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1.  The role of proteomics in defining the human embryonic secretome 
Molecular Human Reproduction  2009;15(5):271-277.
Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers.
PMCID: PMC2666223  PMID: 19223337
proteomics; embryo; secretome; non-invasive assessment
2.  Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle 
PLoS ONE  2012;7(5):e36627.
Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.
PMCID: PMC3348877  PMID: 22590579
3.  In Vitro Fertilization and Multiple Pregnancies 
Executive Summary
The objective of this health technology policy assessment was to determine the clinical effectiveness and cost-effectiveness of IVF for infertility treatment, as well as the role of IVF in reducing the rate of multiple pregnancies.
Clinical Need: Target Population and Condition
Typically defined as a failure to conceive after a year of regular unprotected intercourse, infertility affects 8% to 16% of reproductive age couples. The condition can be caused by disruptions at various steps of the reproductive process. Major causes of infertility include abnormalities of sperm, tubal obstruction, endometriosis, ovulatory disorder, and idiopathic infertility. Depending on the cause and patient characteristics, management options range from pharmacologic treatment to more advanced techniques referred to as assisted reproductive technologies (ART). ART include IVF and IVF-related procedures such as intra-cytoplasmic sperm injection (ICSI) and, according to some definitions, intra-uterine insemination (IUI), also known as artificial insemination. Almost invariably, an initial step in ART is controlled ovarian stimulation (COS), which leads to a significantly higher rate of multiple pregnancies after ART compared with that following natural conception. Multiple pregnancies are associated with a broad range of negative consequences for both mother and fetuses. Maternal complications include increased risk of pregnancy-induced hypertension, pre-eclampsia, polyhydramnios, gestational diabetes, fetal malpresentation requiring Caesarean section, postpartum haemorrhage, and postpartum depression. Babies from multiple pregnancies are at a significantly higher risk of early death, prematurity, and low birth weight, as well as mental and physical disabilities related to prematurity. Increased maternal and fetal morbidity leads to higher perinatal and neonatal costs of multiple pregnancies, as well as subsequent lifelong costs due to disabilities and an increased need for medical and social support.
The Technology Being Reviewed
IVF was first developed as a method to overcome bilateral Fallopian tube obstruction. The procedure includes several steps: (1) the woman’s egg is retrieved from the ovaries; (2) exposed to sperm outside the body and fertilized; (3) the embryo(s) is cultured for 3 to 5 days; and (4) is transferred back to the uterus. IFV is considered to be one of the most effective treatments for infertility today. According to data from the Canadian Assisted Reproductive Technology Registry, the average live birth rate after IVF in Canada is around 30%, but there is considerable variation in the age of the mother and primary cause of infertility.
An important advantage of IVF is that it allows for the control of the number of embryos transferred. An elective single embryo transfer in IVF cycles adopted in many European countries was shown to significantly reduce the risk of multiple pregnancies while maintaining acceptable birth rates. However, when number of embryos transferred is not limited, the rate of IVF-associated multiple pregnancies is similar to that of other treatments involving ovarian stimulation. The practice of multiple embryo transfer in IVF is often the result of pressures to increase success rates due to the high costs of the procedure. The average rate of multiple pregnancies resulting from IVF in Canada is currently around 30%.
An alternative to IVF is IUI. In spite of reported lower success rates of IUI (pregnancy rates per cycle range from 8.7% to 17.1%) it is generally attempted before IVF due to its lower invasiveness and cost.
Two major drawbacks of IUI are that it cannot be used in cases of bilateral tubal obstruction and it does not allow much control over the risk of multiple pregnancies compared with IVF. The rate of multiple pregnancies after IUI with COS is estimated to be about 21% to 29%.
Ontario Health Insurance Plan Coverage
Currently, the Ontario Health Insurance Plan covers the cost of IVF for women with bilaterally blocked Fallopian tubes only, in which case it is funded for 3 cycles, excluding the cost of drugs. The cost of IUI is covered except for preparation of the sperm and drugs used for COS.
Diffusion of Technology
According to Canadian Assisted Reproductive Technology Registry data, in 2004 there were 25 infertility clinics across Canada offering IVF and 7,619 IVF cycles performed. In Ontario, there are 13 infertility clinics with about 4,300 IVF cycles performed annually.
Literature Review
Royal Commission Report on Reproductive Technologies
The 1993 release of the Royal Commission report on reproductive technologies, Proceed With Care, resulted in the withdrawal of most IVF funding in Ontario, where prior to 1994 IVF was fully funded. Recommendations of the Commission to withdraw IVF funding were largely based on findings of the systematic review of randomized controlled trials (RCTs) published before 1990. The review showed IVF effectiveness only in cases of bilateral tubal obstruction. As for nontubal causes of infertility, there was not enough evidence to establish whether IVF was effective or not.
Since the field of reproductive technology is constantly evolving, there have been several changes since the publication of the Royal Commission report. These changes include: increased success rates of IVF; introduction of ICSI in the early 1990’s as a treatment for male factor infertility; and improved embryo implantation rates allowing for the transfer of a single embryo to avoid multiple pregnancies after IVF.
