Triple Transfers Research Articles

P–693 Gonadotropin stimulation reduces the implantation and live birth but not the miscarriage rate – a study based on the comparison of stimulated and unstimulated IVF

Abstract Study question Does gonadotropin stimulation in conventional IVF (cIVF) affect the implantation, miscarriage and live birth rates? Summary answer Gonadotropin stimulation negatively affects the implantation and live birth but not the miscarriage rate in IVF treatments. What is known already Literature hypothesizes that embryos derived from unstimulated, natural cycle IVF (NC-IVF) have a higher implantation potential compared to embryos from cIVF. In NC-IVF, recruitment of the leading follicle is based on natural selection. Hormonal stimulation might not only affect the embryo but also endometrial function. It’s possible to compare outcomes of NC-IVF and cIVF if cIVF is performed without embryo selection, in other words, if only those zygotes, which will be transferred 1–2 days later, are left in culture and all other zygotes are cryopreserved. To test this hypothesis, we compared success rates in NC-IVF and in cIVF. Study design, size, duration We performed a cohort study from 2011–2016 including data on IVF cycles with transfer of fresh embryos on day 2–3 at a University based infertility center. Our sample consisted of 640 women with 1482 embryos transferred in 996 cycles. We defined implantation rate as the number of sonograhically detected amniotic sacs per transferred embryos. Data originated from the Swiss ART registry “FIVNAT” and the Bern IVF Cohort and was completed using medical and delivery records. Participants/materials, setting, methods We defined NC-IVF as IVF without stimulation of follicular growth and cIVF as IVF with gonadotropin stimulation ≥75 IE/d and >3 retrieved oocytes. We performed zygote, but not embryo selection and transferred embryos on day 2–3. We calculated implantation and live birth per transferred embryo as binary outcomes using bi- and multivariable multilevel logistic regression models accounting for two clusters; the women and the cycle; and adjusting for maternal and infertility characteristics using STATA. Main results and the role of chance Age of women (p = 0.531), parity (p = 0.194) and type of infertility (primary vs secondary) (p = 0.463) did not differ between women undergoing NC-IVF or cIVF. In NC-IVF, 468 (31.6%) embryos were transferred, 450 as single, 18 as double transfers. In cIVF, 1014 (68.4%) embryos were transferred, 91 as single, 830 as double and 93 as triple transfers. Implantation rate was higher in NC-IVF. In NC-IVF 80 (17.1%) and in cIVF 132 (13.0%) embryos developed into an amniotic sac (OR 1.58; 95% CI 1.01–2.46; p = 0.042). After adjustment for maternal age (p < 0.001), parity (p < 0.001), type of infertility (p = 0.037), duration of subfertility and indication for IVF, aOR for implantation per transferred embryo increased to 1.87 (95% CI 1.21–2.91; p = 0.005). Miscarriage rate was similar. In NC-IVF and cIVF 25% (n = 20; n = 33) miscarried and 75% (n = 60; n = 99) ended in a live birth, respectively (OR 0.91; 95% CI 0.32–2.60; p = 0.855; aOR 1.0; 95% CI 0.42–2.36; p = 1.000). Live birth rate per transferred embryo was increased in NC-IVF; 60 of 468 (12.8%) embryos in NC-IVF compared to 99 of 1041 (9.8%) embryos in cIVF resulted in a live birth (OR 1.51; 95% CI 0.92–2.49; p = 0.106); and became significantly higher after adjustment (aOR 1.85; 95% CI 1.16–2.95; p = 0.010). Limitations, reasons for caution This study analyses observational data from a clinic offering NC-IVF and cIVF treatment as equivalent options. NC-IVF is a model for natural fertility and allows us to study the impact of gonadotropins. However, it is not a randomised study and therefore prone to selection bias. Wider implications of the findings: The study suggests that gonadotropin stimulation might reduce the implantation potential and subsequently live birth rates, by possibly affecting embryo and endometrium quality. Clinicians should consider lower gonadotropin doses for stimulation. Trial registration number Not applicable

An objective functional evaluation of the flexor carpi ulnaris set of triple tendon transfer in radial nerve palsy.

