The use of embryo transfer and steroid hormone replacement therapy in the study of prenatal mortality

Embryo transfer and steroid hormone replacement therapy have been used to advantage in studies on prenatal mortality, particularly in sheep.The form, timing and magnitude of loss, have been well documented and steroid hormone replacement therapy coupled with embryo transfer has clearly shown the involvement of the ovarian steroid hormones secreted before, during and after estrus in survival and development of embryos.However, little is known of the manner in which the steroid hormones exert their effects on the environment of pre-implantation embryos; a stage when most mortality occurs.     

Quantification of embryo quality by respirometry

It is generally accepted that assessment of embryo metabolism, in particular oxygen consumption, may improve embryo selection by identifying the embryos with higher developmental competence. Several methods have been employed to measure embryonic oxygen consumption, but most of them were detrimental to subsequent embryo development. Recently, we have introduced the Nanorespirometer system, which is a non-invasive and highly sensitive technology developed for the individual measurement of embryonic respiration rates. This technology is able to perform single measurements at a fixed time or stage of embryonic development without adversely influencing embryo viability. Concomitantly, and based on the same principles, a second technology -- the Embryo Respirometer -- has been developed. The Embryo Respirometer allows the continuous measurement of individual respiration rates with simultaneous acquisition of digital images of each embryo, during the entire culture period (6-7 days). In this review, both technologies are described and their potential use as diagnostic tools for improving embryo selection in bovine and human following IVF treatments is discussed. Correlations between respiration rates of individual embryos and other parameters such as morphological quality, sex, stage of development, kinetics, diameter, expression of key metabolic genes and subsequent viability following embryo transfer are also examined. On the basis of the results obtained, it is postulated that assessment of embryonic respiration rates in association with other viability parameters allows for a more accurate embryo evaluation, both under clinical and research conditions.     

Nonsurgical embryo transfer in the scimitar-horned oryx (Oryx dammah): Birth of a live offspring

The objective of this project was to determine if modifications of methods of estrous synchronization, superovulation, embryo recovery, and transfer used successfully in other ungulates, both domestic and nondomestic, could be applied to scimitar-horned oryx (Oryx dammah). Donors were two parous females and recipients were one parous and two nulliparous females that were given a total of two cloprostenol injections at an interval of 0 and 13 or 12 days, respectively. Donors were treated with follicle-stimulating hormone (FSH-P, Schering, Kenilworth, NJ) b.i.d. for 4 days and placed with a fertile male. Seven days after the last FSH-P injection, nonsurgical uterine lavages were performed on both donors. One good-quality embryo at the morula stage was recovered and nonsurgically transferred into the right uterine horn of the parous recipient. A healthy female calf born at 247 days post-transfer represents the first known live birth of scimitarhorned oryx following embryo transfer. These results provide additional evidence that estrous synchronization and embryo transfer techniques used in other ungulates can be applied to endangered antelopes such as the scimitar-horned oryx.     

Recent developments in pig embryo transfer.

Porcine embryo transfer has been performed for approximately 50 years, and surgical methods have proven to be reliable for collection and transfer of embryos. However, surgical collection and transfer have the disadvantage of being less useful on the farm. Recently, new procedures for both collection and transfer of embryos have been developed to improve usefulness. The surgical procedure has been refined to a minimally invasive procedure, using endoscopy for collection and transfer of embryos. A nonsurgical procedure for embryo collection has also been devised, but is limited to use in sows with surgically shunted (shortened) uterine horns. Nonsurgical embryo transfer procedures have been developed recently and have proven to be successful. The nonsurgical procedures are preferable to surgical procedures from an animal welfare point of view and because these procedures can be performed on farms without the need for special facilities.     

Early human embryo metabolism

Non-invasive microanalytical methods have been devised to study the energy metabolism of single human preimplantation embryos. Psyruvate, which is added routinely to all media used to culture human embryos, is consumed throughout the preimplantation period, with glucose assuming an increasing role at embryo compaction and blastocyst formation. All of the glucose consumed may be accounted for by the appearance of lactate in the incubation medium. The enzyme hexokinase my be involved in regulating this aerobic glycolysis. There is cosiderable indirect evidence for the utilisation of endogenous as opposed to exogenous energy substrates, the most likely candidate being protein. Information on early human embryo metabolismis likely to find application in a number of areas: these include the improvement of techniques for assisted human conception, notably in the selection of embryos for transfer following In Vitro Fertilisation; the diagnosis of gentic defects at the preimplantation stage; increased undersding of the causes of implantation failure and miscarriage, and the development of novel post-coital contraceptives.     