Studies After the Royal Commission Report: Review Strategy
Three separate literature reviews were conducted in the following areas: clinical effectiveness of IVF, cost-effectiveness of IVF, and outcomes of single embryo transfer (SET) in IVF cycles.
Clinical effectiveness of IVF: RCTs or meta-analyses of RCTs that compared live birth rates after IVF versus alternative treatments, where the cause of infertility was clearly stated or it was possible to stratify the outcome by the cause of infertility.
Cost effectiveness of IVF: All relevant economic studies comparing IVF to alternative methods of treatment were reviewed
Outcomes of IVF with SET: RCTs or meta-analyses of RCTs that compared live birth rates and multiple birth rates associated with transfer of single versus double embryos.
OVID MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, Cochrane Library, the International Agency for Health Technology Assessment database, and websites of other health technology assessment agencies were searched using specific subject headings and keywords to identify relevant studies.
Summary of Findings
Comparative Clinical Effectiveness of IVF
Overall, there is a lack of well composed RCTs in this area and considerable diversity in both definition and measurement of outcomes exists between trials. Many studies used fertility or pregnancy rates instead of live birth rates. Moreover, the denominator for rate calculation varied from study to study (e.g. rates were calculated per cycle started, per cycle completed, per couple, etc...).
Nevertheless, few studies of sufficient quality were identified and categorized by the cause of infertility and existing alternatives to IVF. The following are the key findings:
A 2005 meta-analysis demonstrated that, in patients with idiopathic infertility, IVF was clearly superior to expectant management, but there were no statistically significant differences in live birth rates between IVF and IUI, nor between IVF and gamete-intra-Fallopian transfer.
A subset of data from a 2000 study showed no significant differences in pregnancy rates between IVF and IUI for moderate male factor infertility.
In patients with moderate male factor infertility, standard IVF was also compared with ICSI in a 2002 meta-analysis. All studies included in the meta-analysis showed superior fertilization rates with ICSI, and the pooled risk ratio for oocyte fertilization was 1.9 (95% Confidence Interval 1.4-2.5) in favour of ICSI. Two other RCTs in this area published after the 2002 meta-analysis had similar results and further confirmed these findings. There were no RCTs comparing IVF with ICSI in patients with severe male factor infertility, mainly because based on the expert opinion, ICSI might only be an effective treatment for severe male factor infertility.
Cost-Effectiveness of IVF
Five economic evaluations of IVF were found, including one comprehensive systematic review of 57 health economic studies. The studies compared cost-effectiveness of IVF with a number of alternatives such as observation, ovarian stimulation, IUI, tubal surgery, varicocelectomy, etc... The cost-effectiveness of IVF was analyzed separately for different types of infertility. Most of the reviewed studies concluded that due to the high cost, IVF has a less favourable cost-effectiveness profile compared with alternative treatment options. Therefore, IVF was not recommended as the first line of treatment in the majority of cases. The only two exceptions were bilateral tubal obstruction and severe male factor infertility, where an immediate offer of IVF/ICSI might the most cost-effective option.
Clinical Outcomes After Single Versus Double Embryo Transfer Strategies of IVF
Since the SET strategy has been more widely adopted in Europe, all RCT outcomes of SET were conducted in European countries. The major study in this area was a large 2005 meta-analysis, followed by two other published RCTs.
All of these studies reached similar conclusions:
Although a single SET cycle results in lower birth rates than a single double embryo transfer (DET) cycle, the cumulative birth rate after 2 cycles of SET (fresh + frozen-thawed embryos) was comparable to the birth rate after a single DET cycle (~40%).
SET was associated with a significant reduction in multiple births compared with DET (0.8% vs. 33.1% respectively in the largest RCT).
Most trials on SET included women younger than 36 years old with a sufficient number of embryos available for transfer that allowed for selection of the top quality embryo(s). A 2006 RCT, however, compared SET and DET strategies in an unselected group of patients without restrictions on the woman’s age or embryo quality. This study demonstrated that SET could be applied to older women.
Estimate of the Target Population
Based on results of the literature review and consultations with experts, four categories of infertile patients who may benefit from increased access to IVF/ICSI were identified:
Patients with severe male factor infertility, where IVF should be offered in conjunction with ICSI;
Infertile women with serious medical contraindications to multiple pregnancy, who should be offered IVF-SET;
Infertile patients who want to avoid the risk of multiple pregnancy and thus opt for IVF-SET; and
Patients who failed treatment with IUI and wish to try IVF.
Since, however, the latter indication does not reflect any new advances in IVF technology that would alter existing policy, it was not considered in this analysis.
Economic Analysis
Economic Review: Cost–Effectiveness of SET Versus DET
Conclusions of published studies on cost-effectiveness of SET versus DET were not consistent. While some studies found that SET strategy is more cost-effective due to avoidance of multiple pregnancies, other studies either did not find any significant differences in cost per birth between SET and DET, or favoured DET as a more cost-effective option.