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A Loop-Tendon Suture for Tendon Transfer or Graft Surgery

We present a loop-tendon suture technique that was designed for easy tension adjustment and early postoperative rehabilitation in tendon transfer or graft surgeries. This study tested the biomechanical strength of the loop-tendon suture by using chicken flexor tendons and we report the preliminary clinical results. We tested the ultimate strength of the loop-tendon suture against the end-weave suture technique in chicken flexor tendons. Forty flexor digitorum longus tendons of chickens were divided into 2 groups according to the suture technique, loop-tendon suture and end-weave suture groups, and then were subjected to linear loading in a tensile load testing machine. From 2000 to 2002 we performed 27 tendon transfer surgeries clinically, including 4 interpositional tendon grafts in 15 patients, using this technique followed by immediate passive motion exercise after surgery. The mean follow-up time was 20 months. The ultimate tensile load of chicken flexor tendons was 31 +/- 6 N for the loop-tendon suture group and 23 +/- 8 N for the end-weave suture group, and the difference was statistically significant. Clinically, 6 patients with an extensor indicis proprius to an extensor pollicis longus transfer showed more than good results in the Geldmacher scheme. Three patients with an extensor indicis proprius to a central tendon transfer achieved proximal interphalangeal extension of less than 15 degrees of extension lag. One patient with a flexor digitorum profundus reconstruction obtained total active motion of 210 degrees. The 5 patients with triple transfers for radial nerve palsy showed more than neutral extension of the wrist and metacarpal joint. There were no tendon ruptures. The loop-tendon suture method has greater strength than the conventional end-weave technique, and can be used for secondary tendon reconstruction surgery with favorable clinical outcomes. Therapeutic IV.

185ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS BY TRIPLE AND SINGLE TRANSFER

Factors that affect the viability of in vitro-produced (IVP) embryos are usually evaluated by comparing pregnancy rates of a treatment and a control group. The ‘er’ model of embryo survival (McMillan WH et al., 1998 Theriogenology 50, 1053–1070) utilizes twin embryo transfer to estimate embryo (‘e’) and recipient (‘r’) contributions to embryo survival, and allows the comparison of treatment effects without using a control group, when treatment is the only change in operations. Application of the model to data of contemporaneous single and twin transfer indicates that ‘e’ and ‘r’ are independent of the number of embryos transferred. Thus, twin transfers enable the efficient use of costly recipients while providing meaningful estimates of single embryo survival rates. The objective of this study was to assess the embryo survival rates of fresh IVP embryos of a newly established IVP lab by applying the model to triple transfers and comparing the expected embryo survival rates with those achieved for single transfers. Cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries of cows of unknown breeds or by ovum pick-up (OPU) from Holstein-Friesian 2- or 3-yr-old donor cows. COCs were matured in 500μL of TCM199+10% FCS (Life Technologies, Auckland, NZ), 10μgmL−1 FSH and LH (ICPBio, Auckland, NZ), 1μgmL−1 estradiol (Sigma, Auckland, NZ), 100μM cysteamine (Sigma) for 24h under 5% CO2 and then fertilized with 1×106 percoll-separated sperm mL−1 from a single bull (Tervit HR and Pugh PA, 2000 14th ICAR 18, 37(abst)). Twenty-four h after insemination, presumptive zygotes were transferred into 500μL mSOF (Pugh A et al., 2001 Theriogenology 55, 314 (abst)) and cultured for 4 days under humidified 5% CO2, 7% O2 and 88% N2. On Day 4, cleaved embryos were transferred into fresh culture medium and culture continued for a further 3 days under the same conditions. Embryo stage and grade were evaluated on Day 7 of culture. Grades 1, 2 and 3 (IETS manual, 2002) compact morulae and blastocysts produced from abattoir-derived COCs were transferred in triplets, while grades 1 and 2 compact morulae and blastocysts from OPU-derived COCs were transferred singly, in 0.25mL insemination straws into synchronized Holstein-Friesian heifers. Recipients received a CIDR (CIDR Cattle Insert, Pharmacia, Auckland, NZ) at Day −12 followed by a prostaglandin (Estroplan, Parnell Laboratories, Auckland, NZ ) injection at Day −6. CIDRs were removed at Day −2, followed by estrus at Day 0 (= day of IVF). Embryos were transferred on Day 7 and recipients received a CIDR after transfer (ET). CIDRs were removed at Day 19 to synchronize any returns. Two experienced practitioners performed all the transfers. Pregnancies (single transfers) and number of live fetuses (triple transfers) were confirmed at Days 60 and 42, respectively. Pregnancies were terminated between Days 62 and 65 by two prostaglandin injections 48h apart. A total of 76 single transfers resulted in 36 pregnancies (47.4%, binomial SD 5.7%). A total of 75 triple transfers (225 embryos) resulted in 98 viable fetuses (44%) and 58 pregnant recipients (77.3%). For triple transfers, the estimates for ‘e’ and ‘r’ were 0.50 and 0.89, respectively, with the product yielding an expected triple embryo survival rate of 44.1%. The actual distribution of 17, 23, 30 and 5 recipients carrying 0, 1, 2, or 3 fetuses, respectively, was not significantly different from the expected values of 16, 25, 25 and 8 estimated from the model (chi-square=2.49, NS). Estimates for ‘e’ and ‘r’ were not significantly different when combined single and triple data were included in the model (‘e’=0.55 and ‘r’=0.90), indicating that embryo survival is independent of the number of embryos transferred. Results indicate that multiple transfers do increase pregnancy rate (from 47.4 to 77.3%), but not embryo survival posttransfer (44.1 v. 47.4%). Although single ET was done with OPU-derived embryos and triple with slaughterhouse-derived embryos and results are not strictly comparable, the similarity of estimates for ‘e’ suggests that using the same in vitro-embryo assessment criteria resulted in embryos of similar intrinsic viability from the two sources. In the near future, we will perform triple transfers of cryopreserved IVP embryos and use the model to estimate embryo and recipient contributions to embryo survival of frozen IVP embryos, without using a fresh control. We will continue to build a dataset based on triple and single transfers to further assess the effect on embryo survival rates of triple and single transfers.

Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donors.

The efficiency of bovine nuclear transfer using recipient oocytes recovered by ultrasound-guided follicle aspiration (ovum pick-up [OPU]) was investigated. Oocyte donors were selected from 2 distinct maternal lineages (A and B) differing in 11 nucleotide positions of the mitochondrial DNA control region. A total of 1342 cumulus-oocyte complexes (COCs) were recovered. The numbers of total COCs and class I/II COCs recovered from donors of lineage A were higher (P < 0.001) than those obtained from lineage B. Follicle aspiration once per week yielded a higher (P < 0.001) total number of COCs per session than aspiration twice per week, whereas the reproduction status of donors (heifer vs. cow) had no effect on OPU results. Of the 1342 oocytes recovered, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) karyoplast-cytoplast complexes (KCCs), resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 84 transferable embryos (15% based on fused KCCs) were obtained. After 38 transfers (10 single, 22 double, and 6 triple transfers), 9 recipients (8 double and 1 triple transfer) were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. The proportion of transferable embryos on Day 7 was significantly (P < 0.05) influenced by maternal lineage of oocyte donors and by the frequency of follicle aspiration. Our study demonstrates the feasibility of generating nuclear transfer embryos with defined cytoplasmic background. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic components on embryonic, fetal, and postnatal development.

Embryo quality and developmental potential is compromised by age.

The aim of this retrospective study was to assess whether and how the age of the woman affects the quality and developmental potential of the oocytes and embryos in an ART program. A total of 878 IVF cycles was included as a consecutive series of single transfers (n=292), dual transfers (n=366) and triple transfers (n=220), where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. We found a highly significant decrease in oocyte recovery with increasing age with about one oocyte per 2.3 years (95% CI 1.8 years to 3.1 years, p<0.0001). Further, we found that the number of oocytes that cleaved declined significantly with increasing age with one per 3.7 years (95% CI 2.7 years to 5.5 years, p<0.0001). This decline was mainly due to the decline in number of oocytes retrieved as the ratio of aspirated oocytes that cleaved with increasing age (approx. -0.04/10 year 95% CI: -0.10; +0.009) was not significantly different (p=0.10). The percentage of transfers using fragmented embryos did not increase significantly with increasing age (p=0.08). The odds of fragmentation increased by 3% per year. The average number of embryos transferred decreased significantly (p=0.03) with age from approximately 2.1 at the age of 25 to approximately 1.8 at the age of 40. In a selected subgroup of embryos all consisting of good quality embryos, a significant decrease was found in implantation rate with increasing age (approx. -0.08/10 years, 95% CI: -1.6; +0.00, p=0.05). Of the 357 pregnancies achieved in this study we found a significantly decreased ongoing pregnancy rate and a significantly increased abortion rate with increasing age (p=0.03). The decrease in the rate of ongoing pregnancies was almost linear, decreasing by approximately 1.5% per year. We conclude that age has an impact throughout a woman's reproductive life and that it is important to realize that the age-related decline in fertility may start already in the late twenties and not in the mid-thirties as is generally assumed.

