The effect of different temperature conditions on human embryosin vitro: two sibling studies.

RESEARCH QUESTION: What temperature is optimal for human embryo development up to day 5 or 6? DESIGN: Two prospective sibling oocyte studies on culture temperature were conducted in a university-based tertiary referral centre. Eligibility critera for both studies: Study 1: 50 cycles between August and October 2015, with culture at a stable temperature (37.0°C ± 0.3°C) or culture using a circadian temperature rhythm (CTR) (1 am to 6 am: 36.6°C, gradual increase to 37.5°C; 11 am to 9 pm: 37.5°C; gradual decrease to 36.6°C); study 2: 99 cycles between April and November 2016, with stable culture at 36.6°C or 37.1°C. PRIMARY OUTCOME MEASURES: fertilization and embryo development (top and good quality) up to day 5 or 6, and utilization rate (number of embryos transferred and cryopreserved per zygote). Secondary outcome measure: clinical pregnancy (number of pregnancies with at least one gestational sac). RESULTS: An incubator with CTR was used for culture. An effect was found on embryo development (utilization rate: 42.1% versus 32.6%; P = 0.014), but not on clinical pregnancy rate (60.0% versus 45.5%; P = 0.670). Stable culture at 36.6°C or 37.1°C did not affect embryo development (utilization rate: 40.0% versus 40.4%; P = 0.905); clinical pregnancy rate was improved by culture at 37.1°C (46.4% versus 74.2%; P = 0.036). CONCLUSION: Culture in an incubator with CTR does not improve fertilization rate or embryo quality. Embryo culture at 36.6°C or 37.1°C showed similar embryo development.

Comparison of the development of human embryos cultured in either an EmbryoScope or benchtop incubator.

PURPOSE: In this current study, our main goal was to establish that EmbryoScope incubation environment is comparable to standard incubation. METHODS: The development of sibling human zygotes was compared after culture in either a benchtop incubator (SI) or an EmbryoScope time-lapse incubator (ES). Between May 2015 to April 2016, a total of 581 normally fertilized 2PN, pronuclear-stage embryos, from 47 patients were allocated to culture in either a benchtop incubator (SI) or an EmbryoScope incubator (ES). RESULTS: The development of embryos to cleavage (up to day 3) and blastocyst stages (day 5/6) was compared between the two different incubators. The proportion of good quality embryos was higher in the ES group compared to the SI on day 2 (66.8 vs. 50.5%, P = 0.014) and on day 3 (75.1 vs. 56.0%, P = 0.006). Those differences were statistically significant. A higher proportion of embryos developed to good quality blastocysts when cultured in the EmbryoScope compared to the benchtop (49.4 vs. 42.0%, P = 0.24), but this was not significant. Finally, no significant differences were noted with the proportion of blastocysts chosen for cryopreservation on day 5/6 in the two incubators. CONCLUSIONS: The findings support the view that the EmbryoScope incubator supports at least equivalent in vitro development of human embryos compared to other standard incubation methods and may promote improved development during early cleavage stages.

An HPLC method for the determination of selected amino acids in human embryo culture medium.

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.

Comparison between paraffin and mineral oil covering on early human embryo culture: a prospective randomized study.

The oil overlay in microdrop culture systems prevents medium evaporation, helps to maintain appropriate pH and osmotic conditions and protects from microbial contamination. In the present study, we prospectively compared covering by Ovoil™, a paraffin oil, and LiteOil®, a mineral oil, on the in vitro development of human embryos and their suitability for transfer/freezing at day 3 and live birth rate. One hundred and one patients undergoing in vitro fertilization (IVF) treatment by  intracytoplasmic sperm injection (ICSI) were enrolled in our study. After ICSI, 1237 oocytes were 1:1 randomly allocated into 2 groups according to the type of overlaying oil: Ovoil™ (616 oocytes) or LiteOil® (621 oocytes). Fertilization rate was assessed around 18 hours post-insemination (hpi) and embryos were checked for early cleavage at 25 hpi. Embryo morphology was recorded on days 2 and 3. A total of 437 (Ovoil™) and 438 day 3 embryos (LiteOil®) were analyzed. There were no differences between the two groups in terms of fertilization rate and occurrence of early cleavage. The proportion of top quality embryos (41.7% vs. 41.2%) and the final utilization rates (92.2% vs. 92.0%) were similar in Ovoil and LiteOil groups, respectively, at day 3. Live birth rate per transfer was essentially the same with Ovoil™ overlay (26.9%) when compared to LiteOil® (26.2%). Live birth rate in patients who simultaneously received  embryos from both overlay types was 17.2%. Despite the different characteristics of these two oils regarding hydrocarbon saturation, packing and temperature storage, Ovoil™ and LiteOil® can be used in parallel in the same IVF protocol. Abbreviations: ART: assisted reproductive technologies; hpi: hours post-insemination; hSA: human serum albumin; HTF: human tubal fluid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MII: metaphase II; MEA: mouse embryo assay; RT: room temperature.

Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development.

UNLABELLED: Histidine-rich glycoprotein (HRG) is an abundant plasma protein involved in multiple biological processes including immunology, vascularisation, and coagulation. These processes are of importance in regulating embryo development and implantation. A specific polymorphism in the HRG gene, HRG C633T, has an impact on various aspects of fertility, such as oocyte quality, endometrial receptivity, and possibly the capacity of the embryo itself to implant. To further examine the potential role of the HRG C633T polymorphism in regulating endometrial angiogenesis and on embryo development, two HRG peptides were constructed. These HRG peptides correspond to the amino acids 169-203 of the protein which, in turn, reflects the C633T polymorphism in the gene. The HRG proline or serine peptides were added to cultures of primary human endometrial endothelial (HEE) cells and to human embryos in vitro. The HRG peptides inhibited vascular endothelial growth factor (VEGF) induced proliferation and migration and promoted tube formation of HEE cells. The embryos were monitored using a time-lapse system (EmbryoScope®). Except for a prolonged time from first cleavage after thawing to development of the morula, no difference in embryo morphokinetics or embryo quality was noted in human embryos cultured in the presence of the HRG proline peptide. Taken together, these results suggest that treatment with a specific HRG peptide might prime the endometrium for implantation and be beneficial for adequate placentation. However, addition of a specific HRG proline peptide to human embryos has no beneficial effects in terms of embryo development. ABBREVIATIONS: HRG: histidine-rich glycoprotein; HEE: human endometrial endothelial; VEGF: vascular endothelial growth factor; TSP: thrombospondin; SNP; single nucleotide polymorphism; IVF: in vitro fertilization; CLESH-1: CD36 LIMPII Emp structural homology domain-1; ECM: endothelial cell medium; FBS: fetal bovine serum; cDNA: complementary DNA.

The Oldham Notebooks: an analysis of the development of IVF 1969-1978. III. Variations in procedures.

A survey is presented of the various technical and scientific challenges that had to be met during the 10-year period before the first successful live birth after IVF and embryo transfer was achieved, and the approaches used to meet these challenges is discussed. Records dated from January 1969 to July 1978 indicate that a minimum of 282 women were involved in 495 cycles scheduled for laparoscopic oocyte recovery, of which 457 cycles (92%) proceeded to attempted egg collection. A total of 1361 eggs were recovered over 388 cycles, of which 1237 (91%) are recorded as having been inseminated in 331 (85%) of these cycles. Approximately 221 embryos were described in 165 (43%) of the 388 cycles. A total of 112 embryo transfers were attempted, which resulted in five clinical pregnancies with two live births. This paper discusses the ways in which hormonal stimulation of follicle growth to the pre-ovulatory stage was varied, and the endocrine monitoring of these variations in blood, urine and follicular fluid, as well as their influence on egg recovery and fertilization rates. Variations in media composition and preparation are also described. It is concluded that, whilst driven by scientific reasoning, the approach adopted in trying to achieve successful IVF was empirical rather than evidence-driven.

Examining the temperature of embryo culture in in vitro fertilization: a randomized controlled trial comparing traditional core temperature (37°C) to a more physiologic, cooler temperature (36°C).

OBJECTIVE: To determine whether culture at a more physiologically cooler temperature, as suggested by limited human and animal data, would improve blastulation and pregnancy rates in human clinical IVF. DESIGN: Paired randomized controlled trial. SETTING: Academic. PATIENT(S): Infertile couples (n=52) with a female partner less than 42 years old with eight or more mature oocytes retrieved. INTERVENTION(S): Mature oocytes obtained from a single cohort of oocytes were randomly divided into two groups; one was cultured at 37°C and the other at 36°C from the time of ICSI to the time of embryo transfer or vitrification. Paired embryo transfers were accomplished by transferring one euploid embryo from each group. DNA fingerprinting was used as needed to determine the outcome for each embryo. MAIN OUTCOME MEASURE(S): Rate of development of expanded blastocysts suitable for transfer or vitrification (primary outcome), fertilization, aneuploidy, and sustained implantation. RESULT(S): A total of 805 mature oocytes were cultured; 399 at 36°C and 406 at 37°C. Paired analysis demonstrated a higher rate of usable blastocyst formation per zygote at 37°C (48.4%) vs. at 36°C (41.2%). Rates of fertilization, aneuploidy, and sustained implantation were equivalent. CONCLUSION(S): IVF culture at 36°C does not improve clinically relevant parameters of embryo development or sustained implantation rates. CLINICAL TRIAL REGISTRATION NUMBER: NCT01506089.