GDF9 concentration in embryo culture medium is linked to human embryo quality and viability.

PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, significantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. CONCLUSION: The GDF9 concentration in culture medium is linked to embryo quality and viability, and exhibited the potential to be a non-invasive biomarker for embryo selection.

Analysis and quantification of female and male contributions to the first stages of embryonic kinetics: study from a time-lapse system.

PURPOSE: The few studies that examined the effect of male and/or female features on early embryo development, notably using the time-lapse system (TL), reported conflicting results. This can be explained by the small number of studies using an adapted model. METHODS: We used two original designs to study the female and male effects on embryo development: (1) based on embryos from donor oocytes (TL-DO), and (2) from donor sperm (TL-DS). Firstly, we analyzed the female and male similarities using an ad hoc intraclass correlation coefficient (ICC), then we completed the analysis with a multivariable model to assess the association between both male and female factors, and early embryo kinetics. A total of 572 mature oocytes (TL-DO: 293; TL-DS: 279), fertilized by intracytoplasmic sperm injection (ICSI) and incubated in a TL (Embryoscope®) were included from March 2013 to April 2019; 429 fertilized oocytes (TL-DO: 212; TL-DS: 217) were assessed. The timings of the first 48 h have been analyzed. RESULTS: The similarities in the timings thought to be related to the female component were significant: (ICC in both DO-DS designs respectively: tPB2: 9-18%; tPNa: 16-21%; tPNf: 40-26%; t2: 38-24%; t3: 15-20%; t4: 21-32%). Comparatively, those related to male were lower. Surprisingly after multivariable analyses, no intrinsic female factors were clearly identified. However, in TL-DO design, oligozoospermia was associated with a tendency to longer timings, notably for tPB2 (p = 0.026). CONCLUSION: This study quantifies the role of the oocyte in the first embryo cleavages, but without identified specific female factors. However, it also highlights that sperm may have an early embryonic effect.

Blastulation rates of sibling oocytes in two IVF culture media: an evidence-based workflow to implement newly commercialized products.

RESEARCH QUESTION: An evidence-based novel commercially available continuous IVF culture medium in compliance with an efficient quality-management system is proposed. DESIGN: Non-interventional study on sibling oocytes. Intracytoplasmic sperm injection cycles among women aged 42 years or younger that used ejaculated spermatozoa and retrieved four to eight oocytes were included. Sibling oocytes were randomized for culture in the novel Geri-medium or continuous single culture medium (CSCM). Primary outcome measure was blastulation rate per cohort of inseminated oocytes; 1182 oocytes were required to outline down to a 7% difference (power = 80%). RESULTS: A total of 181 cohorts of sibling oocytes were included. Geri-medium (n = 631 oocytes) and CSCM (n = 643 oocytes) resulted in similar blastulation rates (mean ± SD: 42.8% ± 30.1% versus 43.1% ± 29.0%; Wilcoxon signed rank test = 0.77). Blastocysts cultured in the former (n = 275 versus n = 277) showed longer timings during preimplantation development (P < 0.01) and were poorer quality (26% versus 18%; P = 0.03). Euploidy rate was no different in cycles that underwent preimplantation genetic testing for aneuploidy (n = 113) (117/237 [49%] versus 117/249 blastocysts [47%]; P = 0.6). Ongoing implantation rate was comparable in the study arms after euploid (29/47 [63%] versus 14/ 34 [41%]; P = 0.1) or untested (12/31 [39%] versus 7/18 [39%]; P = 0.3) transfers. CONCLUSION: Blastulation rate among cohorts of sibling oocytes cultured in the same incubator is a fast, reliable and comprehensive performance indicator to validate novel commercially available culture medium. The media tested were considered similarly efficient. The differences in blastocyst morphology and developmental timings warrant further investigation, although euploidy and ongoing implantation rates were similar.

Impact of in vitro embryo culture and transfer on blood pressure regulation in the adolescent lamb.

