Embryo Transfer Technique Research Articles

Embryo Sexing and Sex Chromosomal Chimerism Analysis by Loop-Mediated Isothermal Amplification in Cattle and Water Buffaloes

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

Delaying embryo development by storing at 4°C for synchronization to recipients in microinjection technique in rabbits

Short-term storage of embryos at low temperature induces developmental arrest of the embryo and would appear to be a valuable aid in embryo-transfer techniques to avoid wasting embryos. Embryo storage at 4°C was examined to allow synchronization with embryo-transfer recipients using the microinjection technique. Superovulation was induced in female Japanese White donor rabbits four days before mating with males. At the same time, control recipients were injected with human chorionic gonadotropin (hCG) to allow synchronization (R1); the hCG injections were delayed by 24 h in the experimental group (R2). DNA constructs for expressing human C-reactive protein or apolipoprotein AII were microinjected into the male pronuclei of the ova. The microinjected embryos were immediately transferred to recipients (R1) or stored at 4°C in phosphate-buffered saline containing 10% fetal bovine serum. After 17-20 h, the stored embryos were incubated at 37°C for one hour, and the morphologically normal embryos were transferred to recipients (R2). In the R1 rabbits, 855 embryos were transferred to 29 recipients, and 72.4% of the recipients became pregnant. Seven of the 84 offspring were transgenic. In the R2 rabbits, 478 embryos were transferred to 16 recipients, and 62.5% of the recipients became pregnant. Two of the 39 offspring were transgenic. There were no differences in pregnancy rate, litter size and transgenic integration rate between R1 and R2. These results suggest that the short-term 4°C storage of microinjected embryos can be a valuable method for synchronization with recipients, and reducing wastage of embryos and the sacrifice of rabbits.

Cryopreservation of Canine Embryos and Resulting Pregnancies

The assisted reproductive techniques used in dogs have strictly limited utility when compared with other mammals. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen has been only readily available method for improved breeding for companion and working dogs. Recently, successful cryopreservation of embryos and subsequent embryo transfer with a non-surgical technique in dog was reported. Application of embryo cryopreservation and transfer technology could contribute to breeding management in companion dogs, working dogs including guide dogs and drug-detecting dogs and quarantine dogs. Such technology would also facilitate the transportation and storage of genetic materials and aid in the elimination of vertically transmitting diseases in the dog.

Induction of superovulation in South American camelids

The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular growth.

Recurrent IVF failure: other factors

IVF failure is a problem for a couple in the singular but can be a tragedy in the plural. Recurrent IVF failure has multiple known causes but many which are not routinely considered as part of the posttreatment analysis. The reason is there are several causes associated with lifestyle and other causes related to pre-existing conditions that have only a tenuous or no apparent connection to fertility. This article examines the impact of obesity, cigarette smoke, uterine anatomy, body mass index, thyroid dysfunction, immune factors, the hereditary and acquired thrombophilias, and embryo transfer technique on recurrent IVF failure.

Standardization of catheter load speed during embryo transfer: comparison of manual and pump-regulated embryo transfer

The position of transfer air bubbles after embryo transfer is related to the pregnancy rate. With the conventional manual embryo-transfer technique it is not possible to predict the final position of the air bubbles. This position mainly depends on the catheter load speed at transfer (injection speed), a parameter that remains uncontrollable with the conventional technique even after standardization of the protocol. Therefore, the development of an automated device that generates a standardized injection speed is desirable. This study aimed to examine the variation in injection speeds in manual embryo transfer and pump-regulated embryo transfer (PRET). Seven laboratory technicians were asked to perform simulated transfers using the conventional embryo-transfer technique. Their injection speeds were compared with that of a PRET device. The results indicate that in manually performed transfers, even after standardization of the protocol, there is still a large variation in injection speed, while a PRET device generates a reliable and reproducible injection speed and therefore brings new possibilities for further standardization of the embryo-transfer procedure. Future research should reveal whether these experiments mimic real clinical circumstances and if a standardized injection speed results in more exact positioning of the transferred embryos and therefore higher pregnancy rates. The position of transfer air bubbles after embryo transfer is related to the pregnancy rate. With the currently used embryo-transfer technique, in which embryos are transferred manually with a syringe, we are not able to predict the final position of the air bubbles. This position depends on the injection speed of the syringe, which remains uncontrollable in embryo transfers that are performed manually even after standardization of the protocol. Therefore we developed an automated device that generates a reliable and reproducible injection speed, a pump-regulated embryo transfer (PRET) device. This study aimed to examine the variation in injection speeds in transfers performed manually and with the PRET device. Our results indicate that in manually performed transfers, even after standardization by protocol, there is still a large variation in injection speed and that the PRET device generates a reliable and reproducible injection speed and therefore brings new possibilities for further standardization of the embryo-transfer procedure. Future research should reveal whether our experiments mimic real clinical circumstances and if a standardized injection speed results in more exact positioning of the transferred embryos and therefore higher pregnancy rates.

