The impact of embryo vitrification on placental histopathology features and perinatal outcome in singleton live births.

STUDY QUESTION: Does embryo vitrification affect placental histopathology pattern and perinatal outcome in singleton live births? SUMMARY ANSWER: Embryo vitrification has a significant effect on the placental histopathology pattern and is associated with a higher prevalence of dysfunctional labor. WHAT IS KNOWN ALREADY: Obstetrical and perinatal outcomes differ between live births resulting from fresh and frozen embryo transfers. The effect of embryo vitrification on the placental histopathology features associated with the development of perinatal complications remains unclear. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study evaluating data of all live births from one academic tertiary hospital resulting from IVF treatment with autologous oocytes during the period from 2009 to 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients had placentas sent for pathological evaluation irrelevant of maternal or fetal complications status. Placental, obstetric and perinatal outcomes of pregnancies resulting from hormone replacement vitrified embryo transfers were compared with those after fresh embryo transfers. A multivariate analysis was conducted to adjust the results for determinants potentially associated with the development of placental histopathology abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1014 singleton live births were included in the final analysis and were allocated to the group of pregnancies resulting from fresh (n = 660) and hormone replacement frozen (n = 354) embryo transfers. After the adjustment for confounding factors the frozen embryo transfers were found to be significantly associated with chorioamnionitis with maternal (odds ratio (OR) 2.0; 95% CI 1.2-3.3) and fetal response (OR 2.6; 95% CI 1.2-5.7), fetal vascular malperfusion (OR 3.9; 95% CI 1.4-9.2), furcate cord insertion (OR 2.3 95% CI 1.2-5.3), villitis of unknown etiology (OR 2.1; 95% CI 1.1-4.2), intervillous thrombi (OR 2.1; 95% CI 1.3-3.7), subchorionic thrombi (OR 3.4; 95% CI 1.6-7.0), as well as with failure of labor progress (OR 2.5; 95% CI 1.5-4.2). LIMITATIONS, REASONS FOR CAUTION: Since the live births resulted from frozen-thawed embryos included treatment cycles with previously failed embryo transfers, the factors over embryo vitrification may affect implantation and placental histopathology. WIDER IMPLICATIONS OF THE FINDINGS: The study results contribute to the understanding of the perinatal future of fresh and vitrified embryos. Our findings may have an implication for the clinical decision to perform fresh or frozen-thawed embryo transfer. STUDY FUNDING/COMPETING INTEREST(S): Authors have not received any funding to support this study. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Identification of the gene FMR2, associated with FRAXE mental retardation.

Five folate-sensitive fragile sites have been characterized at the molecular level (FRAXA, FRAXE, FRAXF, FRA16A and FRA11B). Three of them (FRAXA, FRAXE and FRA11B) are associated with clinical problems, and two of the genes (FMR1 in FRAXA and CBL2 in FRA11B) have been identified. All of these fragile sites are associated with (CCG)n/(CGG)n triplet expansions which are hypermethylated beyond a critical size. FRAXE is a rare folate sensitive fragile site only recently recognized. Its cytogenetic expression was found to involve the amplification of a (CCG)n repeat adjacent to a CpG island. Normal alleles vary from 6 to 25 copies. Expansions of greater than 200 copies were found in FRAXE expressing males and their FRAXE associated CpG island was fully methylated. An association of FRAXE expression with concurrent methylation of the CpG island and mild non-specific mental handicap in males has been reported by several groups. We now report the cloning and characterization of a gene (FMR2) adjacent to FRAXE. Elements of FMR2 were initially identified from sequences deleted from a developmentally delayed boy. We correlate loss of FMR2 expression with (CCG)n expansion at FRAXE, demonstrating that this is a gene associated with the CpG island adjacent to FRAXE and contributes for FRAXE-associated mild mental retardation.

A novel X-linked gene, G4.5. is responsible for Barth syndrome.