Nutrition during the periconceptional period influences postnatal cardiovascular health. We determined whether in vitro embryo culture and transfer, which are manipulations of the nutritional environment during the periconceptional period, dysregulate postnatal blood pressure and blood pressure regulatory mechanisms. Embryos were either transferred to an intermediate recipient ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) and a methyl donor (IVCHS+M) for 6 days. Basal blood pressure was recorded at 19-20 weeks after birth. Mean arterial pressure (MAP) and heart rate (HR) were measured before and after varying doses of phenylephrine (PE). mRNA expression of signaling molecules involved in blood pressure regulation was measured in the renal artery. Basal MAP did not differ between groups. Baroreflex sensitivity, set point, and upper plateau were also maintained in all groups after PE stimulation. Adrenergic receptors alpha-1A (αAR1A), alpha-1B (αAR1B), and angiotensin II receptor type 1 (AT1R) mRNA expression were not different from controls in the renal artery. These results suggest there is no programmed effect of ET or IVC on basal blood pressure or the baroreflex control mechanisms in adolescence, but future studies are required to determine the impact of ET and IVC on these mechanisms later in the life course when developmental programming effects may be unmasked by age.

Day 5 versus Day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes.

STUDY QUESTION: Is there a difference in clinical pregnancy and live birth rates (LBRs) between blastocysts developing on Day 5 (D5) and blastocysts developing on Day 6 (D6) following fresh and frozen transfers? SUMMARY ANSWER: D5 blastocyst transfers (BTs) present higher clinical pregnancy and LBRs than D6 in both fresh and frozen transfers. WHAT IS KNOWN ALREADY: BT is increasingly popular in assisted reproductive technology (ART) centers today. To our knowledge, no meta-analysis has focused on clinical outcomes in both fresh and frozen BT. Concerning frozen blastocysts, one meta-analysis in 2010 found no significant difference in pregnancy outcomes between D5 and D6 BT. Since then, ART practices have evolved particularly with the wide use of vitrification, and more articles comparing D5 and D6 BT cycles have been published and described conflicting results. STUDY DESIGN, SIZE, DURATION: Systematic review and meta-analysis of published controlled studies. Searches were conducted from 2005 to February 2018 on MEDLINE and Cochrane Library and from 2005 to May 2017 on EMBASE, Eudract and clinicaltrials.gov, using the following search terms: blastocyst, Day 5, Day 6, pregnancy, implantation, live birth and embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 47 full-text articles were preselected from 808 references, based on title and abstract and assessed utilizing the Newcastle-Ottowa Quality Assessment Scales. Study selection and data extraction were carried out by two independent reviewers according to Cochrane methods. Random-effect meta-analysis was performed on all data (overall analysis) followed by subgroup analysis (fresh, vitrified/warmed, slow frozen/thawed). MAIN RESULTS AND THE ROLE OF CHANCE: Data from 29 relevant articles were extracted and integrated in the meta-analysis. Meta-analysis of the 23 studies that reported clinical pregnancy rate (CPR) as an outcome, including overall fresh and/or frozen ET cycles, showed a significantly higher CPR following D5 ET compared with D6 ET (risk ratio (RR) = 1.27, 95% CI: 1.15-1.39, P < 0.001). For CPR, calculated subgroup RRs were 2.38 (95% CI: 1.74-3.24, P < 0.001) for fresh BT; 1.27 (95% CI: 1.16-1.39, P < 0.001) for vitrified/warmed BT; and 1.15 (95% CI: 0.93-1.41, P = 0.20) for slow frozen/thawed BT. LBR was also significantly higher after D5 BT (overall RR = 1.50 (95% CI: 1.32-1.69), P < 0.001). The LBR calculated RRs for subgroups were 1.74 (95% CI: 1.37-2.20, P < 0.001) for fresh BT; 1.38 (95% CI: 1.23-1.56, P < 0.001) for vitrified/warmed BT; and 1.44 (95% CI: 0.70-2.96, P = 0.32) for slow frozen/thawed BT. Sensitivity analysis led to similar results and conclusions: CPR and LBR were significantly higher following D5 compared to D6 BT. LIMITATIONS, REASONS FOR CAUTION: The validity of meta-analysis results depends mainly on the quality and the number of the published studies available. Indeed, this meta-analysis included no randomized controlled trial (RCT). Slow frozen/thawed subgroups showed substantial heterogeneity. WIDER IMPLICATIONS OF THE FINDINGS: In regards to the results of this original meta-analysis, ART practitioners should preferably transfer D5 rather than D6 blastocysts in both fresh and frozen cycles. Further RCTs are needed to address the question of whether D6 embryos should be transferred in a fresh or a frozen cycle. STUDY FUNDING/COMPETING INTEREST(S): This work was sponsored by an unrestricted grant from GEDEON RICHTER France. The authors have no competing interests to declare. REGISTRATION NUMBER: CRD42018080151.

