnancies. The pregnancy rate was 17% per cycle initiated and 28% per cycle with embryo transfer. Up until december 2003 29 singletons, 8 sets of twins and 1 set of triplets were born. There were no misdiagnoses and none of the babies had congenital abnormalities. CONCLUSION: PGD was a reliable and successful method, with pregnancy rates similar to those of IVF or intracytoplasmatic sperm injection. PGD should be stated as an alternative during the preconception counselling of couples with an increased genetic risk, especially for disorders where PGD can be routinely applied, such as Huntington's disease, myotonic dystrophy, cystic fibrosis, spinal muscular atrophy, Fragile-X syndrome and structural chromosomal abnormalities.

Accuracy of CO(2) angiography in vessel diameter assessment: a comparative study of CO(2) versus iodinated contrast material in a porcine model.

PURPOSE: To compare, with use of intravascular ultrasound (IVUS) as an internal reference standard in a porcine model, arterial diameters measured from arteriograms obtained with use of CO(2) to those obtained with use of iodinated contrast material (ICM). MATERIALS AND METHODS: In nine pigs, digital subtraction angiograms (DSAs) were obtained in the aorta and iliac arteries to compare vessel diameters measured with use of CO(2) to those measured with use of ICM. These measurements were divided by measurements made with use of intravascular ultrasound (IVUS) to yield a DSA/IVUS ratio. Differences between ICM and CO(2) were compared with analysis of variance to assess the effect of location (aorta vs iliac), contrast material used (ICM vs CO(2)), and position (posteroanterior, right anterior oblique, or left anterior oblique). Secondary analysis compared measurements of dependent and nondependent iliac arteries and compared the use of hand-injected CO(2) to that of CO(2) injected by an injector. RESULTS: The DSA/IVUS ratio was 70.7% +/- 4.4% with ICM use and 69.6% +/- 6.3% with CO(2) use, which did not represent a significant difference (P =.311). Animal position had no effect (P =.477). Underestimation was worse in the iliac arteries than in the aorta (67.4% +/- 1.5% vs 71.4% +/- 1.7%; P =.038). There was no difference in nondependent (P =.163) arteries, but CO(2) underestimated dependent iliac artery size more than ICM did (66.3% +/- 4.8% vs 70.3% +/- 5.4%; P =.051). Vessel diameter was underestimated more with the CO(2) injector than with hand-injected CO(2) (64.3% +/- 2.3% vs 71.7% +/- 1.7%; P <.0001). CONCLUSION: There is no difference in diameter underestimation between CO(2) and ICM in this animal model. Hand-injection of CO(2) causes less underestimation of vessel diameter than does the CO(2) injector.

The effects of a mechanical thrombolytic device on normal canine vein valves.

PURPOSE: To determine if the Arrow-Trerotola Percutaneous Thrombolytic Device (PTD) causes damage to normal vein valves. MATERIALS AND METHODS: Ten lateral saphenous veins in five dogs were studied with descending venography with use of a wedge balloon catheter positioned above 48 valves (demonstrating 51 valves) before and after five antegrade passes each with an over-the-wire (0.025-inch), 6.5-F, 9-mm-diameter PTD. Vein diameters were 3.2-11.4 mm (mean, 5.9 mm). Contrast matter was injected at incremental rates from 3 to 15 mL/min during continuous pressure monitoring. Imaging was performed with digital subtraction angiography at a rate of 1 frame/sec. The time to valve reflux was determined by noting the frame at which reflux was first seen through the valve. The time to reflux and pressure required to reflux were compared before and after the PTD passes. All vessels were explanted and evaluated histologically for presence or absence of endothelial loss, thrombus formation, inflammation, or valve degeneration. Four veins in two animals were studied with venography to determine the variability of the venographic method. These veins thrombosed during venography and therefore served as positive pathologic controls. In two animals, one vein was studied with venography and one was not studied to provide pathologic controls. RESULTS: With use of two physiologic tests of valve function, 77% of valves had minimal or no damage as assessed by valve competency and 80% had minimal or no damage as demonstrated by the change in the pressures the valve can withstand before reflux. Twenty-six of 51 valves (51%) had no difference or later reflux after PTD use. Thirteen (26%) refluxed 1 second earlier after PTD use and 12 (23%) refluxed > or =2 seconds earlier (six at 2, four at 3, and two at 4). Four of the six valves with more than a 2-second difference in reflux times were in valves with diameters less than 4.2 mm. All these vessels were smaller than 7 mm in diameter. Twenty-one of 48 valve levels (44%) had no difference or sustained higher pressures before reflux after PTD use. Seventeen (36%) had a pressure drop of <10 mm Hg; five (10%) had drops of 12-24 mm Hg; and five (10%) had drops of more than 40 mm Hg. There was a significant difference in endothelial loss, thrombus formation, and inflammation between experimental veins, the veins with thrombus, the venography controls, and the normal vein controls. There was significant difference only in terms of inflammation when the experimental group was compared to the thrombosis group. CONCLUSION: The antegrade use of the PTD across normal canine vein valves does not cause physiologically significant damage in valves 7 mm or larger in diameter in this animal model.