Ontario-Based Economic Analysis
An Ontario-based economic analysis compared cost per birth using three treatment strategies: IUI, IVF-SET, and IVF-DET. A decision-tree model assumed three cycles for each treatment option. Two separate models were considered; the first included only fresh cycles of IVF, while the second had a combination of fresh and frozen cycles. Even after accounting for cost-savings due to avoidance of multiple pregnancies (only short-term complications), IVF-SET was still associated with a highest cost per birth. The approximate budget impact to cover the first three indications for IVF listed above (severe male factor infertility, women with medical contraindications to multiple pregnancy, and couples who wish to avoid the risk of multiple pregnancy) is estimated at $9.8 to $12.8 million (Cdn). Coverage of only first two indications, namely, ICSI in patients with severe male factor infertility and infertile women with serious medical contraindications to multiple pregnancy, is estimated at $3.8 to $5.5 million Cdn.
Other Considerations
International data shows that both IVF utilization and the average number of embryos transferred in IVF cycles are influenced by IVF funding policy. The success of the SET strategy in European countries is largely due to the fact that IVF treatment is subsidized by governments.
Surveys of patients with infertility demonstrated that a significant proportion (~40%) of patients not only do not mind having multiple babies, but consider twins being an ideal outcome of infertility treatment.
A women’s age may impose some restrictions on the implementation of a SET strategy.
Conclusions and Recommendations
A review of published studies has demonstrated that IVF-SET is an effective treatment for infertility that avoids multiple pregnancies.
However, results of an Ontario-based economic analysis shows that cost savings associated with a reduction in multiple pregnancies after IVF-SET does not justify the cost of universal IVF-SET coverage by the province. Moreover, the province currently funds IUI, which has been shown to be as effective as IVF for certain types of infertility and is significantly less expensive.
In patients with severe male factor infertility, IVF in conjunction with ICSI may be the only effective treatment.
Thus, 2 indications where additional IVF access should be considered include:
IVF/ICSI for patients with severe male factor infertility
IVF-SET in infertile women with serious medical contraindications to multiple pregnancy
PMCID: PMC3379537  PMID: 23074488
4.  Preimplantation Mouse Embryo Selection Guided by Light-Induced Dielectrophoresis 
PLoS ONE  2010;5(4):e10160.
Selection of optimal quality embryos for in vitro fertilization (IVF) transfer is critical to successful live birth outcomes. Currently, embryos are chosen based on subjective assessment of morphologic developmental maturity. A non-invasive means to quantitatively measure an embryo's developmental maturity would reduce the variability introduced by the current standard. We present a method that exploits the scaling electrical properties of pre-transfer embryos to quantitatively discern embryo developmental maturity using light-induced dielectrophoresis (DEP). We show that an embryo's DEP response is highly correlated with its developmental stage. Uniquely, this technique allows one to select, in sequence and under blinded conditions, the most developmentally mature embryos among a mixed cohort of morphologically indistinguishable embryos cultured in optimized and sub-optimal culture media. Following assay, embryos continue to develop normally in vitro. Light-induced dielectrophoresis provides a non-invasive, quantitative, and reproducible means to select embryos for applications including IVF transfer and embryonic stem cell harvest.
PMCID: PMC2854157  PMID: 20405021
5.  Preimplantation Diagnosis by FISH: The Rambam Experience 
Purpose:Our purpose was to summarize our experience gained using fluorescence in situ hybridization for preimplantation diagnosis at the Rambam Medical Center.
Methods:Seventy-three embryos (29 cycles) were analyzed for preimplantation diagnosis for the following indications: advanced maternal age (>39 years), X-linked diseases, poor-quality embryos, repeated failure in vitro fertilization cycles and fast-dividing embryos. An additional 38 embryos with unequal pronuclei size were examined for ploidy. Biopsy of embryonic blastomeres was performed at the six- to eight-cell stage. Five fluorescence probes, for chromosomes X, Y, 13, 18, and 21, were applied for ploidy detection.
Results:Eighty-four (87%) blastomeres of the 73 embryos analyzed showed clear signals. Six of the blastomeres were lost during spreading. Two of the embryos were destroyed following biopsy. No nucleus was found in five of the blastomeres, while in nine, more than one nucleus was verified. Transfer was performed in 10 patients (32 embryos). Two pregnancies were achieved. Two healthy babies were born. Fifty-seven percent of the fast-dividing embryos demonstrated normal signals. In two groups of embryos of unequal pronuclei size, following conventional insemination and intracytoplasmic sperm injection, 50 and 11.4% demonstrated normal signals.
Conclusions:The efficiency of florescence in situ hybridization for aneuploidy detection is 87 and 97% for autosomes and gonosomes, respectively. The preimplantation genetic diagnosis is suitable for selected in vitro fertilization cases including fast-dividing embryos and embryos with unequal pronuclei size following regular in vitro fertilization.
PMCID: PMC3454750  PMID: 9604765
fluorescence in situ hybridization; preimplantation genetic diagnosis
6.  Assisted reproductive technologies in rhesus macaques 
The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized.
