Severe maternal morbidity during delivery hospitalisation in a large international administrative database, 2008-2013: a retrospective cohort.

OBJECTIVE: This study utilized the Dr. Foster Global Comparators database to identify pregnancy complications and associated risk factors that led to severe maternal morbidity during delivery hospitalisations in large university hospitals based in the USA, Australia, and England. DESIGN: Retrospective cohort. SETTING: Births in the USA, England and Australia from 2008 to 2013. SAMPLE: Data from delivery hospitalisations between 2008 and 2013 were examined using the Dr. Foster Global Comparators database. METHODS: We identified delivery hospitalisations with life-threatening diagnoses or use of life-saving procedures, using algorithms for severe maternal morbidity from the Center for Disease Control. Frequency of severe maternal morbidity was calculated for each country. MAIN OUTCOME MEASURES: Multivariable analysis was used to examine the association between morbidity and socio-demographic and clinical characteristics within each country. Chi-square tests assessed differences in covariates between countries. RESULTS: From 2008 to 2013, there were 516 781 deliveries from a total of 18 hospitals: 24.5% from the USA, 57.0% from England and 18.4% from Australia. Overall severe maternal morbidity rate was 8.2 per 1000 deliveries: 15.6 in the USA, 5.0 in England, and 8.2 in Australia. The most common codes identifying severe morbidity included transfusion, disseminated intravascular coagulation, acute renal failure, cardiac events/procedures, ventilation, hysterectomy, and eclampsia. Advanced maternal age, hypertension, diabetes, and substance abuse were associated with severe maternal morbidity in all three countries. CONCLUSION: Rates of severe maternal morbidity differed by country. Identification of geographical, socio-demographic, and clinical differences can help target modifications of practice and potentially reduce severe maternal morbidity. TWEETABLE ABSTRACT: Rates of severe maternal morbidity vary, but risk factors associated with adverse outcomes are similar in developed countries.

Effect of Cytochalasin B pretreatment on developmental potential of ovine oocytes vitrified at the germinal vesicle stage.

Oocyte cryopreservation remains a challenge in most mammalian species because of their sensitivities to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw viability and pre-implantation embryo development. The aim of this study was to investigate the effect of cytochalasin B (CB) pre-treatment before vitrification on viability, frequencies of in vitro fertilisation (IVF) and subsequent development of ovine cumulus-oocyte complexes (COCs) vitrified at the germinal vesicle (GV) stage using cryoloop. COCs obtained at slaughter were randomly divided into two groups and incubated with or without 7.5µg/mL CB for 60 min. Oocytes from each group were then vitrified using a cryoloop or used as toxicity and controls. Oocytes were then matured, fertilised, and cultured in vitro for 7 days. Viability following vitrifiaction and warming, fertilisation events following IVF and subsequent pre-implantation embryo development were evaluated. No significant differences were observed in survival rates between CB treated and non-treated oocytes in both vitrified and toxicity groups. Frequencies of fertilisation were increased in CB-vitrified group (oocytes pre-treated with CB before vitrification) than those vitrified without CB pre-treatment (57.0% vs 40.7%). Cleavage was significantly lower (P < 0.05) in vitrified and CB-vitrified oocytes at both 24 hpi (12.5% vs 9.1%) and 48 hpi (25.0% vs 16.2%) than in other groups. Based on the numbers of cleaved oocytes, (48 hpi), 16.1% and 18.8% of the cleaved embryos developed to blastocysts in both vitrified and CB-vitrified groups. These values did not differ significantly from those obtained in CB-control group (37.8%). No significant differences were observed in mean cell numbers per blastocyst between all groups. In conclusion, pre-treatment of ovine GV oocytes with cytochalasin B as cytoskeleton stabilizer before vitrification increased frequencies of in vitro fertilisation and subsequently resulted in production of good quality late stage pre-implantation embryos following IVF.

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes.

The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P<0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC's and DO's developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO's prevented this (31.3%, 12.7% and 29.4% respectively (P<0.05)) but had no effect on COC's (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4+/-5.2, 84.7+/-3.7 and 80.4+/-5.8 (P>0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy.

Congenital heart defects in twin gestations.

