Perinatal outcomes in lesbian couples employing shared motherhood IVF compared with those performing artificial insemination with donor sperm.

STUDY QUESTION: In lesbian couples, is shared motherhood IVF (SMI) associated with an increase in perinatal complications compared with artificial insemination with donor sperm (AID)? SUMMARY ANSWER: Singleton pregnancies in SMI and AID had very similar outcomes, except for a non-significant increase in the rate of preeclampsia/hypertension (PE/HT) in SMI (recipient's age-adjusted odds ratio (OR) = 1.9, 95% CI = 0.7-5.2; P = 0.19), but twin SMI pregnancies had a much higher frequency of PE/HT than AID twins (recipient's age-adjusted OR = 21.7, 95% CI = 2.8-289.4; P = 0.01). WHAT IS KNOWN ALREADY: Oocyte donation (OD) pregnancies are associated with an increase in perinatal complications, in particular, preterm delivery and low birth weight, and PE/HT. However, it is unclear to what extent these complications are due to OD process or to the conditions why OD was performed, such as advanced age and underlying health conditions. Unfortunately, the literature concerning perinatal outcomes in SMI is scarce. STUDY DESIGN, SIZE, DURATION: Retrospective study involving 660 SMI cycles (299 pregnancies) and 4349 AID cycles (949 pregnancies) assisted over a 10-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: All cycles fulfilling the inclusion criteria performed in lesbian couples seeking fertility treatment in 17 Spanish clinics of the same group. Pregnancy rates of SMI and AID cycles were compared. Perinatal outcomes were compared: gestational length, newborn weight, preterm and low birth rates, PE/HT rates, cesarean section rates, perinatal mortality, and newborn malformations. MAIN RESULTS AND THE ROLE OF CHANCE: Pregnancy rates were higher in SMI than in AID (45.3% versus 21.8%, P < 0.001). There was a non-significant trend to higher multiple rate in AID (4.7% versus 8.5%, P = 0.08). In single pregnancies, there were no differences between SMI and AID in gestational age (278 days (268-285) versus 279 (272-284), P = 0.24), preterm rate (8.3% versus 7.3%, P = 0.80), preterm <28 weeks (0.6% versus 0.4%, P = 1.00), newborn weight (3195 g (2915-3620) versus 3270 g (2980-3600), P = 0.296), low birth rate (6.4% versus 6.4%, P = 1.00), extremely low birth weight (0.6% versus 0.5%, P = 1.00), and the distribution of newborns by weight groups. Cesarean section rate, newborn malformation rate, and perinatal mortality were also similar in SMI and AID. Additionally, there was non-significant trend in hypertensive disorders to an increase in PE/HT among SMI (recipient's age-adjusted OR = 1.9, 95% CI = 0.7-5.2). Overall, perinatal data are consistent with what is reported in the general population. In twin pregnancies, the aforementioned perinatal parameters were also very similar in SMI and AID. However, SMI twin pregnancies had a very high risk of PE/HT when compared with AID (recipient's age-adjusted OR = 21.7, 95% CI = 2.8-289.4, P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Our data regarding the pregnancy course were obtained from information registered in the delivery report as well as from what was reported by the patients themselves, so a certain degree of inaccuracy cannot be ruled out. Additionally, in some parameters, there was up to 10% of data missing. However, since the methodology of reporting was the same in SMI and AID groups, one should not expect a differential reporting bias. It cannot be ruled out that the risk of PE/HT in simple gestations would be significant in a larger study. Additionally, in the SMI group allocation to the transfer of 2 embryos was not randomized so some bias is possible. WIDER IMPLICATIONS OF THE FINDINGS: SMI, if single embryo transfer is performed, seems to be is a safe procedure. Double embryo transfer should not be performed in SMI. Our data suggest that the majority of complications in OD could be related more with recipient status than with OD itself, since with SMI (performed in women without fertility problems) the perinatal complications were much lower than usually described in OD. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Application of transport phenomena analysis technique to cerebrospinal fluid.