PMCID: PMC441410  PMID: 15200674
7.  Reprogenetics: Preimplantational genetics diagnosis 
Genetics and Molecular Biology  2013;37(1 Suppl):271-284.
Preimplantational Genetics Diagnosis (PGD) is requested by geneticists and reproductive specialists. Usually geneticists ask for PGD because one or both members of the couple have an increased genetic risk for having an affected offspring. On the other hand, reproductive specialists ask for embryo aneuploidy screening (PGS) to assures an euploid embryo transfer, with the purpose to achieve an ongoing pregnancy, although the couple have normal karyotypes. As embryonic aneuploidies are responsible for pre and post implantation abortions, it is logical to considerer that the screening of the embryonic aneuploidies prior to embryo transfer could improve the efficiency of the in vitro fertilization procedures. Nevertheless, it is still premature to affirm this until well-designed clinical trials were done, especially in women of advanced age where the rate of embryos with aneuploidies is much greater. Although the indications of PGD are similar to conventional prenatal diagnosis (PND), PGD has less ethical objections than the PND. As with the PGD/PGS results only unaffected embryos are transferred, both methods can avoid the decision to interrupt the pregnancy due to a genetic problem; this makes an important difference when compared to conventional prenatal diagnosis.
PMCID: PMC3983589  PMID: 24764761
PGD; PGS; PGSS; embryo biopsy; trophectoderm biopsy
8.  Attitudes of fertile and infertile woman towards new reproductive technologies: a case study of Lithuania 
Reproductive Health  2014;11:26.
This article analyzes several key issues in the debate: the acceptability of in vitro fertilization; regulation of assisted reproduction and the possibilities of reimbursement for assisted reproduction treatment in Lithuania.
Two groups of respondents participated in the survey: fertile women and women with fertility disorders. 93 completed questionnaires from women with fertility problems and 146 from women with no fertility problems were analysed.
Fertile respondents more frequently perceived the embryo as a human being (Fertile Individuals – 68.5%; Infertile Individuals – 35.5%; p < 0.05) and more frequently maintained that assisted reproduction treatment should be only partly reimbursed (Fertile Individuals – 71.3%; Infertile Individuals – 39.8%; p < 0.05). Respondents with fertility disorders more frequently thought that artificial insemination procedure could also be applied to unmarried couples (Fertile Individuals – 51.4%; Infertile Individuals – 76.3%; p < 0.05), and more frequently agreed that there should be no age limit for artificial insemination procedures (Fertile Individuals – 36.3%; Infertile Individuals – 67.7%; p < 0.05). The majority of respondents in both groups (Fertile Individuals – 77.4%; Infertile Individuals – 82.8%; p < 0.05) believed that donation of reproductive cells should be regulated by law. Fertile respondents more frequently considered that strict legal regulation was necessary in case of the number of transferred embryos (Fertile Individuals – 69.2%; Infertile Individuals – 39.8%; p < 0.05) and freezing of embryos (Fertile Individuals – 69.9%; Infertile Individuals – 57.0%; p < 0.05).
Fertile respondents were statistically more likely to believe that the IVF procedure should be applied only to married couples or women who had a regular partner, the age limit should be defined and the psychological assessment of the couple’s relationship and their readiness for the IVF procedure was necessary. In contrast, infertile couples were statistically more likely than fertile respondents to maintain that the IVF procedure should be fully reimbursed by the state. Fertile respondents were statistically more likely to be categorical with respect to the number of embryos and the freezing of embryos. Meanwhile there is a statistically significant difference in opinions of infertile respondents who were in favour of stricter regulation on donation of reproductive cells.
PMCID: PMC4017967  PMID: 24684746
Fertile women; Infertile women; New reproductive technologies
9.  The Effects of Di-(2-ethylhexyl)-phthalate Exposure on Fertilization and Embryonic Development In Vitro and Testicular Genomic Mutation In Vivo 
PLoS ONE  2012;7(11):e50465.
The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ – at least in testes, are of great concern to human male reproductive health.
PMCID: PMC3511574  PMID: 23226291
10.  Fluorescence-based visualization of autophagic activity predicts mouse embryo viability 
Scientific Reports  2014;4:4533.
Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.
PMCID: PMC3970120  PMID: 24681842
11.  Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5 
Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5.
For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis.
The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/− 6.5; 87,7%) were cleaved and 195 (+/− 3.2; 54,6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/− 8.9, 84,5%), and these embryos produced 193 (+/− 3.2, 49,7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/− 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity.
This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
PMCID: PMC3447700  PMID: 22823939
BoHV-5; Infection; Bovine embryos; Apoptosis
12.  Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development 
eLife  2014;3:e02181.
Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.
eLife digest
When a fertilized egg develops into an embryo, the expression of many genes must be carefully timed and coordinated. Researchers regularly use a type of fruit fly called Drosophila to study development because it is small, it has a short lifespan, and its whole genome sequence is already known. The development of a Drosophila embryo starts with the nucleus of the fertilized egg, which contains most of the cell’s genetic material, dividing 13 times in quick succession, without the cell itself splitting. These divisions are amongst the fastest known for any animal, and given the fast developmental speed, the embryo must efficiently express all genes it needs to stay alive. Because cell division is known to inhibit gene expression this raises an interesting conundrum about the way fast cell proliferation and gene expression are coordinated.
The first step of gene expression involves a length of DNA being transcribed to produce an intermediate molecule called a messenger RNA (mRNA), which is then translated to produce a protein. However, some mRNA molecules contain regions called ‘introns’ that are not translated and must instead be removed via a time-consuming process called ‘splicing’ before the protein is produced.
At first a Drosophila embryo uses mRNA molecules that were spliced and packaged inside the egg by the mother, but later it starts to make its own mRNA molecules. The very first mRNA molecules made by the early embryo tend to be short and lack introns. The shortness of these molecules is thought to reflect the fact there is not enough time to produce longer mRNA molecules. Is the same ‘need for speed’ also responsible for the lack of introns in these molecules?
Now, Guilgur et al. have tested this hypothesis by manipulating a gene named fandango, which codes for part of the cellular machinery that removes introns from mRNA molecules, in fruit flies. These mutant fruit flies had less of the Fandango protein than wild-type flies and while they passed through the early stages of development normally, they later developed defects—such as abnormally shaped cells. Guilgur et al. revealed that fandango mutants fail to splice out the introns in the mRNA molecules that are made in the early embryo, whereas similar mRNA molecules from the mother were spliced as normal. Further experiments suggested that wild-type embryos struggled to correctly splice an untypical early gene that had multiple introns.
Together the findings of Guilgur et al. suggest that when nuclei (or cells) are dividing rapidly, there is a strong selective pressure to splice mRNA molecules quickly in the short time between the divisions. Furthermore, this pressure appears to have shaped the architecture of the earliest genes expressed in the Drosophila embryo, which is why the first mRNA molecules produced by the embryo itself tend not to contain introns.
PMCID: PMC3989599  PMID: 24755291
fast development; early zygotic genes; mRNA splicing; Prp19/NTC; Syf1/Xab2; D. melanogaster
13.  Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors 
PLoS ONE  2012;7(11):e49328.
Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.
PMCID: PMC3495911  PMID: 23152897
14.  Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos 
PLoS ONE  2013;8(10):e76422.
The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.
PMCID: PMC3797840  PMID: 24146866
15.  Preimplantation genetic diagnosis in Saudi Arabia 
Bioinformation  2013;9(8):388-393.
Preimplantation genetic diagnosis (PGD) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of in vitro fertilization (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PGD has become an increasingly useful adjunct to IVF procedures. The ability to provide couples who are known carriers of genetic abnormalities the opportunity to deliver healthy babies has opened a new frontier in reproductive medicine. The purpose of the PGD is enables us to choose which embryos will be implanted into the mother. In the present study 137 families who had undergone IVF at Habib Medical Centre, were enrolled for the PGD analysis. The couple visited the clinic for the sex selection, recurrent fetal loss and with the recurrent IVF failure. 802 embryos were tested by the biopsy method and 512 are found to be normal and 290 were abnormal embryos. In this study only 24% of the embryos were transferred and the remaining was not transferred because of the abnormalities or undesired sex of the embryos. The structural and numerical abnormalities were found to be 16.8%.
PMCID: PMC3670120  PMID: 23750087
Preimplantation genetic diagnosis (PGD); invitro fertilization (IVF); Biopsy; Saudi Arabia
16.  Cryopreservation of human failed-matured oocytes followed by in vitro maturation: vitrification is superior to the slow freezing method 
Oocyte cryopreservation is an important method used in a number of human fertility circumstances. Here, we compared the survival, in vitro maturation, fertilization, and early embryonic development rates of frozen-thawed human immature oocytes using two different cryopreservation methods.
A total of 454 failed-matured oocytes [germinal vesicle (GV) and metaphase I (MI) stages] were collected from 135 patients (mean age 33.84 +/- 5.0 y) who underwent intracytoplasmic sperm injection (ICSI) cycles between February 2009 and December 2009 and randomly divided into a slow freezing group [1.5 mol/L-1, 2-propanediol (PROH) + 0.2 mol/l sucrose] and vitrification group [20% PROH + 20% ethylene glycol (EG) + 0.5 mol/l sucrose].