Performance of Day 5 KIDScore™ morphokinetic prediction models of implantation and live birth after single blastocyst transfer.

PURPOSE: While several studies reported the association between morphokinetic parameters and implantation, few predictive models were developed to predict implantation after day 5 embryo transfer, generally without external validation. The objective of this study was to evaluate the respective performance of 2 commercially available morphokinetic-based models (KIDScore™ Day 5 versions 1 and 2) for the prediction of implantation and live birth after day 5 single blastocyst transfer. METHODS: This monocentric retrospective study was conducted on 210 ICSI cycles with single day 5 embryo transfer performed with a time-lapse imaging (TLI) system between 2013 and 2016. The association between both KIDScore™ and the observed implantation and live birth rates was calculated, as well as the agreement between embryologist's choice for transfer and embryo ranking by the models. RESULTS: Implantation and live birth rate were both 35.7%. A significant positive correlation was found between both models and implantation rate (r = 0.96 and r = 0.90, p = 0.01) respectively. Both models had statistically significant but limited predictive power for implantation (AUC 0.60). There was a fair agreement between the embryologists' choice and both models (78% and 61% respectively), with minor differences in case of discrepancies. CONCLUSIONS: KIDScore™ Day 5 predictive models are significantly associated with implantation rates after day 5 single blastocyst transfer. However, their predictive performance remains perfectible. The use of these predictive models holds promises as decision-making tools to help the embryologist select the best embryo, ultimately facilitating the implementation of SET policy. However, embryologists' expertise remains absolutely necessary to make the final decision.

Randomized comparison of two commercial culture media (Cook and Vitrolife) for embryo culture after IMSI.

OBJECTIVE: A variety of studies randomizing women/cycles or oocytes/embryos has been carried out to compare different culture media for culturing embryos up to cleavage or blastocyst stages showing controversial results. A recent systematic review suggested that data in the literature are insufficient to conclude the best culture medium for embryo quality, pregnancy and implantation. The objective of this study was to evaluate whether there is any difference between two commercial culture media regarding clinical outcomes after IMSI cycles. METHODS: A total of 120 patients, ≤39 years of age, undergoing ART treatment submitted to the IMSI program were prospectively broken down and randomized into two groups: Group I (Cook media) and Group II (Vitrolife media). RESULTS: Our data demonstrated that there was no difference using all the media from Cook or all the media from Vitrolife, for culturing embryos till day 2, in the bench incubator at low O2 concentration, in relation to fertilization, embryo quality, pregnancy and implantation rates (p>0.05). CONCLUSION: Both culture media used, Cook medium and Vitrolife medium, for the IMSI procedure and for later embryo culture with transfer on the second day, are equally effective and can be used depending on the ease and availability of acquisition.

Blastocyst culture and transfer in clinically assisted reproduction: a committee opinion.