The study of hydrocephalus and the modeling of cerebrospinal fluid flow have proceeded in the past using mathematical analysis that was very capable of prediction phenomenonologically but not well in physiologic parameters. In this paper, the basis of fluid dynamics at the physiologic state is explained using first established equations of transport phenomenon. Then, microscopic and molecular level techniques of modeling are described using porous media theory and chemical kinetic theory and then applied to cerebrospinal fluid (CSF) dynamics. Using techniques of transport analysis allows the field of cerebrospinal fluid dynamics to approach the level of sophistication of urine and blood transport. Concepts such as intracellular and intercellular pathways, compartmentalization, and tortuosity are associated with quantifiable parameters that are relevant to the anatomy and physiology of cerebrospinal fluid transport. The engineering field of transport phenomenon is rich and steeped in architectural, aeronautical, nautical, and more recently biological history. This paper summarizes and reviews the approaches that have been taken in the field of engineering and applies it to CSF flow.

OPTIMIZATION OF A MICROFLUIDIC DEVICE FOR DIFFUSION-BASED EXTRACTION OF DMSO FROM A CELL SUSPENSION.

This study considers the use of a two-stream microfluidic device for extraction of dimethyl sulphoxide (DMSO) from a cryopreserved cell suspension. The DMSO diffuses from a cell suspension stream into a neighboring wash stream flowing in parallel. The model of Fleming et al.[14] is employed to determine and discuss optimal geometry and operating conditions for a case requiring removal of 95% DMSO from suspension streams with volumetric flow rates up to 2.5 ml/min. The effects of Peclet number, flow rate fraction, and cell volume fraction are analyzed, and expansion of the analysis to other applications is discussed.

Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite.

In Leishmania major a 100-kDa heat shock protein, Hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. A replacement of both clpB alleles which encode Hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. In experimental infections of BALB/c inbred mice, the lack of Hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with wild-type L. major. Overexpression of exogenous clpB gene copies can partly restore virulence to the gene replacement mutants. Genetic-selection experiments also reveal a strong pressure for Hsp100 expression in the mammalian stage. This requirement for Hsp100 was also observed in in vitro infection experiments with mouse peritoneal macrophages. These experiments indicated a role for Hsp100 during the development from the promastigote to the amastigote stage. Our results suggest an important role for this parasite heat shock protein during the initial stages of a mammalian infection.

Cell partitioning during the directional solidification of trehalose solutions.

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.

Cryopreservation of hematopoietic and non-hematopoietic stem cells.

Recent studies illustrate the potential for improving the cryopreservation of stem cells. Reduced DMSO concentrations in the cryopreservation medium, post thaw washing of cells and increased cell concentration have been actively studied. Standardization of cell processing has led to the study of liquid storage prior to cryopreservation, validation of mechanical (uncontrolled rate freezing) freezing, and cryopreservation bag failure. Finally, the need for the systematic study and optimization of preservation processes has not been fulfilled. As the sources and applications of stem cells (hematopoietic and non-hematopoietic) continue to be developed, the need for effective preservation methods will only grow.

A member of the ClpB family of stress proteins is expressed during heat shock in Leishmania spp.

We have identified and isolated the Leishmania major homologue to the bacterial ClpB gene and to the yeast Hsp104 gene. ClpB in Leishmania major is a single-copy gene and encodes a low-abundance mRNA which is induced several-fold during a heat stress. We raised antibodies against the product of the recombinant gene and show that the leishmanial ClpB encodes a predominantly cytoplasmic protein of approx. 100 kDa which is detectable in Leishmania promastigotes of various species after exposure to elevated temperatures. We, therefore, term this protein Hsp100.

A Rat Pituitary Tumour K(+) Channel Expressed in Frog Oocytes Induces a Transient K(+) Current Indistinguishable from that Recorded in Native Cells.