As ultrasound technology advances, diagnosis of fetal malformations, particularly congenital heart defects (CHD) is becoming standard practice. Currently, a key element of obstetrical care is the use of ultrasound to diagnose chorionicity in multiple gestations. Given the difference in incidence and types of complications between dichorionic and monochorionic pregnancies, early diagnosis of chorionicity is critical to determine the type of care and counseling a patient receives throughout the pregnancy. Early diagnosis of chorionicity allows investigators to more accurately determine the risk of CHD in monochorionic pregnancies. It has been long known that twin gestations incur a higher risk of congenital malformations, including CHD. However, it was not until recently that the incidence could be determined according to chorionicity. Previous studies looking at risk of malformations including CHD used the like-sex technique as a proxy for chorionicity, thereby overestimating the prevalence of monochorionic twins because roughly two-thirds of all twin gestations (including dichorionic) are the same sex. The rate of multiple gestations is increasing in the developed world. Assisted reproductive technology (ART) is partly responsible for the increased incidence of multiples. While many of the ART conceived pregnancies are dichorionic multiples, there is evidence that ART increases the risk of monochorionic multiple gestations. Presently, it is not technically feasible, nor practical, to screen all pregnancies with fetal echocardiography. Thus, many perinatal ultrasound centers screen women for risk factors that place them at higher risk for having a fetus with CHD. This higher risk' group then receives a fetal echocardiogram. The available literature regarding risk of CHD in monochorionic multiple gestations strongly points to a significant increase over the general population risk of 0.5-0.8%. Fetal echocardiography is technically feasible in twin pregnancies and increasingly available. Monochorionic multiple gestations should be screened with fetal echocardiography.

Somatic cell nuclear transfer: Past, present and future perspectives.

It is now over a decade since the birth, in 1996, of Dolly the first animal to be produced by nuclear transfer using an adult derived somatic cell as nuclear donor. Since this time similar techniques have been successfully applied to a range of species producing live offspring and allowing the development of transgenic technologies for agricultural, biotechnological and medical uses. However, though applicable to a range of species, overall, the efficiencies of development of healthy offspring remain low. The low frequency of successful development has been attributed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Many studies have demonstrated that such reprogramming occurs by epigenetic mechanisms not involving alterations in DNA sequence, however, at present the molecular mechanisms underlying reprogramming are poorly defined. Since the birth of Dolly many studies have attempted to improve the frequency of development, this review will discuss the process of animal production by nuclear transfer and in particular changes in the methodology which have increased development and survival, simplified or increased robustness of the technique. Although much of the discussion is applicable across species, for simplicity we will concentrate primarily on published data for cattle, sheep, pigs and mice.

Healthy ageing of cloned sheep.

The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals.

Nuclear transfer in practice.

The technique of nuclear transfer (NT) allows the production of embryos, fetuses, and offspring from a range of embryonic, fetal, and adult derived cell types in a range of species. Successful development is dependent upon numerous factors, including type of recipient cell, source of recipient cell, method of reconstruction, activation, embryo culture, donor cell type, and donor and recipient cell cycle stages. The present review will discuss the uses of NT, the techniques presently available, and the factors affecting subsequent development.

Embryonic and somatic cell cloning.

Revolutionary opportunities in biology, medicine and agriculture arise from the observation that offspring are obtained after nuclear transfer if somatic donor cells are induced to become quiescent. Exploitation of many of these opportunities will depend upon optimizing procedures for nuclear transfer. This may come about through an understanding of the means by which factors in the oocyte cytoplasm act upon the DNA of the transferred nucleus to regulate gene expression. Similarly, research will extend the procedure to other species. This technology may be used for embryo production, the introduction of genetic change and the derivation of cells needed to treat human diseases. Groups of genetically identical animals will be used in research to control genetic variation and to allow transfer of cells between individuals. In agriculture, production of a small number of clones will separate genetic and environmental effects, whereas production of larger numbers of offspring will disseminate genetic improvement from nucleus herds. Precise genetic modification will be achieved by site specific recombination in the donor cells before nuclear transfer. In all mammals it will become possible to define the role of any gene product and to analyse the mechanisms that regulate gene expression. Medical uses of these techniques will include the production of proteins needed to treat disease and the supply of organs such as hearts, livers and kidneys from pigs. As genome mapping projects identify loci associated with traits of commercial importance in agriculture then gene targeting will be used to study this effect. Finally, cells capable of differentiation into any of the tissues of a patient will provide treatment for diseases reflecting damage to a specific cell population that neither repairs nor replaces itself.

