The two-step process of ovarian follicular growth and maturation in mammals can be compared to a fruit ripening where quality depends on the second step

In human IVF, the main uncertainty factor impacting on success is oocyte quality, which largely depends on the follicular status at the time of collection. Decades of debate ensued to find the perfect stimulation protocol demonstrated the complexity of the ovarian response to exogenous gonadotropins and the dynamic nature of late folliculogenesis. Although several follicular markers, proteins, RNA from granulosa cells or microRNA and follicular fluid metabolites have been associated with outcome, the possibility to influence them during stimulation remains elusive. The heterogeneity of the follicle’s maturity following control ovarian stimulation is also an important factor to explain average poor oocyte quality still observed today. In this review, the analogy between the apple ripening on the tree and follicular development is presented to focus the attention on a biphasic process: growth and differentiation. The molecular analysis of the progressive follicular differentiation indicates 2 competing phenomena: growth and differentiation where a delicate balance must operate from one to the other to ensure proper maturity at ovulation. As long as FSH stimulates growth, follicles remain green, and it is only when FSH is replaced by LH that the ripening process begins, and “apples” become red. Both fruits, follicles and apples, depend on a perfect timing of events to generate offspring.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

O-056 Evaluating the utility of screening human IVF embryos with polygenic risk scores for complex diseases

Study question It is now feasible to screen human IVF embryos with “polygenic risk scores” for predicting complex disease risk. What is the expected risk reduction? Summary answer Under some conditions, prioritizing embryos based on polygenic risk scores can lead to substantial disease risk reductions. However, only excluding high-risk embryos is less effective. What is known already Recent genetic studies have identified numerous mutations associated with complex diseases, leading to the development of accurate polygenic risk scores (PRSs) for disease risk prediction. Given that genomes of human IVF embryos can now be sequenced with relative ease, it has become technically feasible to use PRSs for prioritization of embryos for transfer. Clearly, such use is associated with ethical and social concerns, from inequality to eugenics. Nevertheless, polygenic embryo screening is already offered to consumers, with little research so far on expected outcomes. Our previous evaluation of screening IVF embryos for polygenic traits showed little current utility. Study design, size, duration This is a theoretical/computational study based on statistical genetics theory and simulations. Participants/materials, setting, methods We used the liability threshold model to estimate the disease risk given the PRS. We considered screening for a single disease (with known prevalence and PRS accuracy), and assumed that n viable embryos are available. We calculated the risk of the child given these parameters and the prioritization strategy, either when parents are random or when their disease status is known. We also used simulations based on genomic data from a schizophrenia case-control study. Main results and the role of chance We modeled the disease risk of a hypothetical future child when the PRSs of embryos are used for prioritization, relative to random selection. When selecting an embryo at random among those who do not have an extremely high risk (typically, top 2% of the PRS distribution), the relative risk reduction (RRR) is limited, and is under 10% for currently realistic scenarios. In contrast, selecting the lowest risk embryo for implantation results in substantial RRRs of ∼20-50% already with n = 5 embryos and realistic disease parameters. For example, the RRR for schizophrenia is > 40% with current PRSs, a result we validated with simulated genomes of parents and children based on genotypes from a schizophrenia study. When one of the parents is known to be affected, selecting the lowest risk embryo out of n = 5 may restore the risk of the future child to nearly normal levels. Limitations, reasons for caution Our analytical modeling is based on several simplifying assumptions regarding the dependence of the risk on the PRS and the accuracy of the PRS. Further, the estimated risk reductions depend on the availability of n = 5 embryos that could lead to a live birth. Wider implications of the findings Under some conditions, prioritizing embryos for transfer based on polygenic risk scores could lead to substantial disease risk reductions. However, predicted outcomes vary considerably with prioritization strategies and disease and PRS parameters. The emerging ethical and social concerns and the challenges in communicating these results need to be urgently discussed. Trial registration number Not applicable

A new microfluidic concept for successful in vitro culture of mouse embryos

ABSTRACTInnovative techniques for gene editing have enabled accurate animal models of human diseases to be established. In order for these methods to be successfully adopted in the scientific community, the optimization of procedures used for breeding genetically altered mice is required. Among these, the in vitro fertilization (IVF) procedure is still suboptimal and the culture methods do not guarantee the development of competent embryos. Critical aspects in traditional in vitro embryo culture protocols include the use of mineral oil and the stress induced by repetitive handling of the embryos.A new microfluidic system was designed to allow for efficient in vitro culture of mouse embryos. Harmful fluidic stress and plastic toxicity were excluded by completing the industry gold standard Mouse Embryo Assay. The potential competence of the embryos developed in the device was quantified in terms of blastocyst rate, outgrowth assay, energy substrate metabolism and expression of genes related to implantation potential.Mass spectrometry analyses identified plastic-related compounds released in medium, and confirmed leaching of low molecular weight species into the culture medium that might be associated to un-crosslinked PDMS.Finally, these data show the potential for the system to study preimplantation embryo development and to improve human IVF techniques.

Non-rodent mammalian zygotes assemble dual spindles despite the presence of paternal centrosomes

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded and, instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In non-rodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether non-rodent zygotes assemble a single centrosomal spindle around both genomes, or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form around the parental genomes despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in non-rodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.SummaryThis study investigates spindle assembly during the first embryonic division in bovine zygotes that, like human, inherit centrosomes from the sperm. It shows that two independent microtubule arrays form by self-organization around parental genomes with only loosely connected centrosomes.

Necessity is the mother of invention and the evolutionary force driving the success of in vitro fertilization

During the last few decades, millions of healthy children have been born with the aid of in vitro fertilization (IVF). This success belies the fact that IVF treatment is comprised of a complex series of interventions starting with a customized control ovarian stimulation protocol. This is followed by the induction of oocyte maturation, the retrieval of mature oocytes and in vitro fertilization, which often involves the microinjection of a single sperm into the oocyte. After fertilization, the resulting embryos are cultured for up to 7 days. The best embryos are transferred into the uterus where the embryo implants and hopefully develops into a healthy child. However, frequently the best embryos are biopsied and frozen. The biopsied cells are analyzed to identify those embryos without chromosomal abnormalities. These embryos are eventually thawed and transferred with pregnancy rates as good if not better than embryos that are not biopsied and transferred in a fresh cycle. Thus, IVF treatment requires the coordinated efforts of physicians, nurses, molecular biologists and embryologists to conduct each of these multifaceted phases in a seamless and flawless manner. Even though complex, IVF treatment may seem routine today, but it was not always the case. In this review the evolution of human IVF is presented as a series of innovations that resolved a technical hurdle in one component of IVF while creating challenges that eventually lead to the next major advancement. This step-by-step evolution in the treatment of human infertility is recounted in this review.

Effects of vitrification and the superovulated environment on placental function and fetal growth in an IVF mouse model

In studies of human IVF, as compared to frozen embryo transfer (ET), fresh ET is associated with smaller infants and higher risk of small for gestational age infants. Recent observations suggest that ET using vitrified embryos is associated with higher pregnancy and live birth rates compared to fresh ET, but increased rates of large for gestational age infants. The mechanisms underlying these associations are largely unknown, and available evidence suggests that the influence of IVF, vitrification and the superovulated (SO) uterine environment on placental function and fetal growth is complex. This warrants further investigation given the prevalent practice in human IVF of both fresh ET into a SO uterine environment, and vitrification with ET into a more physiologic uterine environment. Using a mouse model that closely resembles human IVF, we investigated if vitrification of IVF embryos better preserves placental function and results in better pregnancy outcomes as compared to fresh ET because of transfer into a more physiologic endometrium. We found that the SO environment, independent of vitrification status, reduced implantation rates, inhibited placental mechanistic target of rapamycin signaling and induced placental stress signaling, resulting in fetal growth restriction (1.080 ± 0.05 g estrous fresh (n = 17 litters), 1.176 ± 0.05 g estrous vitrified (n = 12), 0.771 ± 0.06 g SO fresh (n = 15), 0.895 ± 0.08 g SO vitrified (n = 10), P < 0.0001). In addition, our study suggests that vitrification impairs the developmental potential of IVF blastocysts that resulted in a significantly smaller litter size (2.6 ± 2.3 fresh estrous vs 2.5 ± 2.4 fresh SO vs 1.6 ± 1.7 estrous vitrified vs 1.7 ± 1.8 SO vitrified, P = 0.019), with no effect on fetal growth or placental function at term. Our findings suggest that vitrification may negatively impact early embryonic viability, while the SO maternal uterine environment impairs both placental development and fetal growth in IVF.

Processing of human IVF/IVM oocytes for single cell RNA sequencing

The protocol describes the step-by-step procedure of isolating and preparing human oocytes 1) oocytes donated in connection with IVF/ICSI treatment and 2) in vitro matured (IVM) oocytes from small antral follicles donated in connection to fertility preservation. It details how cumulus cells and the zona pellucida are enzymatically removed to ensure that only the oocyte is analyzed. We have successfully used this protocol in connection with SMART-Seq2 (Takara).This oocyte preparation has recently been used in Sankar et al. 2020 (Sankar et al. 2020).