Mammalian embryo culture media: now and into the future.

For over 70years, since the culture of the first mammalian embryo in vitro , scientists have undertaken studies to devise and optimise media to support the manipulation and culture of gametes and embryos. This area of research became especially active in the late 1970s onwards following the successful birth of the first human in vitro fertilised embryo. This review summarises some of the key advances in mammalian embryo culture media over time based on a greater understanding of the biochemical milieu of the reproductive tract. It highlights how learnings from studies in mice and agricultural species have informed human culture media compositions, in particular the inclusion of albumin, growth factors, cytokines, and antioxidants into contemporary culture media formulations, and how these advances may then in turn help to inform and guide development of in vitro culture systems used in other arenas, in particular agriculture. Additionally, it will highlight how the introduction of new technologies, such as timelapse, can influence current trends in media composition and usage that may see a return to a single step medium.

Reduction of Mitochondrial Function by FCCP During Mouse Cleavage Stage Embryo Culture Reduces Birth Weight and Impairs the Metabolic Health of Offspring.

The periconceptual environment represents a critical window for programming fetal growth trajectories and susceptibility to disease; however, the underlying mechanism responsible for programming remains elusive. This study demonstrates a causal link between reduction of precompaction embryonic mitochondrial function and perturbed offspring growth trajectories and subsequent metabolic dysfunction. Incubation of embryos with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation, significantly reduced mitochondrial membrane potential and ATP production in 8-cell embryos and the number of inner cell mass cells within blastocysts; however, blastocyst development was unchanged. This perturbed embryonic mitochondrial function was concomitant with reduced birth weight in female offspring following embryo transfer, which persisted until weaning. FCCP-treated females also exhibited increased adiposity at 4 wk, increased adiposity gain between 4 and 14 wk, glucose intolerance at 8 wk, and insulin resistance at 14 wk. Although FCCP-treated males also exhibited reduced glucose tolerance, but their insulin sensitivity and adiposity gain between 4 and 14 wk was unchanged. To our knowledge, this is one of the first studies to demonstrate that reducing mitochondrial function and, thus, decreasing ATP output in the precompacting embryo can influence offspring phenotype. This is of great significance as a large proportion of patients requiring assisted reproductive technologies are of advanced maternal age or have a high body mass index, both of which have been independently linked with perturbed early embryonic mitochondrial function.

Mitochondrial SIRT5 is present in follicular cells and is altered by reduced ovarian reserve and advanced maternal age.

Women with reduced ovarian reserve or advanced maternal age have an altered metabolic follicular microenvironment. As sirtuin 5 (SIRT5) senses cellular metabolic state and post-translationally alters protein function, its activity may directly impact on oocyte viability and pregnancy outcome. Therefore, we investigated the role of SIRT5 in relation to ovarian reserve and maternal age. Women (n=47) undergoing routine IVF treatment were recruited and allocated to one of three cohorts based on ovarian reserve and maternal age. Surplus follicular fluid, granulosa and cumulus cells were collected. SIRT5 mRNA, protein and protein activity was confirmed in granulosa and cumulus cells via qPCR, immunohistochemistry, western blotting and desuccinylation activity. The presence of carbamoyl phosphate synthase I (CPS1), a target of SIRT5, was investigated by immunohistochemistry and follicular-fluid ammonium concentrations determined via microfluorometry. Women with reduced ovarian reserve or advanced maternal age had decreased SIRT5 mRNA, protein and desuccinylation activity in granulosa and cumulus cells resulting in an accumulation of follicular-fluid ammonium, presumably via alterations in activity of a SIRT5 target, CPS1, which was present in granulosa and cumulus cells. This suggests a role for SIRT5 in influencing oocyte quality and IVF outcomes.

Does obesity really matter? The impact of BMI on embryo quality and pregnancy outcomes after IVF in women aged ≤38 years.