The purposes of this Practice Committee Opinion, which replaces the 2013 ASRM Practice Committee Opinion of the same name (Fertil Steril 2013; 99:667-72), are to review the literature regarding the clinical application of blastocyst transfer and identify the potential risks and laboratory issues related to the use of this technology. This document does not apply to patients undergoing blastocyst culture and transfer for preimplantation genetic testing.

Elevated incidence of monozygotic twinning is associated with extended embryo culture, but not with zona pellucida manipulation or freeze-thaw procedure.

OBJECTIVE: To identify the incidence and risk factors associated with IVF-conceived monozygotic twinning (MZT). DESIGN: Retrospective study. SETTING: Academic hospital. PATIENT(S): A total of 3,463 women with clinical pregnancies between January 2014 and February 2015 were analyzed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The measures were the incidence of MZT based on the number of embryos that were replaced, type of insemination method (conventional IVF or intracytoplasmic sperm injection [ICSI]), with or without the use of assisted hatching (AH), and day of embryo transferred in fresh and frozen cycles. RESULT(S): Ninety-three women (2.69%) with MZT were observed. No statistically significant differences were observed in the cycle parameters of fresh or frozen cycles between MZT and other non-MZT pregnancies. Specific IVF procedures or techniques, such as the number of embryo replaced, zona pellucida manipulation (ICSI and AH), and freeze-thaw procedure, did not significantly increase the rate of MZT, except for the day of embryo transferred. Compared with day 3 transferred, day 4 and 5/6 transferred showed an increased probability of MZT (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.16-6.42 for day 4 transferred and OR, 3.68; 95% CI, 2.29-5.93 for day 5/6 transferred). CONCLUSION(S): Extended culture (advanced embryo stage) in fresh and frozen cycles appeared to be associated with increased rates of MZT. The effect of the number of embryos transferred, ICSI and AH, and freeze-thaw procedures on the risk for MZT was not demonstrated.

Single versus sequential culture medium: which is better at improving ongoing pregnancy rates? A systematic review and meta-analysis.

This study aimed to evaluate if single medium is better than sequential medium at improving ongoing pregnancy rates in patients undergoing assisted reproductive technology (ART) procedures. The data featured in this meta-analysis were extracted from four randomized controlled trials yielded from a systematic search carried out on electronic databases. The primary endpoint was ongoing pregnancy rate. Secondary endpoints included clinical pregnancy and miscarriage rates. The endpoints for ongoing pregnancy rate were also analyzed based on the time at which the embryo transfers were performed: cleavage stage (day 2/3) and/or blastocyst stage (day 5/6). There were no significant differences between single and sequential medium for clinical pregnancy (RR=1.09; 95%CI=0.83-1.44; p=0.53), ongoing pregnancy (RR=1.11; 95%CI=0.87-1.40; p=0.39), or miscarriage rates (RR=0.89; 95%CI=0.44-1.81; p=0.74). No significant difference was found for ongoing pregnancy rate (RR=1.29; 95%CI=0.93-1.78; p=0.12) between single and sequential medium when only trials in which embryos were transferred at the blastocyst stage were included. In conclusion, the choice of embryo culture approach - single or sequential medium - did not affect the ongoing pregnancy rates of patients undergoing ART cycles.

Humid versus dry incubator: a prospective, randomized, controlled trial.