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells.

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95-100%), but distributed from GVI to GVIV (GVI 56.1 +/- 9.1%, GVII 15.3 +/- 1.4%, GVIII 21.5 +/- 7.1%, GVIV 7.1 +/- 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 +/- 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1-25 microg mL(-1) resulted in 95-100% oocytes being arrested and synchronized at GVII. With >5 microg mL(-1) CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 microg mL(-1) CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6-12 h, metaphase I (MI) at 10-18 h and anaphase I/telophase I at 16-20 h. After 12 h preincubation with 5 microg mL(-1) CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.

New advances in somatic cell nuclear transfer: application in transgenesis.

The ability to produce live offspring by nuclear transfer from cultured somatic cells provides a route for the precise genetic manipulation of large animal species. Such modifications include the addition, or "knock-in", and the removal or inactivation, "knock-out", of genes or their control sequences. This paper will review some of the factors which affect the development of embryos produced by nuclear transfer, the advantages of using cultured cells as donors of genetic material, and methods that have been developed to enrich gene targeting frequency. Commercial applications of this technology in biomedicine and agriculture will also be addressed.

Physiological temperature variants and culture media modify meiotic progression and developmental potential of pig oocytes in vitro.

Ovarian follicles in vivo are cooler than surrounding abdominal and ovarian tissues. This study investigated whether typical follicular temperatures influence the maturation and developmental potential of pig oocytes in vitro. Oocytes were synchronised at the germinal vesicle (GV) stage and incubated at 39, 37 or 35.5 degrees C. When compared with 39 degrees C, which is often used for in vitro studies, lower temperatures delayed spontaneous progression to the metaphase I and II (MI and MII) stages of meiosis. The MII was delayed by about 12 h per degrees C. All oocytes had normal morphology. Oocytes reaching GV breakdown (GVBD) at 39 degrees C were subsequently unaffected by cooling, demonstrating thermal sensitivity during the pre-GVBD stage only. Simultaneous assay of maturation-controlling kinases (maturation promoting factor (MPF) and MAPK) showed that cooling delayed kinase activation, provided it was applied prior to GVBD. Activity profiles remained coupled to the stage of meiosis. Neither enzyme was directly thermally sensitive over this temperature range. Following in vitro fertilisation, fewer blastocysts developed from embryos derived from 35.5 or 37 degrees C oocytes as compared with those from 39 degrees C oocytes. Manipulation of fertilisation timings to allow for delayed maturation showed that over-maturing or aging at lower temperatures compromises subsequent embryo development, despite normal nuclear maturation; the GV stage was again the thermally sensitive period. Cleavage rates were improved by the culture of oocytes with follicle-stimulating hormone (FSH) at 37 but not at 35.5 degrees C. Inclusion of 20% follicular fluid in the oocyte medium restored the blastocyst rate to that seen at higher temperatures. Thus, FSH and follicular fluid may allow oocytes to achieve normal developmental potential at in vivo temperatures.

Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles.

Follicles of 4-8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 mug/ml) at 39 degrees C in a humidified atmosphere of 45% O2, 5% CO2 and 50% N2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 +/- 8.53 vs 124.6 +/- 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.

Dynamic changes in meiotic progression and improvement of developmental competence of pig oocytes in vitro by follicle-stimulating hormone and cycloheximide.

The effects of FSH, LH, and epidermal growth factor (EGF) on the dynamics of nuclear maturation and subsequent embryo development were examined in pig oocytes cultured either conventionally or after preincubation with cycloheximide (CHX). In conventional culture, FSH or EGF significantly increased the rate of attainment of metaphase II (MII) for both gilt (50.0%+/-4.2% and 54.8%+/-4.3%, respectively; control, 5.8%+/-1.8%; P<0.001) and sow (87.6%+/-3.4% and 78.8%+/-3.9%, respectively; control, 7.8%+/-2.5%; P<0.001) oocytes. Gilt oocytes treated with both FSH and EGF showed an additive response (93.7%+/-2.1%). Treatment with LH had no effect. Preincubation with CHX caused the majority (84-100%) of both gilt and sow oocytes to undergo germinal vesicle breakdown. Compared to those treated with LH and/or EGF (both>80%), fewer FSH-treated oocytes reached metaphase I (43.8%+/-5.3%, P<0.001) by 14 h and MII (48.4%+/-5.9%, P<0.001) by 24 h, although the majority (71%) did mature to MII by 36 h after removal of CHX. After in vitro fertilization, higher proportions of both CHX-pretreated and untreated, FSH-exposed oocytes cleaved (71.3%+/-2.9% and 75.3%+/-3.1%, respectively) compared with those not treated with FSH (37.7%+/-3.0% and 43.0%+/-2.9%, respectively; P<0.001). Pretreatment with CHX significantly increased blastocyst yield for both FSH-treated (32.8%+/-2.0% and 10.3%+/-1.5%, respectively; P<0.001) and untreated (16.7%+/-1.5% and 9.4%+/-1.2%, respectively; P<0.001) oocytes. Polyspermy rates were unaffected. In conclusion, pig oocytes meiotically arrested by CHX before maturation retain and improve their developmental competence. FSH stimulates nuclear maturation but slows meiotic progression.