The vitrification protocol yielded a better survival rate than the slow freezing protocol at each maturation stage assessed. Regardless of the maturation stage (GV + MI), the slow freezing protocol had a significantly lower survival rate than the vitrification protocol (p < 0.001). In addition, a significant difference was found in the survival rates between GV and MI oocytes regardless of the protocol used (90.1 vs. 64.7%, respectively; p < 0.01). We also found that the maturation rates of GV and MI oocytes from the slow freezing and vitrification groups were 16.7 vs. 24.4% and 50.8 vs. 55.4%, respectively. Regardless of the protocol used, the GV oocytes had significantly lower viability than MI oocytes after 36 h of in vitro maturation (21.2 vs. 54.0%, respectively; p < 0.01). In addition, the GV and MI oocytes from the slow freezing group had a markedly lower maturation rate than those from the vitrification group (33.6 vs. 43.1%, respectively), but no statistical difference was found between the two groups (P > 0.05). For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, while a 19.0% (4/21) fertilization rate was observed in the vitrification group. For the MI-matured oocytes, fertilization rates for the slow freezing and vitrified groups were 36% and 61.1%, respectively, but no significant difference was found between the two groups (PIn the Methods section in the MS, all procedures were compliant with ethical guidelines, i.e. approved by the Ethical Committee of our university and Informed Consent signed by each patient. > 0.05). In the GV vitrification group, no embryo formed; however, in the MI slow freezing group, 12 oocytes were fertilized, but only two achieved cleavage and were subsequently blocked at the 2-cell stage. In the MI vitrification group, a total of 22 embryos were obtained, five of which developed to the blastocyst stage.
Vitrification is superior to the slow freezing method in terms of the survival and developmental rates for the cryopreservation of human failed-matured oocytes. In addition, GV oocytes appeared to be more resistant than MI oocytes to the low temperature and cryoprotectant used during cryopreservation.
PMCID: PMC3253057  PMID: 22151887
cryopreservation of human failed-matured oocytes; slow-freezing; vitrification; survival rate; in vitro maturation rate; fertilization; development rates
17.  Current status of assisted reproductive technology in Korea, 2009 
Obstetrics & Gynecology Science  2013;56(6):353-361.
Great advances have been made in the field of assisted reproductive technology (ART) since the first in vitro fertilization (IVF) baby was born in Korea in the year of 1985. However, it deserve to say that the invaluable data from fertility centers may serve as a useful source to find out which factors affect successful IVF outcome and to offer applicable information to infertile patients and fertility clinics. This article intended to report the status of ART in 2009 Korean Society of Obstetrics and Gynecology surveyed. The current survey was performed to assess the status and success rate of ART performed in Korea, between January 1 and December 31, 2009. Reporting forms had been sent out to IVF centers via e-mail, and collected by e-mail as well in 2012. With International Committee Monitoring Assisted Reproductive Technologies recommendation, intracytoplasmic sperm injection (ICSI) and non-ICSI cases have been categorized and also IVF-ET cases involving frozen embryo replacement have been surveyed separately. Seventy-four centers have reported the treatment cycles initiated in the year of 2009, and had performed a total of 27,947 cycles of ART treatments. Among a total of 27,947 treatment cycles, IVF and ICSI cases added up to 22,049 (78.9%), with 45.3% IVF without ICSI and 54.7% IVF with ICSI, respectively. Among the IVF and ICSI patients, patients confirmed to have achieved clinical pregnancy was 28.8% per cycle with oocyte retrieval, and 30.9% per cycle with embryo transfer. The most common number of embryos transferred in 2009 is three embryos (40.4%), followed by 2 embryos (28.4%) and a single embryo transferred (13.6%). Among IVF and ICSI cycles that resulted in multiple live births, twin pregnancy rate was 45.3% and triple pregnancy rate was 1.1%. A total of 191 cases of oocyte donation had been performed to result in 25.0% of live birth rate. Meanwhile, a total of 5,619 cases of frozen embryo replacement had been performed with 33.7% of clinical pregnancy rate per cycle with embryo transfer. When comparing with international registry data, clinical pregnancy rate per transfer from fresh IVF cycles including ICSI (34.1%,) was comparable to clinical pregnancy rate per transfer in European Society for Human Reproduction and Embryology report was 32.5% though lower than 45.0% for USA data. There was no remarkable difference in status of assisted reproductive technology in Korea between the current report and the data reported in 2008. The age of women trying to get pregnant was reconfirmed to be the most important factor that may have impact on success of ART treatment.
PMCID: PMC3859020  PMID: 24396813
International Committee for Monitoring Assisted Reproductive Technologies; Korea; Reproductive techniques; Survey
18.  Manipulation and Imaging of Kryptolebias marmoratus Embryos 
The self-fertilizing mangrove killifish, Kryptolebias marmoratus, is an upcoming model species for a range of biological disciplines. To further establish this model in the field of developmental biology, we examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. These methodological approaches can be divided into two categories: handling of embryos with and without their chorionic membrane. Embryos still enclosed in their chorion can be manipulated using an agarose bed or a methyl cellulose system, holding them in place and allowing their rotation to more specific angles and positions. Using these methods, we demonstrate microinjection of embryos and monitoring of fluorescent yolk syncytial nuclei (YSN) using both stereo and compound microscopes. For higher magnification imaging using compound microscopes as well as time-lapse analyses, embryos were dechorionated and embedded in low-melting-point agarose. To demonstrate this embedding technique, we further examined fluorescent YSN and also analyzed the yolk surface of K. marmoratus embryos. The latter was observed to provide an excellent imaging platform for study of the behavior and morphology of cells during embryonic development, for various types of cells. Our data demonstrate that K. marmoratus is an excellent model species for research in developmental biology, as methodological approaches for the manipulation and imaging of embryos are efficient and readily available.
