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Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice.

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.

Animal Welfare↗

A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.

A prospective randomized trial of in-vitro fertilization and embryo transfer was undertaken to investigate the reported beneficial effects of culturing preimplantation human embryos in groups, rather than individually. A total of 159 treatment cycles, in which the women were matched for age, basal gonadotrophin concentrations and number of previous attempts, were included in the study. Of these, 78 cycles were randomized to the 'individual culture' group, and 81 cycles were randomized to the 'group culture' group. The groups did not differ in terms of the median number of oocytes or embryos obtained per cycle. There was no statistically significant difference between the two groups in terms of treatment outcome, as assessed by pregnancies or clinical pregnancies.

Adult↗

Ultrasonographic-guided embryo transfer does not enhance pregnancy rates compared with embryo transfer based on previous uterine length measurement.

OBJECTIVE: To compare pregnancy rates (PRs) after ultrasound-guided embryo transfer and embryo transfer based on ultrasonographic length measurement. DESIGN: Prospective intervention group combined with retrospective control group. SETTING: University fertility clinic. PATIENT(S): Patients undergoing IVF and intracytoplasmic sperm injection. INTERVENTION(S): Transabdominal ultrasonographic guidance at embryo transfer. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rates. RESULT(S): In 367 ultrasound-guided embryo transfers clinical PR, ongoing PR, and implantation rate were 35.1%, 31.1%, and 24.3%, respectively. In 363 embryo transfers based on previous ultrasonographic length measurement, the rates were 33.9%, 29.5%, and 24.2%, respectively. There were no statistical significant differences between the groups. CONCLUSION(S): Ultrasonographic guidance does not show any benefit in terms of PR and implantation rate compared to previous ultrasonographic length measurement, an other precise and atraumatic transfer technique.

Adult↗

Cervical dilatation with hygroscopic rods prior to ovarian stimulation facilitates embryo transfer.

BACKGROUND: Embryo transfer is a critical factor affecting the success of IVF-the ease of embryo transfer has a direct impact on the success rate. The aim of this study was to assess the value of cervical dilatation with hygroscopic cervical rods (Dilapan-S) in patients with difficult embryo transfer. METHODS: Fifty-four patients undergoing IVF treatment, who either failed to conceive after previous difficult embryo transfer or were noted to have difficult mock embryo transfer were retrospectively included in the study. In this way the patients acted as control for themselves. The Dilapan-S rods were placed intracervically and left for 4 h prior to starting gonadotrophin stimulation as an outpatient procedure. RESULTS: Of the 54 patients who originally had difficult embryo transfer, 43 patients (79.5%) had subsequent easy embryo transfer. Thirty patients managed to conceive, giving a clinical pregnancy rate of 55%. CONCLUSIONS: Cervical dilatation using hygroscopic dilators facilitates difficult embryo transfer and helps to improve the pregnancy rate.

Abortion, Spontaneous↗

Influence of embryo transfer depth on in vitro fertilization and embryo transfer outcomes.

OBJECTIVE: To investigate the influence of transfer distance from the fundus (TDF) on clinical pregnancy rate (PR) and ectopic pregnancy rate. DESIGN: Retrospective cohort. Between January 2000 and December 2001, 699 ultrasound (US)-guided embryo transfers were conducted. Mock transfer was performed to measure uterine cavity depth 1 month before treatment. Cavity depth was measured by abdominal US before the transfer, from the vaginal stripe to the fundus. Transfers were performed with a Wallace embryo transfer catheter (Cooper Surgical, Shelton, CT) using US and physician's judgment of cavity depth. Transfer distance from the fundus was calculated by subtracting the depth of catheter insertion from the cavity depth, as determined by US or by mock transfer. Statistical analyses were performed by building a multivariable logistic regression model to calculate odds ratios and 95% confidence intervals (CI). SETTING: Women aged 23 to 43 years who are in a university-affiliated, community-based IVF program in Springfield, Massachusetts. PATIENT(S): All patients enrolled in IVF program undergoing embryo transfer. INTERVENTION(S): No patient received any additional procedure or intervention. All of the measurements obtained with the embryo catheter and the transvaginal ultrasound were part of the program's protocol for the embryo transfer. MAIN OUTCOME MEASURE(S): Odds ratio examining relationship between embryo transfer depth and PR.Clinical, implantation, and ectopic PR were 37%, 20%, and 2.1%. Cavity depth by US differed from cavity depth by mock by at least 10 mm in >30% of cases. The TDF by US was highly predictive of PR; TDF by mock was not predictive of PR. Increasing the TDF by US resulted in significantly increased PR as well as lower ectopic rates. Using regression analysis, the odds ratio for TDF by US was 1.11 (95% CI: 1.07-1.14). This suggests that for every additional millimeter embryos are deposited away from the fundus, the odds of clinical pregnancy increased by 11%. CONCLUSION(S): After controlling for potential confounders, the clinical PR is significantly influenced by the transfer distance from the fundus. Cavity depth by US is clinically useful to determine the depth beyond which catheter insertion should not occur.