Intercellular communication in the early human embryo

A preliminary study on intercellular communicative devices in the early human embryo has been made using dye-coupling techniques and electron microscopy (EM). Lucifer yellow injected into single blastomeres of embryos at the 4-cell stage up to the late morula stage did not spread to neighbouring cells, indicating that gap junctions and cytoplasmic bridges are not significant pathways for information transfer. Dye spread was first observed in the blastocyst stage, where trophectoderm cells and inner mass cells were shown to be in communication through gap junctions. Studies at the EM level confirmed this finding. Tight junctions and desmosome-like structures, apparent from the 6-cell stage onward, were located both peripherally and centrally and were initially nonzonular. The role of intercellular devices in the primary differentiation of the human embryo is discussed.     

Non-surgical embryo transfer and live birth in a llama

The uteri of two donor female llamas were flushed non-surgically and one viable 7-day-old embryo was recovered. The embryo was transferred non-surgically within four hours into a recipient female llama whose estrous cycle was synchronized with the donor by injection of gonadotropin releasing hormone (GnRH). Pregnancy was confirmed 20 days after transfer, at which time the level of serum progesterone was determined and found to be consistent with that of gestation (>/=100 ng/100 ml). A normal and healthy male weighing 33 1bs was born on October 8, 1984, 326 days following transfer of the embryo to the recipient.     

Improving successful pregnancies after embryo transfer

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo.We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7-10 days after transfer.     

Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Defining human embryo phenotypes by cohort-specific prognostic factors

Background

Hundreds of thousands of human embryos are cultured yearly at in vitro fertilization (IVF) centers worldwide, yet the vast majority fail to develop in culture or following transfer to the uterus. However, human embryo phenotypes have not been formally defined, and current criteria for embryo transfer largely focus on characteristics of individual embryos. We hypothesized that embryo cohort-specific variables describing sibling embryos as a group may predict developmental competence as measured by IVF cycle outcomes and serve to define human embryo phenotypes.

Methodology/Principal Findings

We retrieved data for all 1117 IVF cycles performed in 2005 at Stanford University Medical Center, and further analyzed clinical data from the 665 fresh IVF, non-donor cycles and their associated 4144 embryos. Thirty variables representing patient characteristics, clinical diagnoses, treatment protocol, and embryo parameters were analyzed in an unbiased manner by regression tree models, based on dichotomous pregnancy outcomes defined by positive serum ß-human chorionic gonadotropin (ß-hCG). IVF cycle outcomes were most accurately predicted at ∼70% by four non-redundant, embryo cohort-specific variables that, remarkably, were more informative than any measures of individual, transferred embryos: Total number of embryos, number of 8-cell embryos, rate (percentage) of cleavage arrest in the cohort and day 3 follicle stimulating hormone (FSH) level. While three of these variables captured the effects of other significant variables, only the rate of cleavage arrest was independent of any known variables.

Conclusions/Significance

Our findings support defining human embryo phenotypes by non-redundant, prognostic variables that are specific to sibling embryos in a cohort.     

Improving the safety of embryo technologies: possible role of genomic imprinting

Although developments in mammalian in vitro embryo technologies have allowed many new clinical and agricultural achievements, their application has been hindered by limitations in the developmental potential of resulting embryos. Low efficiencies of development to the pre-implantation blastocyst stage have been consistently observed in most species, including humans, rabbits, pigs and ruminants. Furthermore, in cattle and sheep a wide range of congenital abnormalities currently termed "Large Offspring syndrome" (LOS) are commonly observed as a result of several embryo culture and manipulation procedures. This paper reviews the hypothesis that at least some of the problems associated with embryo technologies may result from disruptions in imprinted genes. Several imprinted genes (i.e. genes which express only the maternal or paternal allele) are known to have significant effects on fetal size and survival in other species and are possible candidates for involvement in livestock LOS. Major changes in putative imprinting mechanisms such as DNA methylation of imprinted genes occur in the mouse embryo during pre-implantation development. Alterations in DNA methylation are stabley transmitted through repeated cell cycles such that changes in the embryo may still act at the fetal stages. Thus any disruption in establishment and/or maintenance of imprinting during the vulnerable periods of embryo culture or manipulation is a plausible candidate mechanism for inducing fetal loss and Large Offspring Syndrome. Identification of these disruptions may provide crucial means to improve the success of current procedures.     