PMCID: PMC3501096  PMID: 22593557
19.  Assessment and treatment of repeated implantation failure (RIF) 
Repeated implantation failure (RIF) is determined when embryos of good quality fail to implant following several in vitro fertilization (IVF) treatment cycles. Implantation failure is related to either maternal factors or embryonic causes. Maternal factors include uterine anatomic abnormalities, thrombophilia, non-receptive endometrium and immunological factors. Failure of implantation due to embryonic causes is associated with either genetic abnormalities or other factors intrinsic to the embryo that impair its ability to develop in utero, to hatch and to implant. New methods of time-lapse imaging of embryos and assessment of their metabolic functions may improve selection of embryos for transfer, and subsequent outcomes for IVF patients, as well as for those diagnosed with RIF. This review discusses the various causes associated with RIF and addresses appropriate treatments.
PMCID: PMC3510376  PMID: 22976427
Implantation; Recurrent implantation failure; Repeated implantation failure; RIF; In vitro fertilization; IVF failure
20.  Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications 
Human Reproduction Update  2010;17(2):272-290.
The first week of human embryonic development comprises a series of events that change highly specialized germ cells into undifferentiated human embryonic stem cells (hESCs) that display an extraordinarily broad developmental potential. The understanding of these events is crucial to the improvement of the success rate of in vitro fertilization (IVF). With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos, and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development.
In order to understand the complex genetic network that controls the first week of embryo development, we performed a systematic review and study of this issue. We performed a literature search using PubMed and EMBASE to identify all relevant studies published as original articles in English up to March 2010 (n=165). We also analyzed the transcriptome of human oocytes, embryos and hESCs.
Distinct sets of genes were revealed by comparing the expression profiles of oocytes, embryos on day-3 and hESCs, that are associated with totipotency, pluripotency and reprogramming properties, respectively. Known components of two signalling pathways (WNT and transforming growth factor-beta) were linked to oocyte maturation and early embryonic development.
Omics analysis provides tools for understanding the molecular mechanisms and signalling pathways controlling early embryonic development. Furthermore, we discuss the clinical relevance of using a non-invasive molecular approach to embryo selection for the single embryo transfer (SET) program.
PMCID: PMC3189516  PMID: 20716614
Embryonic Development; genetics; Embryonic Stem Cells; metabolism; Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Humans; Maternal Age; Oocytes; metabolism; Polycystic Ovary Syndrome; genetics; metabolism; Pregnancy; Pregnancy Outcome; oocyte genes; embryo genes; embryo stem cells; microarray; non-invasive approach
21.  Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain 
Theriogenology  2012;79(2):234-241.
Preimplantation genetic diagnosis (PGD) is considered highly successful in respect to its accuracy in detecting genetic anomalies but the effects of embryo biopsy on embryonic/fetal growth and development are less known, particularly in conjunction with in vitro fertilization (IVF). Here, we compared biopsied (B) and non-biopsied (NB) mouse embryos for their developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the 4-cell stage, and were either cultured for 120 h and subjected to differential inner cell mass (ICM) and trophoblast (T) staining, or were transferred into the uterine tubes of surrogate mothers after 72 h of culture, to examine their pre- and post-implantation development, respectively. Non-biopsied embryos from the same IVF cohorts served as controls. Embryo biopsy negatively affected preimplantation development to blastocyst in C57BL/6 (69 vs 79%, P<0.01) but not in B6D2F1 mice (89 vs 91%, P=NS). Although B6 embryos had lower total cell number than F2 (B6: 47 and 61 vs. F1: 53 and 70; B and NB, respectively, P<0.05) there were no differences between B and NB blastocysts in %ICM (B6: 19.8 vs 19.8; F2: 20.9 vs 20.4, P=NS) and ICM:T ratio (B6: 4.7 vs 4.7; F2: 4.4 vs. 4.7) in both mouse strains. Post-implantation development to live fetuses of B embryos as compared to NB counterparts was impaired in C57BL/6 (6 vs 18%, P<0.001) but not in B6D2F1 mice (26 vs 35%, P=NS). We conclude that blastomere biopsy impairs embryonic/fetal development in mice known to be sensitive to in vitro culture and manipulations. Such mice model infertile couples with poor quality gametes seeking help in assisted reproduction technologies (ART) clinics.