Barth syndrome is a severe inherited disorder, often fatal in childhood, characterized by cardiac and skeletal myopathy, short stature and neutropenia. The disease has been mapped to a very gene-rich region in distal portion of Xq28. We now report the identification of unique mutations in one of the genes in this region, termed G4.5, expressed at high level in cardiac and skeletal muscle. Different mRNAs can be produced by alternative splicing of the primary G4.5 transcript, encoding novel proteins that differ at the N terminus and in the central region. The mutations introduce stop codons in the open reading frame interrupting translation of most of the putative proteins (which we term 'tafazzins'). Our results suggest that G4.5 is the genetic locus responsible for the Barth syndrome.

Fragile X syndrome without CCG amplification has an FMR1 deletion.

We describe a patient with typical clinical features of the fragile X syndrome, but without cytogenetic expression of the fragile X or an amplified CCG trinucleotide repeat fragment. The patient has a previously uncharacterized submicroscopic deletion encompassing the CCG repeat, the entire FMR1 gene and about 2.5 megabases of flanking sequences. This finding confirms that the fragile X phenotype can exist, without amplification of the CCG repeat or cytogenetic expression of the fragile X, and that fragile X syndrome is a genetically homogeneous disorder involving FMR1. We also found random X-inactivation in the mother of the patient who was shown to be a carrier of this deletion.

Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDL; MIM 313400) is an X-linked recessive osteochondrodysplasia that occurs in approximately two of every one million people. This progressive skeletal disorder which manifests in childhood is characterized by disproportionate short stature with short neck and trunk, barrel chest and absence of systemic complications. Distinctive radiological signs are platyspondyly with hump-shaped central and posterior portions, narrow disc spaces, and mild to moderate epiphyseal dysplasia. The latter usually leads to premature secondary osteoarthritis often requiring hip arthroplasty. Obligate female carriers are generally clinically and radiographically indistinguishable from the general population, although some cases have phenotypic changes consistent with expression of the gene defect. The SEDL gene has been localized to Xp22 (refs 8,9) in the approximately 2-Mb interval between DXS16 and DXS987 (ref. 10). Here we confirm and refine this localization to an interval of less than 170 kb by critical recombination events at DXS16 and AFMa124wc1 in two families. In one candidate gene we detected three dinucleotide deletions in three Australian families which effect frameshifts causing premature stop codons. The gene designated SEDL is transcribed as a 2.8-kb transcript in many tissues including fetal cartilage. SEDL encodes a 140 amino acid protein with a putative role in endoplasmic reticulum (ER)-to-Golgi vesicular transport.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Abnormal expression of the KLF8 (ZNF741) gene in a female patient with an X;autosome translocation t(X;21)(p11.2;q22.3) and non-syndromic mental retardation.

Non-syndromic X linked mental retardation (MRX) is a heterogeneous group of conditions in which all patients have mental retardation as the only constant phenotypic feature. We have identified a female patient with mental retardation and a balanced translocation involving chromosomes X and 21, t(X;21)(p11.2;q22.3). Physical mapping of the translocation breakpoint on the human X chromosome was performed using fluorescence in situ hybridisation. We have mapped the X chromosome breakpoint to a 21 kb DNA fragment upstream of the first exon of the KLF8 (ZNF741) gene in Xp11.21. We have subsequently shown that the KLF8 transcript is no longer detected in cells from the patient, although KLF8 expression is otherwise normally present in control lymphoblasts. Mutation screening of probands from 20 unrelated XLMR families linked to the proximal short arm of the human X chromosome failed to show any mutation in the coding region of the KLF8 gene.

Principal mutation hotspot for central core disease and related myopathies in the C-terminal transmembrane region of the RYR1 gene.

The congenital myopathies are a group of disorders characterised by the predominance of specific histological features observed in biopsied muscle. Central core disease and nemaline myopathy are examples of congenital myopathies that have specific histological characteristics but significantly overlapping clinical pictures. Central core disease is an autosomal dominant disorder with variable penetrance which has been linked principally to the gene for the skeletal muscle calcium release channel (RYR1). Two recent reports have identified the 3' transmembrane domain of this gene as a common site for mutations. Two other studies have reported single families that have features of both central core disease and nemaline myopathy (core/rod disease) caused by mutations in RYR1. Screening of the 3' region (exons 93-105) of the RYR1 gene for mutations in 27 apparently unrelated patients with either central core disease or core/rod disease by single strand conformation polymorphism analysis and DNA sequencing identified three described and nine novel mutations in 15 patients.