Cloning: eight years after Dolly.

It is now 8 years since the birth of Dolly, the first animal produced by nuclear transfer using a donor cell population established from an adult animal. During this time, the technique of nuclear transfer has been successfully applied to a range of mammalian species for the production of offspring using a plethora of donor cell types derived from both foetal and adult tissues. In addition, when coupled with genetic manipulation of the donor cells, transgenic offspring have been produced with a range of genetic modifications including gene knockouts and gene knockings. Despite the apparent successes of the technology, the efficiency of development to live offspring has remained low and developmental abnormalities still occur. The objectives of this paper are to review some of the successes and failures of the nuclear transfer procedure since the production of Dolly. In particular, we will review the major steps in the procedure and discuss studies from our laboratory and others which have modified the procedure in ways which may impact on development.

Nuclear transfer in farm animal species.

CLONE 'a group of two or more individuals with identical genetic makeup derived, by asexual reproduction, from a single common parent or ancestor' (The Chambers Dictionary 1993, Chambers Harrap). The term clone was originally applied to plants but has subsequently been used in a much broader context to include a person or thing closely similar to another, a copy or replica. In animals, true clones, as defined above, may be produced by embryo splitting or blastomere separation either artificially, or as occurs naturally in the production of identical twins. In these individuals all of the components making up the individual, including nuclear genetic material (the genome) and other maternally derived factors are derived from a single unique embryo which is the result of sexual reproduction. The term clone has been applied to animals produced by the technique of nuclear transfer. In this asexual process, nuclear genetic material is transferred from a donor cell (karyoplast) into a recipient cell (cytoplast) from which the genetic material has been removed. In farm animals the cytoplast of choice is the matured oocyte (or unfertilised egg) thus the animals developing from this technique are not true clones as each cytoplast is often derived from a different animal. The resultant animals may therefore be more aptly described as 'genomic copies'. In mammals, successful development of embryos reconstructed by nuclear transfer was originally restricted to using early embryos as nuclear donors, however, recent progress has demonstrated successful development using nuclei from embryonic, foetal and adult derived cell populations. Numerous factors affect the development of embryos reconstructed by nuclear transfer including; the cell cycle stage of the recipient cell, the cell cycle stage of the donor nucleus, the differentiated state of the donor nucleus, activation of the recipient cell, the culture method. In addition, there are variations in success between species, these may be related to differences in organisation of the cytoskeleton and/or the meiotic spindle in the recipient cell,differences in cell cycle control during early development, the onset of zygotic transcription or differences in the metabolic requirements of early embryos in vitro. The aim of this article is to describe and discuss some of these factors in relation to the successful development of nuclear transfer reconstructed embryos and in particular to the 'reprogramming' or 'remodeling' of the donor genetic material to attain successful development.

Nuclear equivalence, nuclear transfer, and the cell cycle.

The last 20 years have seen the development of techniques for the production of mammals by nuclear transfer. Originally limited to the swapping of pronuclei and the use of early cleavage-stage embryos as nuclear donors, nuclear transfer came of age in 1995 with the birth of 2 Welsh Mountain lambs, Megan and Morag, that were produced using cultured differentiated cells as donors of genetic material. In 1996, Dolly was the first animal to be produced using the genetic material from an adult-derived somatic cell. The techniques used in the production of these animals have now been reproduced in both sheep and cattle, and as predicted, successful development has been obtained using donor cells taken directly ex vivo. This article reviews the current status of mammalian nuclear transfer and the biological background to these successes.