中国的动物胚胎技术研究进展

胚胎移植技术最早是由英国的Walter Heape率先研究的,随后经过了胚胎学家几十年的努力才在上个世纪来期逐渐开始广泛应用于医学生殖,畜牧业,动物保种等领域。本文集中论述中国的胚胎移植技术发展以及它在畜牧业领域的应用。 在中国商业性的动物胚胎移植正在蓬勃发展,其成功率已经和国外同行相差不大。通过胚胎移植工作者的努力,一些从事商业性胚胎移植的公司已经能够做到鲜胚移植后奶牛的妊娠率55％～70％,冷冻胚胎的妊娠率达到45％～60％。不仅如此,在各个省还建立了自己的专门的冷冻胚胎中心,为本省的畜牧产业提供胚胎或者相关的服务。 在动物保种方面,胚胎技术给人们提供了一种全新的思路：通过胚胎技术将活的胚胎冷冻在液氮罐以实现永久保存。例如中国科学院动物所陈大元先生提出通过胚胎移植技术实现对大熊猫的物种保存,并取得了一定的成果。 随着经济和社会的进一步发展,人民生活水平的提高对于肉和奶的需求会进一步提高。依靠常规的选育技术来改良牲畜是无法满足人民对于畜牧产品急切要求,要实现畜牧业的跨越式发展,就必须依靠科技的力量来实现畜牧业的大发展,满足人民生活需要。胚胎技术等新技术会为我国的畜牧业的产业提高提供了一个新的机遇,可以相信,在不远的将来,动物胚胎技术必将在中国有一个较大发展。     

The ART of studying early embryo development: Progress and challenges in ruminant embryo culture

The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.     

Genetic aspects of embryo transfer

Use of embryo transfer can lead to increases in rates of genetic improvement from selection programs from as little as 5% to a maximum of near 100%, depending on species, trait, and extent of use of other tools such as A.I. In general, embryo transfer will have much less impact on rates of genetic improvement than A.I., and in a dairy cattle program where A.I. is used effectively, embryo transfer is likely to add less than 10% to rate of genetic improvement. The potential for increasing rate of genetic improvement appears to be greater in beef cattle. In any species with low reproductive rate, embryo transfer offers a potential means of rapidly increasing numbers of animals of a breed, strain, mutant genotype or group exceeding a stringent threshold; such use may be of considerable value to a specific genetic research or multiplication program. Maximizing selection intensity through combined use of A.I. and embryo transfer can lead to a rapid increase in inbreeding, and steps should be taken to avoid this in any population which it is desired to maintain in the long term. Embryo transfer offers an effective tool for research on maternal-fetal and fetal-fetal interactions, and in this way can make important indirect contributions to more efficient breeding programs. With improved embryo storage capability, embryo transfer has the potential for useful contributions in the areas of transfer of germ plasm between countries, preservation of rare breeds, and provision of genetically stable control populations.     

Dissecting why superovulation and embryo transfer usually work on some farms but not on others

Bovine embryo transfer is a well-established commercial industry that is often associated with veterinary practices. Practitioners offering embryo transfer services may possess a very high standard of technical expertise; however, success in the production of embryos and the impregnation of recipients cannot be achieved unless the cattle are healthy and maintained in a well-managed cattle operation. In addition to appropriate gonadotropin treatments of donor cattle, the use of highly fertile semen, known to have been properly stored and handled is required for success. Recipient cattle must be managed with the same attention to detail as donors. Traditionally, PGF has been used for the synchronization of recipients. However, PGF is limited in its effectiveness early and late in the bovine estrus cycle. Recipient estrus synchronization with progesterone releasing intravaginal inserts has been successful and high pregnancy rates have resulted following embryo transfer.     

Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle

The objectives of this experiment were to determine the effects of corpus luteum characteristics, progesterone concentration, donor-recipient synchrony, embryo quality, type, and developmental stage on pregnancy rates after embryo transfer. We synchronized 763 potential recipients for estrus using one of two synchronization protocols: two doses of PGF2alpha (25 mg i.m.) given 11 d apart (Location 1); and, a single norgestomet implant for 7 d with one dose of PGF2alpha (25 mg i.m.) 24 h before implant removal (Location 2). At embryo transfer, ovaries were examined by rectal palpation and ultrasonography. Of the 526 recipients presented for embryo transfer, 122 received a fresh embryo and 326 received a frozen embryo. Pregnancy rates were greater (P < 0.05) with fresh embryos (83%) than frozen-thawed embryos (69%). Pregnancy rates were not affected by embryo grade, embryo stage, donor-recipient synchrony, or the palpated integrity of the CL. Corpus luteum diameter and luteal tissue volume increased as days post-estrus for the recipients increased. However, pregnancy rates did not differ among recipients receiving embryos 6.5 to 8.5 days after estrus (P > 0.1). There was a significant, positive simple correlation between CL diameter or luteal tissue volume and plasma progesterone concentration (r = 0.15, P < 0.01 and r = 0.18, P < 0.01, respectively). There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality.