Paolo F. Rinaudo

Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics

The assessment of oocyte quality in human in vitro fertilization (IVF) is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF) is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics

Symptomatic patients with an early viable intrauterine pregnancy: HCG curves redefined.

OBJECTIVE: To analyze the change in serial human chorionic gonadotropin (hCG) levels in women symptomatic with pain or bleeding who presented with nondiagnostic ultrasonography but were ultimately confirmed to have a viable intrauterine pregnancy. METHODS: The rise in serial hCG measures were modeled over time, with the start point defined in 2 ways: by last menstrual period and by date of presentation for care. Both semiparametric (spline) curves and linear random-effects models were explored. The slope and projected increase of hCG were calculated to define 99% of viable intrauterine pregnancies. RESULTS: A total of 287 subjects met inclusion criteria and contributed 861 measurements of hCG. On average, these subjects contributed 3.00 observations and were followed up for 5.25 days. A linear increase in log hCG best described the pattern of rise. Curves derived from last menstrual period and day of presentation do not differ substantially. The median slope for a rise of hCG after 1 day was 1.50, (or a 50% increase); 2.24 after 2 days (or a 124% rise), and 5.00 after 4 days. The fastest rise was 1.81 at 1 day, 3.28 at 2 days, and 10.76 at 4 days. The slowest or minimal rise for a normal viable intrauterine pregnancy was 24% at 1 day and 53% at 2 days. CONCLUSION: These data define the slowest rise in serial hCG values for a potentially viable gestation and will aid in distinguishing a viable early pregnancy from a miscarriage or ectopic pregnancy. The minimal rise in serial hCG values for women with a viable intrauterine pregnancy is “slower” than previously reported, suggesting that intervention to diagnosis and treat an abnormal gestation should be more conservative. LEVEL OF EVIDENCE: II-2

Fetal Programming and Metabolic Syndrome

Metabolic syndrome is reaching epidemic proportions, particularly in developing countries. In this review, we explore the concept-based on the developmental-origin-of-health-and-disease hypothesis-that reprogramming during critical times of fetal life can lead to metabolic syndrome in adulthood. Specifically, we summarize the epidemiological evidence linking prenatal stress, manifested by low birth weight, to metabolic syndrome and its individual components. We also review animal studies that suggest potential mechanisms for the long-term effects of fetal reprogramming, including the cellular response to stress and both organ- and hormone-specific alterations induced by stress. Although metabolic syndrome in adulthood is undoubtedly caused by multiple factors, including modifiable behavior, fetal life may provide a critical window in which individuals are predisposed to metabolic syndrome later in life.

Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model

BACKGROUND Abnormal placentation is a potential mechanism to explain the increased incidence of low birthweight observed after IVF. This study evaluates, in a mouse model, whether the method of conception and embryo transfer affect placentation and fetal development. METHODS IVF blastocysts (CF1 x B6D2F1/J) were cultured in Whittens medium (IVF(WM), n = 55) or K modified simplex optimized medium with amino acids (IVF(KAA), n = 56). Embryos were transferred to the uteri of pseudo-pregnant recipients. Two control groups were created: unmanipulated embryos produced by natural mating (in vivo group, n = 64) and embryos produced by natural mating that were flushed from uterus and immediately transferred to pseudo-pregnant recipients (flushed blastocysts, FB group, n = 57). At gestation age 12.5 days, implantation sites were collected and fixed; fetuses and placentas were weighed and their developmental stage (DS) evaluated. Placental areas and vascular volume fractions were calculated; parametric statistics were applied as appropriate. RESULTS IVF fetuses showed a modest but significant delay in development compared with FB mice (P < 0.05). In addition, IVF conceptuses were consistently smaller than FB (P < 0.05). Importantly, these differences persisted when analyzing fetuses of similar DS. The placenta/fetus ratio was larger in the IVF group (IVF(WM) 0.95; IVF(KAA) = 0.90) than the FB group (0.72) (P < 0.05 for all comparisons). Gross morphology of the placenta and ratio labyrinth/fetal area were equivalent in the IVF and FB groups, as were percentage of fetal blood vessels, maternal blood spaces and trophoblastic components. CONCLUSIONS In vitro embryo culture affects fetal and placental development; this could explain the lower birthweight in IVF offspring.

A quantitative assessment of follicle size on oocyte developmental competence

OBJECTIVE To quantitatively assess the impact of follicle size on oocyte maturation, fertilization, and embryo quality. DESIGN Prospective study. SETTING Academic medical center. PATIENT(S) Couples undergoing ovarian stimulation and in vitro fertilization (IVF). INTERVENTION(S) A total of 235 cycles were monitored prospectively, and 2934 oocytes were collected from five groups of follicle size. Repeated measures multivariate analyses were used to compare the smaller follicle sizes with the lead follicle. MAIN OUTCOME MEASURE(S) Oocyte maturation, fertilization, and embryo quality. RESULT(S) Compared with the lead follicular group (>18 mm), the odds of a mature oocyte from a 16 to 18 mm size follicle were 37% and declined progressively with each size. The odds of fertilization of oocytes from follicles 16 to 18 mm in size was 28% less than the lead group and decreased with each size. The rate of polyspermy with conventional insemination was increased for the smaller follicular groups (adjusted odds ratio = 2.37). Follicle size did not predict embryo cell number, but embryos from smaller follicles had a statistically significantly higher fragmentation compared with the lead group. CONCLUSION(S) The lead follicular group was most likely to have a mature oocyte that was capable of fertilization and best suited for development into a high-quality embryo. The smaller follicles were capable of producing metaphase II oocytes that could fertilize, but at rates approaching only 60% that of the lead follicular group.

Impaired Placental Nutrient Transport in Mice Generated by in Vitro Fertilization

More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.

Use of a Mouse In Vitro Fertilization Model to Understand the Developmental Origins of Health and Disease Hypothesis

The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.

In Vitro Fertilization Affects Growth and Glucose Metabolism in a Sex-Specific Manner in an Outbred Mouse Model

ABSTRACT The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O2 (IVFWM group, less optimal) or K simplex optimized medium with amino acids and 5% O2 (IVFKAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVFKAA offspring did not differ significantly from FB offspring. However, male IVFWM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVFWM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

Effect of ICSI on gene expression and development of mouse preimplantation embryos

BACKGROUND In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whittens medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)). CONCLUSIONS Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.