Adult↗

Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program.

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.

Animals↗

Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial.

BACKGROUND: With the aim of reducing the number of multiple pregnancies after IVF we investigated the effectiveness of two cycles with single embryo transfer (SET) and one cycle with double embryo transfer (DET) after IVF and calculated the cost-effectiveness of both strategies. METHODS: A randomized controlled trial was performed in 107 women, aged <35 years, in their first IVF cycle, with at least one good quality embryo. They were randomized to the SET (n = 54) or DET (n = 53) group using a computer-generated random block number table, stratified for primary or secondary infertility. RESULTS: The cumulative live birth rates per woman randomized of two consecutive cycles of SET [41%; 95% confidence interval (CI) 27-54] versus one cycle of DET (36%; 95% CI 23-49) were comparable, whereas the multiple pregnancy rate was significantly higher: 37% (95% CI 15-59) in the DET and 0% in the in the SET group (P = 0.002). Combining the medical costs of the IVF treatments (where 1.5 more SET cycles were required to achieve each live birth) and of pregnancies up to 6 weeks after delivery, the total medical costs of DET per live birth were 13,680 and 13,438 for SET. CONCLUSIONS: Two cycles with SET were equally effective as one cycle with DET, and the medical costs per live birth up to 6 weeks after delivery were the same. However, if lifetime costs for severe handicaps are included, more than 7000 per live birth will be saved after implementing SET. Because of the high probability of multiple pregnancies in this group of IVF patients, only SET should be performed.

Adult↗

Effectiveness of two-step (consecutive) embryo transfer in patients who have two embryos on day 2: comparison with cleavage-stage embryo transfer.

OBJECTIVE: To evaluate the effectiveness, for patients who have only two embryos on day 2, of a two-step (consecutive) embryo transfer (ET) procedure in which a cleaved embryo is transferred on day 2 and a single blastocyst is transferred on day 5. DESIGN: Observational comparative study. SETTING: Private IVF clinic. PATIENT(S): Ninety two-step ET cycles were performed in patients who had two embryos on day 2 (two-step group). Ninety day-2 ET cycles were performed in age- and infertility-matched patients who had two embryos on day 2 (control group). INTERVENTION(S): Cleaved-ET, extended culture of one embryo, and a second transfer of a blastocyst. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): The pregnancy and implantation rates in the two-step group (respectively 33.3% and 17.2%) were significantly higher than those in the control group (18.9% and 9.4%). Thirty-nine of the patients in the two-step group (43.3%) could not proceed to the second step of ET because no viable blastocyst could be obtained, but four of them conceived anyway. CONCLUSION(S): Taking advantage of both day-2 ET and blastocyst transfer, two-step ET may be an effective option for ET in patients who have an insufficient number of embryos.

Adult↗

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.

Animal Welfare↗

Serum prolactin response to embryo transfer during human in vitro fertilization and embryo transfer.

Serum prolactin (PRL) concentrations around the time of embryo transfer (ET) have not been studied, despite the fact that transient hyperprolactinemia regularly occurs in response to laparoscopy for oocyte recovery and ET itself may be stressful enough to induce a PRL rise. Hyperprolactinemia might compromise luteal support for implantation and contribute to the limited success of ET. We measured serum PRL concentrations in 10 normoprolactinemic women immediately before, during, and after ET and compared the PRL response around ET to that induced by laparoscopy as a measure of the competency of the stress-prolactin axis. Nine of ten patients demonstrated a significant PRL response to surgery. The mean (+/- SEM) intraoperative PRL concentration (124.0 +/- 19.6 ng/ml) was significantly higher than the preoperative level (12.3 +/- 2.4 ng/ml) (P less than 0.01). Three hours after surgery PRL levels had decreased (44.8 +/- 11.5 ng/ml) but remained above baseline. All subjects were normoprolactinemic 48 hr after laparoscopy. Serum PRL concentration did not change significantly in response to ET, with levels of 10.4 +/- 1.7, 12.4 +/- 1.1, and 10.6 +/- 1.8 ng/ml immediately before, during, and 3 hr after ET, respectively. While laparoscopy for in vitro fertilization--embryo transfer commonly induces hyperprolactinemia, the PRL rise is transient, with no carryover to the time of ET. Embryo transfer itself does not induce a significant PRL rise.