Preclinical in vivo testing of the Arrow-Trerotola percutaneous thrombolytic device for venous thrombosis.

PURPOSE: To test the safety and efficacy of using the Arrow-Trerotola percutaneous thrombolytic device (PTD) for treating deep vein thrombosis (DVT) in an animal model. MATERIALS AND METHODS: An established canine model of iliocaval subacute thrombosis was used. Thrombosis was caused by balloon occlusion of the infrarenal inferior vena cava (IVC) for 7 (n = 12), 10 (n = 1), or 17 (n = 1) days. Treatment was performed with use of an 8-F, over-the-wire (0.035-inch) PTD with a 15-mm-diameter basket. The procedure was performed without IVC filtration. Two acute procedures were performed and 12 procedures were intended as survival procedures with 30-day follow-up. Pulmonary arteriography, blood gases, and pulmonary artery pressure measurement were performed before and after the procedure, and at follow-up. The animals were killed after the follow-up procedure and their IVC, iliac veins, and lungs were removed and examined histologically. Heparin was used intraprocedurally but thrombolytic agents were not used. Low-molecular-weight heparin was given daily after the procedure. RESULTS: Thrombolysis was completely (12 of 13) or partially (one of 13) successful in all animals in the 7- and 10-day groups, but was unsuccessful in the animal in the 17-day group (n = 1). Variable amounts of segmental and subsegmental pulmonary emboli were found in all animals with small increases in pulmonary artery pressure. Two animals died within 6 days of the procedure, possibly due to pulmonary emboli. At 30-day follow-up, IVC patency was preserved in 80% (eight of 10) of animals, but significant caval narrowing due to intimal hyperplasia was noted at follow-up. All pulmonary emboli had resolved angiographically at follow-up, but evidence of recanalized or resolving pulmonary thromboemboli was found in seven of the 12 surviving animals. No acute vascular injury (eg, perforation) occurred. CONCLUSION: The modified PTD used in this study is effective in treating subacute (<7 days old) venous thrombosis, but temporary filtration will probably be necessary to keep pulmonary emboli to a minimum during the procedure. The 30-day patency is encouraging. The results in this animal model indicate that the Arrow-Trerotola PTD may be useful in the percutaneous treatment of DVT in humans.

Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis.

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.

Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos.

In this study, the use of Ca2+/Mg2+-free medium for biopsy of human embryos at the 4- to 10-cell stage on the third day of development was evaluated. When compared with control medium containing normal concentrations of Ca2+ and Mg2+ ions, the use of Ca2+/Mg2+-free medium allows an easier removal of blastomeres as illustrated by a lower rate of cell lysis as well as by a shorter time needed to perform the procedure. Subsequent embryo development to the blastocyst stage is not affected by the choice of biopsy medium, not even when embryos are exposed to the medium for 45 min. The use of Ca2+/Mg2+-free medium thus allows for an easier biopsy procedure during pre-implantation genetic diagnosis, while it does not result in a loss of developmental potential of the embryo to the blastocyst stage.

Preimplantation genetic diagnosis of spinal muscular atrophy.

After Duchenne muscular dystrophy, spinal muscular atrophy (SMA) is the most common severe neuromuscular disease in childhood. Since 1995, homozygous deletions in exon 7 of the survival motor neuron (SMN) gene have been described in >90-95% of SMA patients. However, the presence of a highly homologous SMN copy gene complicates the detection of exon 7 deletions. This paper describes the adjustment and evaluation of an established SMN exon 7 polymerase chain reaction (PCR) protocol at the single cell level, and the first preimplantation genetic diagnosis (PGD) of SMA with this PCR protocol. To determine PCR efficiency and allelic loss, 200 leukocytes of normal individuals, SMA carriers and patients, and 25 blastomeres were tested. The PCR efficiency of the SMN exon 7 and the adjacent copy gene sequence, tested in the leukocytes, were 90% and 91% respectively. No allelic loss was detected. One out of 25 blastomeres tested revealed a negative PCR signal for the SMN exon 7 sequence. All 25 showed the copy gene sequence. PGD of SMA was offered to a couple with an affected child homozygous for the SMN exon 7 deletion. After intracytoplasmic sperm injection, four and five embryos could be genotyped for the SMN exon 7 in two cycles respectively. After embryo transfer in the second PGD cycle an ongoing gemelli pregnancy was achieved. This study demonstrates that PGD for SMA is feasible when a previous child is homozygous for the SMN exon 7 deletion.

[Genetic diagnosis of IVF embryos: preliminary results from 'preimplantation genetic diagnoses' in the Netherlands]. / Genetische diagnostiek bij IVF-embryo's: eerste ervaringen met 'preïmplantatiegenetische diagnostiek' in Nederland.