X-linked mental retardation with dystonic movements of the hands (PRTS): revisited.

A family with a syndrome of mental retardation, dystonic movements of the hands, and dysarthria (MIM no. 309510) was described and mapped to Xp22 by Partington et al. (Am J Med Genet 1988; 30:251-262). The original localization encompassed the distal half of the short arm of the X chromosome, with a peak lod score of 2.1 at the DXS41 locus. The gene localization for this disorder (PRTS) has now been refined using recently characterized dinucleotide repeat markers. The PRTS gene maps between DXS365 and DXS28, an interval estimated to be less than 15 cM. A peak lod score of 3.01 at a recombination fraction of zero was generated by 2-point linkage analysis with the marker DXS989. Dystonic movements may be progressive and could be overlooked in children. Clinical assessments of affected men who are mentally retarded should be critically evaluated for this manifestation, where they belong to families in which the gene localization overlaps with PRTS.

X-linked mental retardation with heterozygous expression and macrocephaly: pericentromeric gene localization.

A family is described with X-linked mental retardation (XLMR) with affected males in 2 generations. The manifestations are macrocephaly and heterozygous expression. Linkage analysis gives a 2-point lod score of 3.31 (theta = 0.0) at the AR, DXS991, and MAOB marker loci. The gene is localized by recombination events between DXS1068 (Xp) and DXS1125 (Xq). This condition in this family may be similar to that described by Atkin et al., 1985 (Am J Med Genet 21:697-705).

Localization of non-specific X-linked mental retardation genes.

Gene localization was determined by linkage analysis in 5 families with non-specific X-linked mental retardation (MRX) and were MRX1, Xp11.4-q21.31; MRX10, Xp21.3-p11.4; MRX11, Xp21.3-p11.22; MRX12, Xp21.3-q21.1; and MRX13, Xp22.3-q21.22. Four of these localizations cross the dystrophin brain promoter, a candidate locus for MRX. None of the affected individuals who were tested showed variation suggestive of a deletion. No consistent clinical features were observed between or within 4 of the 5 families. In MRX12, prematurity or low birth weight, hypotelorism and short stature were seen in several affected males. Heterozygote manifestations occurred in 3 families. There was no evidence to suggest involvement of the same gene in more than one family, nor to clinically separate these families into distinct genetic entities. Non-overlapping localizations for MRX1 and MRX10 demonstrate the existence of at least 2 separate loci among these 5 families.

Pericentromeric genes for non-specific X-linked mental retardation (MRX).

Extensive linkage analyses in three families with non-specific X-linked mental retardation (MRX) have localized the gene in each family to the pericentromeric region of the chromosome. The MRX17 gene is localized with a peak lod of 2.41 (theta = 0.0) with the trinucleotide repeat polymorphism at the androgen receptor (AR) gene locus. This gene lies in the interval between the markers DXS255 and DXS990, as defined by recombinants. The MRX18 gene maps to the interval between the markers DXS538 and DXS1126, with a peak lod score of 2.01 (theta = 0.0) at the PFC gene locus. In the third family (Family E) with insufficient informative meioses for assignment of an MRX acronym, the maximum lod score is 1.8 at a recombination fraction of zero for several marker loci between DXS207 and DXS426. Exclusions from the regions of marker loci spanning Xq support the localization of the MRX gene in Family E to the pericentromeric region. Localizations of these and other MRX genes have determined that MRX2 and MRX19 map to distal Xp, MRX3, and MRX6 map to distal Xq, whilst the majority cluster in the pericentromeric region. In addition, we confirm that there are at least two distinct MRX genes near the centromere as delineated by the non-overlapping regional localizations of MRX17 and MRX18. Determination of these non-overlapping localizations is currently the only means of classifying non-syndromal forms of mental retardation and determining the minimum number of MRX loci.

