Electron microscopic analysis of two-dimensional crystals of the Ca2+-transport ATPase — a freeze-fracture study

Summary Two distinct forms of Ca²⁺-ATPase crystals have been analysed in sarcoplasmic reticulum (SR) membranes. The E₁-type crystals, induced by Ca²⁺ or lanthanide ions, consist of single chains of ATPase monomers, and the E₂-type crystals, induced by vanadate ions, consist of dimer chains. Using improved freeze-fracture techniques we have obtained high-resolution images of complementary surface replicas of SR membranes containing these crystal forms. In E₁ crystals, the concave fracture (P) faces display obliquely oriented rows of intramembrane particles (IMPs) spaced at - 6–7 nm along both crystal axes, while the convex fracture (E) faces show corresponding rows of pits. In E₂ crystals, regular arrays of oblique parallel ridges with spacing of - 10.5–11 nm appear on the P-faces and complementary grooves or furrows on the E-faces. In many instances the ridges break up into elongated particles repeating every 5.5 nm. When the direction of the shadow is almost parallel to the axis of the ridges, these 9.5 nm particles can be resolved into two domains, which represent intramembranous contacts between the two monomers of the two adjacent dimer chains. Complementary grooves on the E-faces can also be resolved into rows of pits complementary to the particles of the ridges on the P-faces. In the control SR membranes, randomly dispersed IMPs and corresponding pits are observed on the P- and E-faces, respectively. The data suggest that transport of Ca²⁺ involves significant structural changes of the enzyme molecule, reflected in the ATPase-ATPase interactions both on the cytoplasmic surface and in the lipid bilayer.     

Successful outcome with day 4 embryo transfer after preimplantation diagnosis for genetically transmitted diseases

Preimplantation genetic diagnosis was performed in 61 day 3 embryos obtained by in-vitro fertilization from seven patient carriers of haemophilia, Marfan's syndrome, Bloch-Sulzemberg syndrome (incontinentia pigmentosa) or X chromosome-linked immune deficiency, retinitis pigmentosa, and FG syndrome, which is characterized by mental retardation and hypotonia. After multiplex polymerase chain reaction, 16 embryos were diagnosed as being unaffected, and these were transferred to the uterus on the following day (day 4). Of these embryos, six (37.5%) implanted, resulting in the delivery of a singleton and a twin pregnancy, a late second trimester miscarriage (twins at week 20) and a first trimester miscarriage at week 8. All the diagnoses were confirmed by amniocentesis. We report for the first time a late day 4 transfer of biopsied human embryos undergoing preimplantation genetic diagnosis. This transfer schedule allows an extra day to perform genetic analyses on single blastomeres and to monitor any adverse effect of the biopsy procedure.      

How to use Chlamydia antibody testing in subfertility patients

Screening for tubal factor subfertility by means of Chlamydia antibody testing (CAT) was introduced into the initial work-up of subfertile couples several years ago. The results reported, however, are heterogeneous, and no uniformity exists in cut-off levels of titres, or in definitions of tubal factor subfertility. We performed a prospective cohort study to evaluate the implications of varying the definitions of tubal pathology and of modifying the cut-off levels on the clinical impact of CAT in predicting tubal factor subfertility. In 227 consecutive patients who attended our fertility clinic, the Chlamydia IgG antibody titre was determined and related to tuboperitoneal abnormalities at laparoscopy as a reference standard. According to received operating characteristic (ROC) curve analysis, a titre of 16 is the optimum cut-off level. Increasing the cut-off level improves specificity and positive likelihood ratio (LR+), at the expense of sensitivity and negative LR (LR-). Changing the definition of tubal factor subfertility from unspecified tuboperitoneal abnormalities into extensive adhesions and/or bilateral distal tubal occlusion improves LR+, LR- and kappa significantly. We conclude that CAT is more accurate in predicting severe distal tubal pathology than unspecified tuboperitoneal abnormalities. Although from a statistical point of view a titre of 16 is the optimum cut-off level, from a clinical point of view 32 or 64 may be preferable, depending on the aim of screening and the inception cohort.      

Anxiety during pregnancy and fetal attachment after in-vitro fertilization conception

The aim of this study was to compare 70 couples who had conceived by in- vitro fertilization (IVF) with 63 matched controls for the prevalence of anxiety and quality of attachment to the baby during pregnancy. Results for mothers showed no group differences using a global measure of anxiety, the Spielberger State-Trait Anxiety Inventory. However, pregnancy-specific measures revealed significantly higher levels of anxiety in IVF mothers about the survival and normality of their unborn babies, about damage to their babies during childbirth and about separating from their babies after birth. When IVF mothers were differentiated according to the number of treatment cycles, more differences in anxiety level were revealed, with most increases occurring in mothers who had experienced two or more treatment cycles. IVF fathers did not differ from controls on the global anxiety measure. No data on pregnancy-specific anxiety were available for fathers. Neither IVF mothers nor IVF fathers differed from controls on measures of attachment to the baby during pregnancy. Results are discussed in the context of the need for researchers to employ differentiated and issue-specific measures to identify concerns that may be unique to IVF couples. Clinical implications regarding the need for psychological support during pregnancy are also discussed.