Preimplantation genetic diagnosis (PGD) is a very early form of genetic testing. It involves testing one or two cells taken from a recent embryo of eight cells produced by in vitro fertilization, and selective transfer of genetically normal embryos. So far in the Academic Hospital Maastricht, the Netherlands, 20 couples have undergone PGD, resulting in 6 ongoing pregnancies (one twin pregnancy). In three women the indications for PGD were: cystic fibrosis, sex-linked Pelizaeus-Merzbacher disease and chromosomal translocation, respectively. In the Netherlands PGD is only allowed if there is a high risk of a serious genetic disease. PGD can be carried out in Maastricht for: cystic fibrosis, sex-linked diseases, chromosomal abnormalities, fragile X syndrome, spinal muscular atrophy and myotonic dystrophy. The advantage of PGD is that it excludes the necessity of a therapeutic abortion. Disadvantages ages are the requirement of in vitro fertilization, which has only a 15-20% pregnancy rate, and the experimental nature of the PGD procedure. To date, about 200 children have been born worldwide following PGD.

Photon-absorbed fractions for cylindrical geometry: a TLD photon-absorbed fraction model.

The purpose of this study is to develop a mathematical model and calculate photon-absorbed fractions in a homogeneous nonradioactive cylinder placed inside off-center and outside a cylindrical homogeneous distribution of activity. In the second case, both the radioactive cylinder and the nonradioactive one are placed in a tissue-equivalent nonradioactive medium. The values of the photon-absorbed fractions are investigated for various geometrical configurations using water as the material filling the cylinders and the medium in between and an isotope commonly used in Nuclear Medicine, 99mTc. The calculations for off-center cylinders allows for modeling inhomogeneous distributions of activity within a tumor by placing several "cold" cylinders of various sizes in a radioactive finite cylinder. This three-dimensional model calculates photon-absorbed fractions for inhomogeneous activity distributions that can be used in quantitative nuclear medicine for self-absorption correction, thus introducing a more realistic correction than the one-dimensional corrections. These calculations are also used to model the response of a cylindrical TLD (thermoluminiscent dosimeter) placed inside a homogeneous radioactive cylinder and outside the homogeneous radioactive cylinder, in an absorbing nonradioactive surrounding medium. The purpose of these calculations is to evaluate the photon-absorbed fraction in the TLD as an instrument of measuring the time-integrated activity of a homogeneous radioactive source versus an inhomogeneous one. The dependence of the TLD-absorbed fraction on the position of the TLD with respect to the radioactive cylinder is investigated.

Photon absorbed fractions for cylindrical geometry: homogeneous nonradioactive cylinder containing a homogeneous cylindrical distribution of activity.

The purpose of this study was to calculate photon absorbed fractions in tissue surrounding a radioactive source where both the source and the surrounding tissue are assumed to have cylindrical geometry. Specifically, we treated two cases: the case of a cylindrical source of homogeneous activity placed in air, and second, the case of a cylindrical source of homogeneous activity placed in a cylindrical nonradioactive absorbing material. In this study we offer an analytical solution to these problems followed by numerical integration. The computer program allowed for very general calculations, e.g., different tissues, different geometrical setups. Tables of absorbed fractions have been developed for commonly used radionuclide energies and tissue-equivalent material. A comparison between our results and the results of other related studies showed the advantages and limitations of this approach.

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome.

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR-1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.

Multiplex polymerase chain reaction for sex determination of single mouse blastomeres.

Using a multiplex nested polymerase chain reaction (PCR) method with single copy genes and a dinucleotide repeat locus for the mouse Y and X chromosomes respectively, it was possible to discriminate between single cells derived from male and female embryos. Using single cells, amplification of Sry and Zfy sequences was not evident in all cases. It could be calculated that, with the PCR method used, 0.04% [95% (confidence interval 0.00-2.03)] of the male embryos would erroneously be diagnosed as female if analysis is performed on two cells. The calculated chance for total amplification failure, if two cells are used for analysis, would be 1.4% [95% (confidence interval 0.04-8.04)]. The mouse embryo model proved to be a helpful tool to develop skills in the application of PCR for preimplantation genetic diagnosis at the single cell level.

Prediction of mental status in carriers of the fragile X mutation using CGG repeat length.

For genetic counseling in fragile X (fra(X)) families, it is important to know the diagnostic impact of the CGG repeat length in carriers of the fragile X mutation. We have analyzed the CGG repeat length in 106 males and 73 females who had inherited the maternal fra(X) mutation. The sensitivity, specificity, and predictive value of the CGG repeat analyses, as measured on Southern blots of PstI/EcoRI digests, were calculated. In males the sensitivity, specificity and negative predictive value were 99%, 100% and 94%; respectively. In females the specificity (60%) and, consequently, the positive predictive value (82%) was reduced. Therefore, it remains impossible to predict accurately whether a female fetus demonstrating a full mutation will be affected. On the other hand, the negative predictive value of the CGG test in females was 100%. We have no evidence, as yet, that for female carriers with the full mutation, cytogenetic analysis is of additional value to predict the mental status. However, we have observed one mentally retarded fra(X) case with extreme mosaicism in which a typical premutation fragment was the predominant DNA species. Therefore, until more data and better DNA tests are available, we would like to advocate additional cytogenetic investigation if in a fetus a CGG repeat length in the premutation range is found.