New X-linked syndrome of mental retardation, gynecomastia, and obesity is linked to DXS255.

We describe 14 males from 3 successive generations in a family who have X-linked mental retardation (XLMR), obesity, gynecomastia, speech difficulties, emotional lability, tapering fingers, and small feet. Linkage analysis using markers spread along the X chromosome demonstrated a gene localisation close to the centromere. Maximum lod scores for markers near the centromere, all at theta = 0.00, were 1.36 for DXS72, and 1.46 for DXYS1. The closest flanking markers which showed recombination were DXS84 and DXS94, defining the physical localisation within Xp21.1-q22. DXS255 was fully informative with lod-1 confidence interval for theta of 0.00-0.12. Clinical findings and linkage data in this family distinguish it from the Börjeson-Forssman-Lehmann syndrome and other previously described XLMR syndromes.

Characterization of new PCR based markers for mapping and diagnosis: AC dinucleotide repeat markers at the DXS237 (GMGX9) and DXS102 (cX38.1) loci.

Genomic insert DNAs from 45 probes representing 113.4 kb of the X chromosome were screened for AC dinucleotide repeat sequence. Two new AC repeat sequences were identified with length polymorphism based on variation in repeat copy number. One at DXS237 exhibits 44% heterozygosity and is potentially useful for rapid diagnosis and mapping of X-linked disorders in Xp22.3. The other, at DXS102 in Xq26, has 71% heterozygosity. This marker will improve accuracy of diagnoses by linkage for families with Börjeson-Forssman-Lehmann syndrome. Review of the literature has identified 31 PCR based markers on the X chromosome, with minimum heterozygosity of 50%, applicable to the mapping and diagnosis of X-linked disorders.

Use of linkage data obtained in single families: prenatal diagnosis of a new X-linked mental retardation syndrome.

Prenatal diagnosis was requested by an obligate carrier of a new syndrome of X-linked mental retardation. There was close linkage between the disease gene and the hypervariable VNTR marker DXS255 with a lod score of 4.82 at o = 0 (90% support interval 0.00-0.12). When the request for prenatal diagnosis was made, additional family members were examined, resulting in an amended lod score of 6.71 at o = 0.0 (90% support interval 0.00-0.09). There were no informative flanking markers at the time of the request for prenatal diagnosis; hence it proceeded by 2 point linkage analysis. The fetus was female with a carrier risk in the interval of 91-100%. Given the limitations of the mapping data available for this disorder at the time of the request, the options of accepting or rejecting this as a case for prenatal diagnosis were carefully considered. Whilst prenatal diagnosis based on fetal sexing would be sufficient to prevent the birth of an affected child, the magnitude of the known two-point lod score between DXS255 and the disease gene provided a means for diagnosis with an accuracy between 91 and 100%.

Localisation of the gene for X-linked reticulate pigmentary disorder with systemic manifestations (PDR), previously known as X-linked cutaneous amyloidosis.

X-linked reticulate pigmentary disorder (PDR), previously reported as X-linked cutaneous amyloidosis (MIM#301220), is characterized by brown pigmentation of the skin which follows the lines of Blaschko in females but appears as reticulate sheets in males. Males may suffer severe gastrointestinal disorders in infancy with failure to thrive and early death. Nowadays symptomatic treatment allows survival and other manifestations may appear such as corneal dystrophy with severe photophobia or chronic respiratory disease. Amyloid deposition in the skin may be no more than an age-dependent secondary manifestation. The PDR gene was localised by linkage analysis to Xp21-p22. The background genetic map is Xpter-DXS996-22.5-DXS207-3.3-DXS999-3.3-DXS36 5-14.2-DXS989-4.1-3'DMD-3.5- DXS997-1.0-STR44-9.3-DYSI-2.3-DXS1068-11.0-DX S228 with distances between markers given in cM. Recombinants detected with DXS999 distally and DXS228 proximally, define the limits to the localisation. Linkage was found with several markers within this interval. Peak lod scores of 3.21 at theta = 0.0 were obtained between PDR and DXS989 and between PDR and 5'DYSI within the dystrophin locus.

Linkage and genetic counselling for the fragile X using DNA probes 52A, F9, DX13, and ST14.

Linkage data using the markers DXS51, F9, DXS15, and DXS52 are presented from 14 pedigrees segregating with the fragile X. Cytogenetic and DNA data were combined by two- or three-point linkage analysis for estimation of lod scores and carrier probabilities in potential carriers. Recombination frequencies (theta) corresponding to maximum z scores (zeta) were obtained for DXS51 (zeta = 3.45, theta = 0.0), DXS15 (zeta = 0.40, theta = 0.06), F9 (zeta = 3.15, theta = 0.09), and DXS52 (zeta = 3.60, theta = 0.11) with the fragile X. Considerable alterations to carrier probabilities occurred in some cases, especially when flanking markers were informative. The chance of mentally impaired offspring was reduced to 1% for five of eight women with prior carrier probabilities of 32%. Three pedigrees were identified in which mutation had possibly occurred. An alternative explanation for two of these was inheritance of the fragile X from normal males and for the other inheritance from a clinically normal woman. Probabilities were computed for each of these alternatives.

Linkage studies with the gene for an X-linked syndrome of mental retardation, microcephaly and spastic diplegia (MRX2)

A family in which a gene (MRX2) is segregating for an X-linked syndrome of mental retardation, short stature, microcephaly, brachycephaly, spastic diplegia, small testes and possible intra-uterine growth retardation is described. There are 7 clearly affected males and one possibly affected infant in the family. The obligate carriers are normal. Linkage studies show a suggestion of linkage to loci near the centromere. The maximum lod score was 2.10 at theta = 0.11 for DXYS1, assuming the possibly affected male carried the MRX2 gene. There were lower lod scores suggestive of linkage with DXS7 (theta = 0.14; z = 1.29) and DXS94 (theta = 0.11; z = 1.22).

Genetic localisation of MRX27 to Xq24-26 defines another discrete gene for non-specific X-linked mental retardation.

A large family with non-specific X-linked mental retardation (MRX) was first described in 1991 [Glass et al., 1991], with a suggestion of linkage to Xq26-27. The maximum lod score was 1.60 (theta = 0.10) with the F9 locus. The localisation of this MRX gene has now been established by linkage to microsatellite markers. Peak pairwise lod scores of 4.02 and 4.01 (theta = 0.00) were attained at the DXS1114 and DXS994 loci respectively. This MRX gene is now designated MRX27 and is localised to Xq24-26 by recombination events detected by DXS424 and DXS102. This regional localisation spans 26.2 cM on the genetic background map and defines another distinct MRX interval by linkage to a specific region of the X chromosome.

Börjeson-Forssman-Lehmann syndrome: clinical manifestations and gene localization to Xq26-27.

We have studied 7 males in one family with mild/moderate intellectual handicap, long thick ears, deep-set eyes, small testes, and post pubertal gynecomastia. The affected males and some of the heterozygous females also had tapering fingers and short, widely spaced flexed toes. The pedigree demonstrates X-linked recessive inheritance. The clinical manifestations are similar to those described in the Börjeson-Forssman-Lehmann (BFL) syndrome but differ in the degree of mental handicap and the absence of "dwarfism" and microcepaly. This milder manifestation may represent either phenotypic or genotypic variation. DNA marker studies demonstrated linkage to the DXS86, DXS51, and F9 cluster at Xq26-q27. The maximum lod score was 2.1 with DXS51, at theta = 0.0. Definite recombinants were observed between DXS10 (at Xq26 but proximal to DXS86), DXS105 (at Xq27 but distal to F9), and BFL. Thus, the regional localization for BFL is Xq26-q27 between DXS10 and DXS105.