BACKGROUND: The increasing prevalence of obesity in women of child-bearing age is of growing concern in the health community. Obesity is associated with sub-optimal reproductive performance; therefore, it is understandable that the number of young women with elevated body mass index (BMI) accessing assisted reproductive treatment (ART) is on the rise. Consequently, this study not only assessed the impact of BMI on fertilisation rates, embryo development and freezing during ART in women aged ≤38 years but also determined their subsequent pregnancy and delivery rates. METHODS: Data were retrospectively analysed from all cycles initiated in 2006/2007 for women aged ≤38 years. The BMI categorisations were as follows: normal - 18.5-24.9 kg/m(2) ; overweight - 25-29.9 kg/m(2) ; obese - 30-34.9 kg/m(2) ; morbidly obese class I - 35-39.9 kg/m(2) ; morbidly obese class П -≥40 kg/m(2) . RESULTS: Obese and morbidly obese women required a significantly higher follicle stimulating hormone start dose than normal BMI women; however, they obtained significantly fewer oocytes (P < 0.05). Although BMI did not affect embryo development, morbidly obese class Π women had significantly reduced pregnancy rates compared to normal BMI women (30.5 vs 41.7%, respectively; P < 0.05). Furthermore, increasing BMI was positively correlated to increasing rates of preterm delivery (P < 0.05). Increasing BMI was also positively correlated to increasing delivery rates of singleton term macrosomic offspring (≥4000 g). CONCLUSION: Obesity in women aged≤38 years does not affect embryo development; however, it does reduce clinical pregnancy rates in women with a BMI≥40 and increases rates of preterm labour and delivery of macrosomic offspring.

Single blastocyst embryo transfer maintains comparable pregnancy rates to double cleavage-stage embryo transfer but results in healthier pregnancy outcomes.

BACKGROUND: The optimal outcome after IVF is a live, healthy, singleton term baby. This can be achieved by transferring a single embryo, but at the possible expense of reducing pregnancy rates. Recent studies suggest that delaying transfer of embryos to the blastocyst stage (day 4/5), rather than the more traditional cleavage stage (day 2-3), allows for better selection of the best embryo, maximising pregnancy rates from a single embryo transfer (SET). The aim of this study was to assess pregnancy outcomes in relation to changing embryo transfer practices. METHODS: A retrospective analysis of pregnancy outcomes was made between IVF cycles conducted in 2007 when blastocyst SET became standard practice, with IVF cycles in 2003 when double cleavage-stage embryo transfer was the norm. RESULTS: The implementation of a blastocyst SET policy resulted in a significant decrease in multiple birth rates, while maintaining live birth delivery rate comparable to double cleavage-stage transfer (27.2% versus 24.8%, respectively, N.S.). CONCLUSION: Improvements in culturing protocols have facilitated extended culture, increasing embryo selection capability. These results indicate that it is now possible to maintain excellent pregnancy rates with SET blastocyst culture, while decreasing complications related to multiple births.

Alterations in mouse embryo intracellular pH by DMO during culture impair implantation and fetal growth.

The preimplantation embryo is highly susceptible to in-vitro stress, and although this does not necessarily perturb blastocyst development, it can significantly affect embryo physiology and the ability to form a viable pregnancy. This study determined that the preimplantation mouse embryo is highly sensitive to a small decrease in intracellular pH (<0.2 pH units). Embryos cultured in media containing a weak acid (5,5-dimethyl-2,4-oxazolidinedione; DMO) formed blastocysts with decreased cell number and inner cell mass number, as well as increased apoptosis, even though blastocyst development and morphology were unchanged. Interestingly, the effects were similar regardless of whether the pH stress was present for a short-term 'acute' exposure (during the zygote to 2-cell, or 2-cell to 8-cell division) or an extended 'chronic' period of time (continually from the zygote to the blastocyst stage). Exposure to DMO during the first cleavage division did not alter implantation; however, fetal weight and crown-rump length were significantly decreased (P<0.05). In contrast, continuous exposure to DMO throughout preimplantation development reduced not only implantation but also fetal weight and crown-rump length. This study highlights the importance of correct intracellular pH and demonstrates that slight deviations can significantly impact embryo development and viability.

Dietary Micronutrient Supplementation for 12 Days in Obese Male Mice Restores Sperm Oxidative Stress.

Male obesity, which often co-presents with micronutrient deficiencies, is associated with sub-fertility. Here we investigate whether short-term dietary supplementation of micronutrients (zinc, selenium, lycopene, vitamins E and C, folic acid, and green tea extract) to obese mice for 12 days (designed to span the epididymal transit) could improve sperm quality and fetal outcomes. Five-week-old C57BL6 males were fed a control diet (CD, n = 24) or high fat diet (HFD, n = 24) for 10 weeks before allocation to the 12-day intervention of maintaining their original diets (CD, n = 12, HFD n = 12) or with micronutrient supplementation (CD + S, n = 12, HFD + S, n = 12). Measures of sperm quality (motility, morphology, capacitation, binding), sperm oxidative stress (DCFDA, MSR, and 8OHdG), early embryo development (2-cell cleavage, 8OHdG), and fetal outcomes were assessed. HFD + S males had reduced sperm intracellular reactive oxygen species (ROS) concentrations and 8OHdG lesions, which resulted in reduced 8OHdG lesions in the male pronucleus, increased 2-cell cleavage rates, and partial restoration of fetal weight similar to controls. Sub-fertility associated with male obesity may be restored with very short-term micronutrient supplementation that targets the timing of the transit of sperm through the epididymis, which is the developmental window where sperm are the most susceptible to oxidative damage.