OBJECTIVE: To evaluate the efficacy of a dry versus humidified incubator on human embryo development ex vivo. DESIGN: Prospective, double-blind, randomized, controlled trial. SETTING: Private fertility centers. PATIENT(S): A total of 297 women undergoing in vitro fertilization randomized into two groups. INTERVENTION(S): From days 0 to day 5 or 6 of culture, intervention group embryos exposed to dry culture and control group embryos exposed to humidified culture. MAIN OUTCOME MEASURE(S): Subsequent ongoing pregnancy rate. RESULT(S): After transfer of embryos, there were statistically significantly lower rates of clinical and ongoing pregnancy in the dry culture arm than in the humidified culture arm (odds ratio [OR] 0.57; 95% confidence interval [CI], 0.36-0.91; versus OR 0.54; 95% CI, 0.34-0.85). On day 3 of culture, embryo quality and compaction were lower in the dry culture group (OR 0.38; 95% CI, 0.32-0.45) than in the group exposed to humidified culture (OR 0.23; 95% CI, 0.19-0.27). On day 5 of culture, embryos in dry culture had a lower rate of blastocyst formation (OR 0.39; 95% CI, 0.33-0.46), quality (OR 0.34; 95% CI, 0.29-0.40), and cryopreservation (OR 0.41; 95% CI, 0.35-0.48). CONCLUSION(S): In this study, human embryos cultivated ex vivo in a dry incubator had statistically significantly decreased implantation and clinical and ongoing pregnancy rates. Our findings indicate the need for larger multicenter, randomized, controlled trials. CLINICAL TRIAL REGISTRATION NUMBER: NCT01695096.

A History of Developments to Improve in vitro Fertilization.

Methods of in vitro fertilization (IVF) have advanced dramatically since the first IVF baby was born in 1978. Originally yielding single-digit success rates, IVF is now successful in nearly 50% of cases in which the woman is younger than 35 years. Here, we describe the improvements in laboratory techniques and advances in our abilities to manipulate reproductive physiology that have facilitated this improvement. Additionally, we describe efforts to ensure safety standards in this competitive field.

Evaluation of a father and son with atypical chronic myeloid leukemia with SETBP1 mutations and a review of the literature

We report the case of a father and son diagnosed with atypical chronic myeloid leukemia (aCML). Both patients harbored SETBP1 mutations, which are present in 24.3% of aCML patients. Moreover, both shared the variant encoding p.Pro737His, but the aCML severity was greater in the son because of the presence of two other missense mutations causing p.Asp868Asn and p.Ser885Arg alterations. SETBP1 mutations may be associated with an adverse prognosis, so their detection would help in the diagnosis of aCML and the determination of a patient's prognosis.

Single vitrified blastocyst transfer maximizes liveborn children per embryo while minimizing preterm birth.

OBJECTIVE: To compare live-birth rates, blastocyst to live-birth efficiency, gestational age, and birth weights in a large cohort of patients undergoing single versus double thawed blastocyst transfer. DESIGN: Retrospective cohort study. SETTING: Assisted reproduction technology (ART) practice. PATIENT(S): All autologous frozen blastocyst transfers (FBT) of one or two vitrified-warmed blastocysts from January 2009 through April 2012. INTERVENTION(S): Single or double FBT. MAIN OUTCOME MEASURE(S): Live birth, blastocyst to live-birth efficiency, preterm birth, low birth weight. RESULT(S): Only supernumerary blastocysts with good morphology (grade BB or better) were vitrified, and 1,696 FBTs were analyzed. No differences were observed in patient age, rate of embryo progression, or postthaw blastomere survival. Double FBT yielded a higher live birth per transfer, but 33% of births from double FBT were twins versus only 0.6% of single FBT. Double FBT was associated with statistically significant increases in preterm birth and low birth weight, the latter of which was statistically significant even when the analysis was limited to singletons. Of the blastocysts transferred via single FBT, 38% resulted in a liveborn child versus only 34% with double FBT. This suggests that two single FBTs would result in more liveborn children with significantly fewer preterm births when compared with double FBT. CONCLUSION(S): Single FBT greatly decreased multiple and preterm birth risk while providing excellent live-birth rates. Patients should be counseled that a greater overall number of live born children per couple can be expected when thawed blastocysts are transferred one at a time.

Assisted hatching: trends and pregnancy outcomes, United States, 2000-2010.