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF 2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0–3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.

Effects of Embryo Transfer Catheters on the Endometrial Surface Noted at Hysteroscopy

Embryo transfer (ET) technique including degree of difficulty has been recognized as an important determinant of IVF success. Using hysteroscopy, we report the effects of 3 different ET catheter systems on the endometrial cavity in those with cervical stenosis.

Does it really matter how far the embryos are transferred from the fundus?

Proper embryo transfer (ET) technique contributes to IVF success. The literature is split on how far the embryos should be transferred (assessed by the location of the air bubble) from the fundus. Our aim was to assess the effect of ET location on ongoing pregnancy (P) rates.

Artificial Shrinkage Improves Embryo Survival and Qualities of Cryopreserved Bovine Blastocysts.

Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification and slow freezing of bovine blastocysts on the survival rate and apoptosis of frozen-thawed embryos. Bovine blastocysts were vitrified and slow-freezed after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. The shrunken (Group A) and not shrunken (Group B) vitrified embryos were exposed to cryoprotectant 15% ethylene glycol-15% DMSO and 0.5 M sucrose for 1min, and the shrunken (Group C) and not shrunken (Group D) slow-freezed embryos were exposed to cryoprotectant 1.5 M ethylene glycol and 0.1 M sucrose for 5 min. Blastocysts (Group A and B) were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. Also blastocysts (Group C and D) were loaded 0.25 ml plastic straws and transferred into a controlled rate freezer and plunging into liquid nitrogen for storage. Then, after thawing, blastocysts derived from Group A and B were diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min, and blastocysts derived from Group C and D were washed three times each in Hepes buffered TCM-199 for 5min, respectively. After thawing, differences in survival and hatching rates of blastocysts derived from shrunken and not shrunken embryos were found in both vitrification (Group A vs B: 95.3±4.3 vs 81.1±7.1% for survival rate, 72.8±10.3 vs 58.9±8.3% for hatching rate P<0.05) and slow freezing (Group C vs D: 89.6±7.6 vs 53.1±12.4% for survival rate, 41.6±9.7 vs 21.4±12.9% for hatching rate, P<0.05) groups. Furthermore, we also found that the total cell numbers of blastocysts in artificial shrinkage groups were increased (Vitrification: 130.5±2.6 vs 113.7±3.5, Slow freezing: 116.7±2.4 vs 101.5±4.3, P<0.05) and the numbers of blastomeres undergoing apoptosis in artificial shrinkage groups were decreased (Vitrification: 8.5±1.5 vs 14.5±1.4, P<0.05; Slow freezing: 13.4±1.0 vs 14.3±2.6). Consistent with the results, qPCR data showed that the expression of anti-apoptotic Bcl-xl gene was greatly increased by shrunken groups, whereas the expression of pro-apoptotic Bax was decreased. Our results showed that survival and qualities in the both vitrified and slow-freezed bovine blastocysts could be improved by reducing the fluid content after artificial shrinkage. Therefore, we suggest that artificial shrinkage with pulled pasteur pipet is an effective pretreatment technique for the both vitrification and slow freezing methods of bovine blastocysts. (poster)

Polymorphisms of the bovine luteinizing hormone/choriogonadotropin receptor (LHCGR) gene and its association with superovulation traits