To QC or not to QC: the key to a consistent laboratory?

A limiting factor in every embryology laboratory is its capacity to grow 'normal' embryos. In human in vitro fertilisation (IVF), there is considerable awareness that the environment of the laboratory itself can alter the quality of the embryos produced and the industry as a whole has moved towards the implementation of auditable quality management systems. Furthermore, in some countries, such as Australia, an established quality management system is mandatory for clinical IVF practice, but such systems are less frequently found in other embryology laboratories. Although the same challenges of supporting consistent and repeatable embryo development are paramount to success in all embryology laboratories, it could be argued that they are more important in a research setting where often the measured outcomes are at an intracellular or molecular level. In the present review, we have outlined the role and importance of quality control and quality assurance systems in any embryo laboratory and have highlighted examples of how simple monitoring can provide consistency and avoid the induction of artefacts, irrespective of the laboratory's purpose, function or species involved.

Peri-conception parental obesity, reproductive health, and transgenerational impacts.

Maternal over-nutrition during pregnancy is a risk factor for pregnancy complications and is increasingly associated with adverse childhood outcomes such as increased propensity for obesity and metabolic disease. However, there is emerging evidence that parental lifestyle factors prior to and at conception have a powerful impact on the health of the offspring for more than one generation. Maternal and paternal obesity prior to conception alters the molecular composition of both oocytes and sperm, which can partly escape epigenetic reprogramming at fertilization, altering the developmental trajectory of the resultant embryo, ultimately increasing the incidence of obesity and metabolic disorders in offspring. Understanding the molecular underpinning of these changes may help create interventions to reduce the risk of disease in future generations.

Oxidative stress in mouse sperm impairs embryo development, fetal growth and alters adiposity and glucose regulation in female offspring.

Paternal health cues are able to program the health of the next generation however the mechanism for this transmission is unknown. Reactive oxygen species (ROS) are increased in many paternal pathologies, some of which program offspring health, and are known to induce DNA damage and alter the methylation pattern of chromatin. We therefore investigated whether a chemically induced increase of ROS in sperm impairs embryo, pregnancy and offspring health. Mouse sperm was exposed to 1500 µM of hydrogen peroxide (H2O2), which induced oxidative damage, however did not affect sperm motility or the ability to bind and fertilize an oocyte. Sperm treated with H2O2 delayed on-time development of subsequent embryos, decreased the ratio of inner cell mass cells (ICM) in the resulting blastocyst and reduced implantation rates. Crown-rump length at day 18 of gestation was also reduced in offspring produced by H2O2 treated sperm. Female offspring from H2O2 treated sperm were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared to control female offspring. Interestingly male offspring phenotype was less severe with increases in fat depots only seen at 4 weeks of age, which was restored to that of control offspring later in life, demonstrating sex-specific impacts on offspring. This study implicates elevated sperm ROS concentrations, which are common to many paternal health pathologies, as a mediator of programming offspring for metabolic syndrome and obesity.

Women with reduced ovarian reserve or advanced maternal age have an altered follicular environment.

OBJECTIVE: To determine whether altered follicular environment is associated with ovarian reserve or maternal age. DESIGN: Prospective study examining follicular fluid (FF) composition and follicular cell metabolism. SETTING: University research department and private IVF clinic. PATIENT(S): Women (n = 54) undergoing routine IVF treatment were allocated to one of three groups based on ovarian reserve and maternal age. INTERVENTION(S): Surplus FF, granulosa cells (GC), and cumulus cells (CC) were collected. MAIN OUTCOME MEASURE(S): Follicular fluid concentrations of carbohydrates, hormones, and selected ions. Metabolic analysis and gene expression of GCs and CCs. RESULT(S): Compared to women <35 years with normal ovarian reserve, FF glucose levels were significantly decreased and lactate and progesterone (P4) concentrations significantly increased in women with reduced ovarian reserve or advanced maternal age, whereas GC and CC glucose uptake, lactate production, and phosphofructokinase platelet gene expression were significantly increased. Granulosa cell P4 production from women with reduced ovarian reserve or advanced maternal age was decreased; however, in CCs the reverse was observed with increased gene expression in P4 receptor, prostaglandin E receptor-2, cytosolic phospholipase A2, and tumor protein 53. CONCLUSION(S): Women with either reduced ovarian reserve or advanced maternal age have altered follicular cell metabolism, FF metabolites, and P4 production. This perturbed environment may be responsible for impaired oocyte developmental competence and subsequent embryo development.