A voltage-gated K(+) channel protein has been cloned from a cDNA library derived from poly(A)(+) RNA of the rat pituitary tumour cell line GH(3) /B(6) by the polymerase chain reaction technique. The clone referred to as RGHK9 encodes a protein sequence very similar to a recently cloned K(+) channel protein from rat brain and heart, with deviations in a few amino-acid positions. In situ hybridization experiments show that RGHK9 mRNA is also present in the anterior pituitary as well as in other brain regions and that it is particularly abundant in the hippocampus. After injection of cRNA transcribed from the RGHK9 cDNA clone into Xenopus oocytes, the expressed protein induces a transient K(+) current. Except for the activation kinetics the properties of this current are indistinguishable from that of the native transient K(+) current measured in GH(3) /B(6) cells, e.g. both K(+) currents are blocked by 4-aminopyridine and show the same voltage dependence and slope of steady state activation and inactivation as well as identical time constants of, and slow recovery from, inactivation. Taken together, these data show that the outward-rectifying voltage-gated K(+) channel protein encoded by the RGHK9 cDNA correlates well in its functional properties with that of a very similar, if not identical, K(+) channel present in GH(3) /B(6) cells.

In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix.

The objective of this investigation was to demonstrate the effectiveness of a tissue-engineered collagen sponge as a substrate for the culture of human corneal cells. To that end, human kerotocyte, epithelial, and endothelial cells were cultured separately on collagen sponges composed of native fibrillar collagen with a pore size of approximately 0.1 mm. Co-culture experiments were also performed (epithelial/endothelial and epithelial/keratocyte cultures). Proliferation of keratocytes and matrix production was assessed. The morphology of the epithelial and endothelial cell cultures was characterized by histology and scanning electron microscopy. Keratocytes cultured on collagen sponges exhibited increased matrix synthesis over time as well as proliferation and repopulation of the matrix. Epithelial and endothelial cells showed the ability to migrate over the collagen sponge. The thickness of the epithelial layer was influenced by soluble factors produced by endothelial cells. The morphology of the bottom layer of epithelial cells was influenced by the presence of keratocytes in the culture. These studies indicate that human corneal cells exhibit normal cell phenotype when cultured individually on an engineered collagen sponge matrix and co-culture of different cell types in the cornea can influence cell behavior.

Parameters of cell freezing: implications for the cryopreservation of stem cells.

The overall objective of this review has been to discuss specific parameters that may influence the ability to successfully cryopreserve stem cells and, more importantly, stem-cell-based therapies. This discussion of factors is in no way complete. Specifically, the effect of temperature and the duration of storage, sensitivities to cryopreservation of bone marrow cells from patients with specific disorders (for example, chronic myelogenous leukemia), and the postfreeze processing of cells are factors of clinical significance that, for the sake of brevity, have been omitted. As new stem-cell-based therapies (gene, stem cell transplant, or immunotherapy) become the standard of care for a wide variety of diseases, appropriate cryopreservation protocols will be necessary to increase patient access, reduce cost, and enhance the safety and effectiveness of these therapies. Appropriate protocols must include methods and reagents appropriate for human use. The cryopreservation protocols developed must also reflect the biological and physical properties of the cells that can be altered significantly by the culture process. Finally, cryopreservation studies should be performed concurrently with in vitro culture studies to reduce the overall cost and time required for the development or validation of a cryopreservation protocol.

A model of low-temperature water transport for hepatocyte spheroids.

Spheroids are multicellular aggregates that exhibit a more tissue-like morphology and function when compared to monolayer cultures of the same cells. Hepatocyte spheroids are presently under investigation for use of an artificial liver. The ability to cryopreserve hepatocyte spheroids is essential for their clinical and commercial application. A multicompartment model was formulated to predict water content as a function of temperature during freezing. The theoretical predictions of water transport indicate that there will be spatial differences in water content of the spheroid during freezing and that due to the rapid decrease in water transport with decreasing temperature, the undercooling of the intracellular solution during freezing will increase steadily. These results indicate that conventional freezing of hepatocyte spheroids will be difficult to accomplish due to transport limitations in the spheroids.

Freezing characteristics of isolated pig and human hepatocytes.