PMCID: PMC3534949  PMID: 23174776
embryo manipulation; assisted reproductive technology; development; in vitro fertilization; embryo biopsy; C57BL/6
22.  In vitro fertilization: a potential means for toxicity testing 
Uses and potential uses of in vitro fertilization are: (1) a research tool for investigating biochemistry of fertilization, (2) an assay for fertilizing ability, (3) a potentially useful clinical approach for certain cases of infertility, and (4) a potentially useful means for improving animal breeding. In vitro fertilization methodology is sufficiently advanced for gametes of several mammalian species, especially mouse, rabbit, and rat, for use in evaluating effects imposed by toxic agents of environmental or genetic origin. Alteration of the normal events of fertilization and/or embryonic development following transfer of in vitro fertilized embryos into surrogate dams can serve as end point(s) in applying this means for toxicity testing. In vitro fertilization of mouse and rat ova has been explored as an alternative to in vivo fertilization in male contraceptive development studies. Original observations on toxicity of abnormal O2 concentrations for rabbit fertilization in vitro are reported here. Ova were fertilized under 0, 20, 48, and 95% O2, but toxicity from the extreme conditions, 0 and 95% was apparent from comparison of proportions of fertilized ova reaching the 4-cell stage during the 24-hr incubation. Toxicity was further evidenced by inability of embryos fertilized under 0 and 95% O2 tensions, in contrast to those fertilized under 20% O2, to sustain normal gestation following their transfer into recipient does. Recent success in the fertilization of cow ova in vitro in this laboratory provides encouragement to develop a useful means for testing normalcy of gametes in this species. Such studies might lead to useful screening procedures for avoidance of human infertility resulting from hazardous environmental conditions.
PMCID: PMC1637203  PMID: 17539157
23.  In vitro and in vivo development of mice morulae after storage in non-frozen conditions 
Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment.
Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR.
In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C.
Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.
PMCID: PMC3517773  PMID: 22913368
Embryo; Mouse; Storage
24.  Genomic RNA profiling and the programme controlling preimplantation mammalian development 
Molecular Human Reproduction  2008;14(12):691-701.
Preimplantation development shifts from a maternal to embryonic programme rapidly after fertilization. Although the majority of oogenetic products are lost during the maternal to embryonic transition (MET), several do survive this interval to contribute directly to supporting preimplantation development. Embryonic genome activation (EGA) is characterized by the transient expression of several genes that are necessary for MET, and while EGA represents the first major wave of gene expression, a second mid-preimplantation wave of transcription that supports development to the blastocyst stage has been discovered. The application of genomic approaches has greatly assisted in the discovery of stage specific gene expression patterns and the challenge now is to largely define gene function and regulation during preimplantation development. The basic mechanisms controlling compaction, lineage specification and blastocyst formation are defined. The requirement for embryo culture has revealed plasticity in the developmental programme that may exceed the adaptive capacity of the embryo and has fostered important research directions aimed at alleviating culture-induced changes in embryonic programming. New levels of regulation are emerging and greater insight into the roles played by RNA-binding proteins and miRNAs is required. All of this research is relevant due to the necessity to produce healthy preimplantation embryos for embryo transfer, to ensure that assisted reproductive technologies are applied in the most efficient and safest way possible.
PMCID: PMC2639449  PMID: 19043080
gene expression; embryo culture; in vitro fertilization; oocyte; blastocyst
25.  Follicular fluid insulin like growth factor-1 (FF IGF-1) is a biochemical marker of embryo quality and implantation rates in in vitro fertilization cycles 
Insulin-like growth factor-1 (IGF-1) has been reported to play a role in human follicular and embryonic development. However, earlier studies carried out mostly in animal models or in culture mediums supplemented with IGF-1 have been unable to directly link IGF-1 with embryo quality. Results correlating IGF-1 with pregnancy outcome have also been ambiguous so far.
The aim of this study is to find if in situ follicular-fluid level of IGF-1 is predictive of embryo quality and implantation rates in in vitro fertilization (IVF) cycles.
Prospective study involving 120 cycles of conventional IVF-embryo transfer in infertile women.
IGF-1 concentrations were estimated in pooled follicular-fluid on the day of oocyte-pickup. Embryo quality was assessed daily at different developmental stages. Cycles were sorted into low and high follicular fluid insulin-like growth factor-1 (FF IGF-1) groups according to the median value of measurement. Embryo quality, clinical pregnancy and implantation rate were the main outcome measures.
Graph-pad Prism 5 statistical package.
FF IGF-1 correlates with embryo quality (Pearson r = 0.3894, r2 = 0.1516, P > 0.0001) and clinical pregnancy (Pearson r = 0.5972, r2 = 0.36, P > 0.0001). High FF IGF-1 group shows significantly higher rates of fertilization, cleavage, blastocyst formation and top grade embryos compared with low FF IGF-1 group. Clinical pregnancy rates (38.33 vs. 20%, P = 0.0272) and embryo implantation rates (21.6 vs. 10.32%, P = 0.0152) are also significantly higher in the high versus low FF IGF-1 group. Threshold value of FF IGF-1 for clinical pregnancy is <58.50 ng/mg protein (receiver operating characteristics AUC : 0.85 ± 0.03, 95% CI: 0.78-0.91).
FF IGF-1 is a plausible biochemical marker of embryo quality and implantation rate and correlates with clinical pregnancy rates in conventional IVF cycles.
PMCID: PMC3778604  PMID: 24082656
Clinical pregnancy; embryo quality; FF IGF-1; implantation rate; IVF cycles

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