The major limitation to the development of embryo transfer technique in cattle is the highly variable between individuals in ovulatory response to FSH-induced superovulation. The objective of this study was to identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, variation in the bovine luteinizing hormone/choriogonadotropin receptor (LHCGR) gene was investigated using PCR-single-strand conformational (PCR-SSCP) and DNA sequencing. Four single nucleotide polymorphisms (SNPs) of G51656T, A51703G, A51726G and G51737A were identified at the intron 9 of the LHCGR gene in 171 Chinese Holstein cows treated for superovulation, and evaluated its associations with superovulatory response. Association analysis showed that these four SNPs had significant effects on the total number of ova (TNO) (P < 0.05). Moreover, the A51703G and A51726G polymorphisms significantly associated with the number of transferable embryos (NTE) (P < 0.05). In addition, significant additive effect on TNO was detected in polymorphisms of G51656T (P < 0.05) and A51703G (P < 0.01), and the A51703G polymorphism also had significant additive effects on NTE (P < 0.01). These results indicate that LHCGR gene is a potential marker for superovulation response and can be used to predict the most appropriate dose of FSH for superovulation in Chinese Holstein cows.

Myometrial response to neurotropic and musculotropic spasmolytic drugs in an extracorporeal perfusion model of swine uteri

To compare the effects of neurotropic and musculotropic spasmolytic drugs in isolated swine uterus specimens, 80 swine uteri were perfused using an established model for preserving a viable organ that responds to oxytocic hormones and spasmolytic drugs. An intrauterine catheter was used to record pressure changes. Following initiation of rhythmic uterine contractions and recording of spontaneous rhythmic contractions, spasmolytic drugs (butylscopolamine, atropine, denaverine, morphine, metamizole, pethidine and celandine) were administered at various concentrations. The musculotropic relaxant denaverine in particular showed significant results ( P ⩽ 0.05) for all dosages and parameters investigated. In terms of muscle physiology, musculotropic agents (denaverine and celandine) have clear advantages in comparison with neurotropic (butylscopolamine and atropine) or musculoneurotropic (morphine, metamizole and pethidine) spasmolytic drugs for inhibiting contractions. Experiments with pethidine (Dolantin) also showed promising results; with celandine (Paverysat), an initial increase in contractions was observed that may suggest ways of promoting rapid directed sperm transport. Denaverine and pethidine in particular may in the future be able to play an important role in improving the pregnancy rate after IVF. Uterine contractions are important not only for labour, but also for the transport of semen and the oocyte. Contraction inducing-medicaments, such as oxytocin or the various prostaglandins, are used in everyday practice. These medicaments, however, interact with the arterial pressure which can be of utmost importance in hypertonic or pre-eclamptic patients. Using perfused swine uteri, which we obtained from the slaughterhouse, we were able to examine which substances can be safely used to influence the uterine contractility with the lowest rise in the arterial pressure. In these experiments we examined a group of drugs known as spasmolytic drugs. Denaverine and pethidine (Dolantin) showed promising results. In view of the latest research, the physiology of the menstrual cycle and the standard embryo-transfer technique, it appears that a combination of different drugs that are capable of positively reducing uterine contractions currently provides the best conditions for preparing the uterus sufficiently for embryo implantation. Denaverine and pethidine may in the future be candidates for improving the pregnancy rate after IVF.

Traversing the ovine cervix – a challenge for cryopreserved semen and creative science

This review brings together research findings on cervical relaxation in the ewe and its pharmacological stimulation for enhancement of the penetration needed for transcervical insemination and embryo transfer. On the basis that the success of artificial insemination is the percentage of ewes lambing, a review is made of recent research aimed at understanding and minimising the sub-lethal effects of freezing and thawing on the viability of spermatozoa, their membrane integrity and their ability to migrate through cervical mucus, as these characteristics have a major influence on fertility, particularly when semen is deposited, artificially, in the os cervix. Milestones of achievement are given for transcervical intrauterine insemination, embryo recovery and transfer and the birth of lambs of pre-determined sex, firstly following intracytoplasmic sperm injection, then laparoscopic intrauterine insemination using highly diluted flow-cytometrically sorted fresh semen and subsequently by os cervix insemination using sexed semen that had been frozen and thawed. Diversity of research endeavour (applied, cellular, molecular), research discipline (anatomy, histology, immunology, endocrinology) and research focus (cell, tissue, organ, whole animal) is embraced within the review as each has significant contributions to make in advancing recent scientific findings from the laboratory into robust on-farm transcervical insemination and embryo transfer techniques.