OBJECTIVE: To assess trends and outcomes of assisted hatching among assisted reproductive technology (ART) cycles. DESIGN: Retrospective cohort analysis using National ART Surveillance System (NASS) data. SETTING: U.S. fertility centers reporting to NASS. PATIENT(S): Fresh autologous noncanceled ART cycles conducted from 2000-2010. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation, clinical pregnancy, live-birth, miscarriage, multiple gestation. RESULT(S): Assisted hatching use statistically significantly increased in absolute number (from 25,724 to 35,518 cycles), percentages of day-3 (from 50.7% to 56.3%) and day-5 transfers (from 15.9% to 22.8%), and percentage of transfers among women ≥38 years (from 17.8% to 21.8%) or women with ≥2 prior ART cycles and no live birth(s) (from 4.3% to 7.4%). Both day-3 and day-5 cycles involving assisted hatching were associated with lower odds of implantation (adjusted odds ratios [aOR] 0.7 and 0.6, respectively), clinical pregnancy (aOR 0.8 and 0.7, respectively), live birth (aOR 0.8 and 0.7, respectively), and increased odds of miscarriage (aOR 1.4 and 1.4, respectively), as compared with cycles without assisted hatching. Assisted hatching was associated with lower odds of multiple gestation in day-5 cycles (aOR 0.8). In cycles for women with a "poor prognosis," the association of assisted hatching with pregnancy outcomes was not statistically significant. CONCLUSION(S): Assisted hatching use had an increasing trend but was not associated with improved pregnancy outcomes, even in poor-prognosis patients. Prospective studies are needed to identify the patients who may benefit from assisted hatching.

In vitro culture increases the frequency of stochastic epigenetic errors at imprinted genes in placental tissues from mouse concepti produced through assisted reproductive technologies.

Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (~20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid.

OBJECTIVE: To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. DESIGN: Experimental laboratory study. SETTING: University-based laboratory. ANIMAL(S): FVB and CF1 mice crossed to create embryos used in experiments. INTERVENTION(S): Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). MAIN OUTCOME MEASURE(S): Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). RESULT(S): The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were <50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C. CONCLUSION(S): The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.

Trophectoderm grade predicts outcomes of single-blastocyst transfers.

OBJECTIVE: To estimate the effect of the embryo stage, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on live birth in single-blastocyst transfers. DESIGN: Retrospective cohort study. SETTING: Large private assisted reproductive technologies (ART) practice. PATIENT(S): Fresh autologous ART cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): A total of 694 single-blastocyst transfers met the inclusion criteria. Univariate regression analysis showed embryo stage and TE score to be correlated with implantation and live birth. Live birth rates were 57%, 40%, and 25% for TE grades A, B, and C, respectively. There was no significant association between ICM grade and implantation or live birth. Live birth rates were 53%, 52%, and 0% for ICM grades A, B, and C respectively. Multiple logistic regression analysis showed that only patient age and TE grade were significantly associated with implantation and live birth, whereas ICM grade was not significantly associated with outcome. The TE score had the strongest correlation with live birth. CONCLUSION(S): TE grading, but not ICM grading, significantly correlated with implantation and live birth for single-blastocyst transfers.

Human embryo culture media comparisons.

Every program of assisted reproduction strives to maximize pregnancy outcomes from in vitro fertilization and selecting an embryo culture medium, or medium pair, consistent with high success rates is key to this process. The common approach is to replace an existing medium with a new one of interest in the overall culture system and then perform enough cycles of IVF to see if a difference is noted both in laboratory measures of embryo quality and in pregnancy. This approach may allow a laboratory to select one medium over another but the outcomes are only relevant to that program, given that there are well over 200 other variables that may influence the results in an IVF cycle. A study design that will allow for a more global application of IVF results, ones due to culture medium composition as the single variable, is suggested. To perform a study of this design, the center must have a patient caseload appropriate to meet study entrance criteria, success rates high enough to reveal a difference if one exists and a strong program of quality assurance and control in both the laboratory and clinic. Sibling oocytes are randomized to two study arms and embryos are evaluated on day 3 for quality grades. Inter and intra-observer variability are evaluated by kappa statistics and statistical power and study size estimates are performed to bring discriminatory capability to the study. Finally, the complications associated with extending such a study to include blastocyst production on day 5 or 6 are enumerated.