Embryo quality and developmental potential is compromised by age

Aims. The aim of this retrospective study was to assess whether and how the age of the woman affects the quality and developmental potential of the oocytes and embryos in an ART program. Method and material. A total of 878 IVF cycles was included as a consecutive series of single transfers (n=292), dual transfers (n=366) and triple transfers (n=220), where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. Results. We found a highly significant decrease in oocyte recovery with increasing age with about one oocyte per 2.3 years (95% CI 1.8 years to 3.1 years, p<0.0001). Further, we found that the number of oocytes that cleaved declined significantly with increasing age with one per 3.7 years (95% CI 2.7 years to 5.5 years, p<0.0001). This decline was mainly due to the decline in number of oocytes retrieved as the ratio of aspirated oocytes that cleaved with increasing age (approx. −0.04/10 year 95% CI: −0.10; +0.009) was not significantly different (p=0.10). The percentage of transfers using fragmented embryos did not increase significantly with increasing age (p=0.08). The odds of fragmentation increased by 3% per year. The average number of embryos transferred decreased significantly (p=0.03) with age from approximately 2.1 at the age of 25 to approximately 1.8 at the age of 40. In a selected subgroup of embryos all consisting of good quality embryos, a significant decrease was found in implantation rate with increasing age (approx. −0.08/10 years, 95% CI: −1.6; +0.00, p=0.05). Of the 357 pregnancies achieved in this study we found a significantly decreased ongoing pregnancy rate and a significantly increased abortion rate with increasing age (p=0.03). The decrease in the rate of ongoing pregnancies was almost linear, decreasing by approximately 1.5% per year. Conclusions. We conclude that age has an impact throughout a woman’s reproductive life and that it is important to realize that the age-related decline in fertility may start already in the late twenties and not in the mid-thirties as is generally assumed.

Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization.

This retrospective study of 1001 in-vitro fertilization (IVF) cycles included a consecutive series of single transfers (n = 341), dual transfers (n = 410) and triple transfers (n = 250) where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. In our 2 day culture system, transfer of 4-cell embryos resulted in a significantly higher implantation rate and pregnancy rate (23 and 49%) compared with 2-cell embryos (12 and 22%) and 3-cell embryos (7 and 15%). Furthermore, the transfer of 4-cell embryos resulted in a significantly higher pregnancy rate compared with embryos that had cleaved beyond the 4-cell stage (28%). The implantation rate (21%) and pregnancy rate (43%) after transfer of embryos of score 1.0 were significantly higher than after transfer of embryos of score 2.0 (14 and 32% respectively). Transferring embryos of score 2.1 resulted in significantly higher implantation rates (26%) and similar pregnancy rates compared with score 1.0. Transferring embryos of score 2.2-3.0 resulted in a significantly lower implantation rate (5%) and pregnancy rate (15%). A striking finding was that embryos of quality score 2.0 had a significantly lower implantation rate compared with embryos of quality score 1.0 and 2.1 and a significantly lower pregnancy rate compared to embryos of quality score 1.0. We also found a lower implantation rate and pregnancy rate when transferring 3-cell embryos. These findings may indicate periods of increased sensitivity to damage during the cell cycle. In conclusion, these results substantiate the idea of the superiority of 4-cell embryos and demonstrate that minor amounts of fragments in the embryo may not be of any importance. These findings may call for a shift when weighing the two main morphological components (quality score and cleavage stage) in the sense that reaching a 4-cell cleavage stage even with the presence of a minor amount of fragments should be preferred to a 2-cell embryo with no fragments.

Ambulation After Transfer of Adductors, External Oblique, and Tensor Fascia Lata in Myelomeningocele

Forty-seven patients with myelomeningocele underwent triple (adductor, external oblique, and tensor fascia lata) transfers or double (adductor and external oblique) transfers and were retrospectively reviewed. Six patients lost significant neurologic function during the 4-years 6-month follow-up. Thirty-seven of the remaining 41 patients had improved gait pattern. Seventeen required less bracing after the muscle transfers. The need for assistive devices decreased in 21 patients. Twenty-seven were able to ambulate independently postoperatively as compared with seven preoperatively. The muscle transfers are indicated in midlumbar and lowlumbar spina bifida patients to improve hip stability, control, balance, and gait pattern.

Embryo scoring as a prognostic tool in IVF treatment.

A semi-quantitative and non-invasive method for scoring embryos obtained after in-vitro fertilization (IVF) has been defined, aiming at selection of embryos before transfer and at prognostic evaluation of IVF trials. Grading of embryos observed on the inverted microscope was essentially based on the amount of anucleate fragments expelled during early cleavage and on developmental speed. Embryos endowed with a high score were more often associated with pregnancy and in particular with the occurrence of multiple pregnancy. No difference was observed between scores attributed to embryos related to ongoing, aborted or chemical pregnancies. Average embryonic scores corresponding to double and triple transfers differed significantly in failures as well as pregnancies. The better quality of embryos replaced in triple transfers was also apparent from the significantly higher implantation rate per embryo observed in this group. From our results, five criteria including clinical data and embryonic scores can be derived for defining a high risk of multiple pregnancy prior to transfer. It might be warranted to replace only two embryos when these conditions are fulfilled.