Embryo Transfer↗

Is elective single embryo transfer a cost-effective alternative to double embryo transfer?

Elective single embryo transfer (eSET) is increasingly being considered as a means to reduce twin pregnancies associated with in vitro fertilisation treatment. However, it is important to consider the cost-effectiveness of alternative strategies when considering a change in policy. A review of the literature showed only five studies assessing both costs and consequences of strategies involving eSET compared with double embryo transfer. Several limitations in these studies prevent a definitive conclusion on the cost-effectiveness of eSET being reached. Future economic evaluations need to compare strategies relevant to routine practice, include all relevant costs, measure and value longer term outcomes appropriately, and assess the cost-effectiveness of eSET across different subgroups of women.

Cost-Benefit Analysis↗

[Comparison of pregnancies issued from frozen embryo transfers and pregnancies issued from fresh embryo transfers in fertilization in vitro. FIVNAT].

The French in vitro fertilization registry FIVNAT allowed us to describe the most important cohort of pregnancies resulting from the replacement of frozen-thawed embryos, and to compare them with pregnancies issued from fresh embryos transfers. From 1987 to 1992, 465 and 8,757 clinical pregnancies were respectively collected, and resulted in the birth of 386 et 8729 babies. Whereas the miscarriage rate was higher, pregnancy evolution was more normal after transfer of frozen-thawed embryos. Multiple pregnancies were less frequent; preterm birth rates and low birth rate were also significantly lower. Nevertheless, after cryopreservation of embryos, more complications were noted. However, further analysis involving more pregnancies after transfer of frozen-thawed embryos is necessary before reaching conclusion. Finally, congenital malformation rate was not different between the two groups.

Abortion, Spontaneous↗

Ultrasound-guided embryo transfer maximizes the IVF results on day 3 and day 4 embryo transfer but has no impact on day 5.

BACKGROUND: The use of ultrasound-guided embryo transfer has been reported to affect success rates in some centres but not others. In a prospective study, we examined the influence of ultrasound guidance in embryo transfer performed on different days after oocyte retrieval. METHODS: Two different methods of embryo transfer were evaluated in 1069 consecutive transfers. The ultrasound-guided embryo transfer was used in 433 cases, whereas 636 embryo transfers were performed with the tactile assessment ('clinical feel') method. RESULTS: Ultrasound-guided embryo transfer yielded a higher overall pregnancy rate than the 'clinical feel' approach, 47 versus 36% (P < 0.001). This difference was statistically significant where embryos were transferred after 3 or 4 days of culture, 45.9 versus 37.1% (P = 0.001) and 42.3 versus 27% (P = 0.035) respectively but not significant (P = 0.112) on day 5 embryo transfer (56.3 versus 45.7%). Likewise, the implantation rate was significantly different between the two groups on day 3 and 4 embryo transfer, 23.3 versus 15.8% (P < 0.01) and 21.6 versus 15.7% (P < 0.05%) respectively but no statistical difference was noted on day 5 embryo transfer, 26.7 versus 23.6%. CONCLUSION: Ultrasound assistance in embryo transfer on day 3 and 4 significantly improved pregnancy rates in IVF but had no impact on day 5.

Culture Techniques↗

Ultrasound-guided embryo transfer and the accuracy of trial embryo transfer.