Assisted Reproductive Technologies (ART) With Baboons Generate Live Offspring: A Nonhuman Primate Model for ART and Reproductive Sciences

Human reproduction has benefited significantly by investigating nonhuman primate (NHP) models, especially rhesus macaques. To expand the Old World monkey species available for human reproductive studies, we present protocols in baboons, our closest Old World primate relatives, for assisted reproductive technologies (ART) leading to live born offspring. Baboons complement rhesus by confirming or modifying observations generated in humans often obtained by the study of clinically discarded specimens donated by anonymous infertility patient couples. Here, baboon ART protocols, including oocyte collection, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), preimplantation development to blastocyst stage, and embryo transfer techniques are described. With baboon ART methodologies in place, motility during baboon fertilization was investigated by time-lapse video microscopy (TLVM). The first ART baboons produced by ICSI, a pair of male twins, were delivered naturally at 165 days postgestation. Genetic testing of these twins confirmed their ART parental origins and demonstrated that they are unrelated fraternal twins not identicals. These results have implications for ART outcomes, embryonic stem cell (ESC) derivation, and reproductive sciences.

Opinion

Opinion

Improving of pregnancy rate by modifying embryo transfer technique

Improving of pregnancy rate by modifying embryo transfer technique

A new safe embryo transfer technique and its impact on ICSI outcome

To assess and modify the after load technique of embryo transfer developed by Adrienne et al. [Adrienne BN, James HS, Sasha H, Aidita NJ, Jeffrey LM. Embryo afterloading: a refinement in embryo transfer technique that may increase clinical pregnancy. Fertil Steril 2005;83:710–4] and to study its impact on ICSI outcome. Prospective comparative analysis. A private referral IVF center. One hundred and forty-four ICSI–ETs were performed by a single provider and a single embryologist over a 1-year period of time in a private IVF center. Ninety-nine ICSI–ETs were performed as the classic ETs technique and forty-five ICSI–ETs were performed by the external sheath 1st technique. Primary outcome: – the degree of presence of mucous and blood in the embryo transfer catheter. Secondary outcome: – clinical pregnancy. The average age of all the patients was 30.4±4.3years, with a range of 21–38years. The overall pregnancy rate was 32.6%. The external sheath 1st technique group showed highly significant easy technique and higher clinical pregnancy than the classic technique ( P -value=0.0023, 0.0025, respectively), also the external sheath 1st technique showed a significantly lower mucous, blood in the inner sheath catheter and the outer sheath catheter over the classic embryo transfer group. External sheath 1st-embryo transfer is a refinement of standard ET technique and may improve clinical pregnancy rates by facilitating the ease of embryo transfer.

Advances in Swine In Vitro Embryo Production Technologies

Recent advances in new technologies to produce cloned and genetically modified pigs involve manipulating oocytes and/or embryos in vitro. Although a great deal of progress has been made, the current IVM-IVF systems still result in major problems: a high rate of polyspermy; and a low development rate and low quality of blastocysts for in vitro compared with the in vivo-produced embryos. This study summarizes recent advancements in IVM-IVF-IVC porcine systems. Recent methods to select monospermic embryos are also discussed. Finally, achievements in vitrification and in somatic cell nuclear transfer are discussed.

What can the clinician do to improve implantation?

Implantation is a complicated process that requires the orchestration of a series of events involving both the embryo and the endometrium. Even with the transfer of high quality embryos, implantation rates remain relatively low. The growing tendency towards transferring fewer embryos provides further incentives to improve implantation rates. In this article, the various clinical strategies employed to increase the chance of implantation are reviewed. Embryo transfer technique is a critical step in assisted reproductive technology cycles. Recent studies have shown significant improvements in clinical pregnancy rates resulting from careful embryo transfer technique, appropriate catheter type and placing for embryo transfer. Increasingly, adjuvant pharmaceutical therapies are also being applied with the aim of improving embryo implantation. However, the evidence for their efficacy and safety is limited. Recent evidence suggests that adoption of milder ovarian stimulation regimens may provide a more effective clinical approach to improving implantation, since beneficial effects have been shown for both endometrial receptivity and embryo quality.

Improvement of pregnancy rate by modification of embryo transfer technique: a randomized clinical trial

In this randomized trial study, two groups of infertile women (n=55) aged≤40 years underwent in vitro fertilization or intracytoplasmic sperm injection treatment cycles with or without 0.2 mL of air pushed into the catheter after embryo transfer. The implantation and clinical pregnancy rates were statistically significantly higher in the study group than in the controls. This improvement on standard ET technique may advance clinical pregnancy rates.