The cellular response of isolated hepatocytes from pigs, humans, and human hepatoblastoma cells to freezing was characterized using cryomicroscopy and analyzed using a thermodynamic model for water transport and Intracellular Ice Formation (IIF). The value for the reference permeability, Lpg, was found to be 5.8(10)-13, 1.62(10)-13, and 2.7(10)-14 m/Ns for pig, human, and Hep G2/C3A cells, respectively. The activation energy, Elp, was found to be 480 kJ/mol for pig hepatocytes, 216 kJ/mol for human, and 121 kJ/mol for Hep G2/C3A cells. The average temperature at which IIF (T(avg)IIF) occurs was calculated to be -7.24 +/- 2.3 degrees C for pig hepatocytes, -8.5 +/- 2.6 degrees C for human hepatocytes, and -9.6 +/- 4.5 degrees C for Hep G2/C3A cells. These results indicate that the freezing characteristics of pig and human cells are distinct and that the specific freezing characteristics need to be understood for the development of appropriate freezing protocols.

Freezing characteristics of genetically modified lymphocytes for the treatment of MPS II.

The freezing characteristics of genetically modified lymphocytes obtained from a donor with mucopolysaccharidosis type II (MPS II) were determined using cryomicroscopy and controlled rate freezing studies to determine postthaw viability. The cells from a donor with MPS II used in this investigation were cultured and transduced with a retroviral vector for the iduronate-2-sulfatase (IDS) enzyme for clinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics of the cells were determined after completion of the culture and transduction protocol. The water transport parameters, I(pg) and E(lp), for the cultured and transduced cells were determined to be 4.4 +/- 1.3 x 10(-14) m3/Ns and 173 +/- 25 kJ/mol, respectively. The IIF nucleation parameters, kappa and omega, were 5.5 x 10(10) K5 and 3.5 x 10(11) (l/m2 s), respectively. The postthaw viability of the genetically modified cells was less than the viability of the freshly isolated cells from the same donor. The postthaw viability of the cultured and transduced cells from a donor with MPS II was also less than that observed with cells from a normal donor that were frozen and thawed under the same conditions. These studies are essential in understanding the biophysical changes resulting from the ex vivo culture of cells and the manner in which these changes influence the ability of the cells to be cryopreserved.

Water content in an engineered dermal replacement during permeation of Me2SO solutions using rapid MR imaging.

The successful cryopreservation of cell and tissues typically requires the use of specialized solutions containing cryoprotective agents. At room temperature, the introduction of a cryopreservation solution can result in cell damage/death resulting from osmotic stresses and/or biochemical toxicity of the solution. For tissues, the permeation and equilibration of a cryoprotective solution throughout the tissue is important in enhancing the uniformity and consistency of the postthaw viability of the tissue. Magnetic resonance (MR) is a common nondestructive technique that can be used to quantitate the temporal and spatial composition of water and cryoprotective agents in a three-dimensional system. We have applied a recently developed rapid NMR imaging technique to quantify the transport of water in an artificial dermal replacement upon permeation of dimethyl sulfoxide (Me2SO) solutions. Results indicate that the rate of water transport is slower in the presence of Me2SO molecules. Furthermore, the transport is concentration-dependent, suggesting that Me2SO tends to retain bound water molecules in the tissue. Moreover, water transport decreases with decreasing temperature, and the presence of cells tends to increase water transport.

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.

Thermoregulation and heat exchange in a nonuniform thermal environment during simulated extended EVA. Extravehicular activities.