BACKGROUND: Studies have suggested that ultrasound-guided embryo transfer (UG-ET) may improve the outcome in IVF; however, several factors may account for the improvement in pregnancy rate. This study examines the use of ultrasound to determine the accuracy of trial transfer (TT) in preparation for ET. METHODS: Sixty-seven consecutive patients prospectively underwent UG-ET over a 2 month period. Total cavity length by US was compared with the length noted by TT. A difference of > or = 1 cm was considered significant. All embryos were placed within 1-2 cm of the fundus by US. RESULTS: Twenty patients (29.9%) had a difference of > or = 1 cm and 13 patients (19.4%) had a difference of < or = 1.5 cm. Patients with a difference of > or = 1 or > or = 1.5 cm had a significantly greater depth at transfer (P < 0.001) and uterine cavity length (P < 0.001) when compared with patients without a difference. Clinical pregnancy, implantation, delivery and overall miscarriage rates did not differ between patients with a difference of > or = 1 or > or = 1.5 cm versus no difference. There were no ectopic pregnancies. CONCLUSIONS: Nineteen percent of patients had a discrepancy of > or = 1.5 cm and approximately 30% had a difference of > or = 1 cm from TT at UG-ET, suggesting a benefit to UG-ET. A large prospective randomized trial comparing UG-ET with blind transfer is required to assess further if UG-ET should be used in all cases of ET.

Abortion, Spontaneous↗

[Influence of the depth of embryo transfer on pregnancy outcome in in vitro fertilization-embryo transfer].

OBJECTIVE: To detect whether the depth of embryo transfer has influence on pregnancy outcome in in vitro fertilization-embryo transfer (IVF-ET). METHODS: The distance between the high echogenic transfer dot and the fundal endometrium was measured under guidance of transabdominal ultrasound. The average distance 0.75 cm was used to divide patients into two groups. Group 1 included 44 patients with a distance > 0.75 cm, while group 2 had 48 patients with a distance </= 0.75 cm. RESULTS: There were no significant differences in the average age (32 +/- 3 versus 31 +/- 4), the mean number of oocytes retrieved (14 +/- 7 versus 14 +/- 6), and the mean number of embryos transferred (2.3 +/- 0.6 versus 2.2 +/- 0.5) between groups 1 and 2. The distance to the fundal endometrium in group 2 was (0.50 +/- 0.21) cm, significantly smaller than that of group 1 (1.02 +/- 0.21) cm (P < 0.01). However, pregnancy rate was significantly higher (P < 0.05) in group 2 (58.3%, 28/48) than in group 1 (36.4%, 16/44). Although implantation rate in group 2 (33.0%, 35/106) was higher than group 1 (22.2%, 22/99), the difference was not significant. CONCLUSION: Pregnancy outcome may be affected by the depth of embryo transfer, and the fundal endometrium is a favorable region for embryo deposition.

Abdomen↗

In vitro production of bovine embryos and their application for embryo transfer.

This review introduces newly developed serum-free media (IVD101 and IVMD101), that are effective for producing high yields of transferable embryos of good quality from in vitro-matured and -fertilized oocytes. Both serum-free media produced better results than serum-containing medium, including increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. In addition, reduced risks of calf mortality and large calf syndrome were also observed for the serum-free-derived embryos. Serum-derived embryos contained a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality. A non-invasive technique using scanning electrochemical microscopy was successful in quantitatively measuring oxygen consumption of single embryos. This technique may prove to be reliable for predicting embryo viability and subsequent developmental ability.

Animals↗

An assessment of alternative policies for embryo transfer in an in vitro fertilization-embryo transfer program.

Conventionally, in in vitro fertilization (IVF) programs, all embryos obtained up to three are freshly transferred in the IVF cycle and supernumerary embryos frozen if cryopreservation facilities exist. This study was concerned with assessing alternative policies for embryo transfer (ET). When three or fewer embryos were obtained in the IVF cycle, fresh embryo transfer was either excluded (group I, n = 69) or only one embryo was immediately transferred (group II, n = 46), the remainder being transferred after freezing and thawing. The pregnancy rate in these two policies was compared to that in a control group of patients (group III, n = 115) in whom all the embryos were transferred in the IVF cycle. The ongoing pregnancy rate was similar in all three groups. No multiple pregnancies occurred in groups I and II compared with one in the controls. The consequences of these policies for pregnancy rates and the incidence of multiple gestation are discussed. It is concluded that the deliberate limitation of fresh ET to a maximum of one embryo followed by one or more cycles of thawed frozen ET is not detrimental to the pregnancy rate and is less likely to be associated with multiple gestations. Conversely, this policy increases the workload of the laboratory staff and raises certain ethical questions.

Chorionic Gonadotropin↗