BACKGROUND: Nonuniform heating and cooling of the body, a possibility during extended duration extravehicular activities (EVA), was studied by means of a specially designed water circulating garment that independently heated or cooled the right and left sides of the body. The purpose was to assess whether there was a generalized reaction on the finger in extreme contradictory temperatures on the body surface, as a potential heat status controller. METHOD: Eight subjects, six men and two women, were studied while wearing a sagittally divided experimental garment with hands exposed in the following conditions: Stage 1 baseline--total body garment inlet water temperature at 33 degrees C; Stage 2--left side inlet water temperature heated to 45 degrees C; right side cooled to 8 degrees C; Stage 3--left side inlet water temperature cooled to 8 degrees C, right side heated to 45 degrees C. RESULTS: Temperatures on each side of the body surface as well as ear canal temperature (Tec) showed statistically significant Stage x Side interactions, demonstrating responsiveness to the thermal manipulations. Right and left finger temperatures (Tfing) were not significantly different across stages; their dynamic across time was similar. Rectal temperature (Tre) was not reactive to prevailing cold on the body surface, and therefore not informative. Subjective perception of heat and cold on the left and right sides of the body was consistent with actual temperature manipulations. CONCLUSIONS: Tec and Tre estimates of internal temperature do not provide accurate data for evaluating overall thermal status in nonuniform thermal conditions on the body surface. The use of Tfing has significant potential in providing more accurate information on thermal status and as a feedback method for more precise thermal regulation of the astronaut within the EVA space suit.

The characterization of arachnoid cell transport II: paracellular transport and blood-cerebrospinal fluid barrier formation.

We used an immortalized arachnoid cell line to test the arachnoid barrier properties and paracellular transport. The permeabilities of urea, mannitol, and inulin through monolayers were 2.9 ± 1.1 × 10(-6), 0.8 ± .18 × 10(-6), 1.0 ± .29 × 10(-6)cm/s. Size differential permeability testing with dextran clarified the arachnoidal blood-cerebrospinal fluid (CSF) barrier limit and established a rate of transcellular transport to be about two orders of magnitude slower than paracellular transport in a polyester membrane diffusion chamber. The theoretical pore size for paracellular space is 11Å and the occupancy to length ratio is 0.8 and 0.72 cm(-1) for urea and mannitol respectively. The permeability of the monolayer was not significantly different from apical to basal and vice versa. Gap junctions may have a role in contributing to barrier formation. Although the upregulation of claudin by dexamethasone did not significantly alter paracellular transport, increasing intracellular cAMP decreased mannitol permeability. Calcium modulated paracellular transport, but only selectively with the ion chelator, EDTA, and with disruption of intracellular stores. The blood-CSF barrier at the arachnoid is anatomically and physiologically different from the vascular-based blood-brain barrier, but is similarly subject to modulation. We describe the basic paracellular transport characteristics of this CSF "sink" of the brain which will allow for a better description of mass and constitutive balance within the intracranial compartment.

Immortalization and functional characterization of rat arachnoid cell lines.

Modeling the behavior of mammalian arachnoid cells is critical to understand hydrocephalus and other brain disorders involving abnormal flow of cerebrospinal fluid, yet relatively little is known about the physiology of arachnoid cells due to lack of a robust three-dimensional model system. Explanted primary cultures have been the only option to study transport across arachnoid cell membranes, but practical limitations of primary culture include slow growth, early senescence, and poor reproducibility. The purpose of this study was to create immortalized rat arachnoid cell lines to permit in vitro study of arachnoid granulations and properties of cerebrospinal fluid (CSF) flow. We established and partially characterized two immortalized cell lines generated from primary rat arachnoid cells, using retroviral gene transfer of SV40 large T antigen (SV40 LTAg) either with or without human telomerase (hTERT). The established cell lines stably express either SV40 LTAg alone, or SV40 LTAg and hTERT, and demonstrate high proliferative rate, contact inhibition at confluence, and stable expression of protein markers characteristic of native arachnoid cells over more than 160 passages.

Correlated electron tunneling through two separate quantum dot systems with strong capacitive interdot coupling.

A system consisting of two independently contacted quantum dots with a strong electrostatic interaction shows an interdot Coulomb blockade when the dots are weakly tunnel coupled to their leads. How the blockade can be overcome by correlated tunneling when tunnel coupling to the leads increases is studied experimentally. The experimental results are compared with numerical renormalization group calculations using predefined (measured) parameters. Combining our experimental and theoretical results we identify transport through Kondo correlations due to the electrostatic interaction between the two dots.