Cumulus and mural granulosa cells of the ovarian follicle surround and interact with the developing oocyte. These follicular cells reflect the oocyte's overall health and may indicate subsequent developmental competence of embryos. Biomarkers of granulosa cells associated with individual oocytes could potentially be used in assisted reproduction to indicate which embryos have the best chance of implanting in the uterus and completing gestation. In this review, we have performed a comprehensive assessment of the recent literature for human cumulus and mural granulosa cell mRNA biomarkers as they relate to pregnancy and live birth. A critical discussion of variables affecting granulosa gene expression profiles for in vitro fertilization patients, including patient demographics and ovarian stimulation regimens, is presented. Although studies with microarray data were evaluated, this synopsis focuses on expressed genes that have been validated by quantitative RT-PCR. Furthermore, we summarize the current published data that support or refute identified granulosa expressed genes as potential biomarkers of embryos that give rise to ongoing pregnancy and live birth. Finally, we review studies that offer predictive models for embryo selection for uterine transfer based on biomarkers that show differential gene expression. Reproduction (2017) 153 R69-R83

Purpose To describe the ultrastructure of spermatozoa from a patient with complete asthenozoospermia that resulted in live births following blastocyst culture. Materials and methods Analyses of spermatozoa from a 36 year old patient were performed using light and electron microscopy. The hypo-osmotic swelling test was used to select spermatozoa for intracytoplasmic sperm injection. Embryos were cultured to the blastocyst stage. Results 100% of the spermatozoa had dynein arm deficiency with secondary defects varying from 3-17%. Six oocytes were injected; five fertilized normally and one was digynic. All five zygotes formed good quality blastocysts. Three blastocysts were cryopreserved and two blastocysts were transferred. Twin females were born at 37 weeks. Conclusions The hypo-osmotic swelling test can be used to select viable immotile ejaculated spermatozoa from a patient with dynein arm deficiency and can produce excellent fertilization rates and blastocyst development resulting in live births.

GATA4 and GATA6 are zinc-finger transcription factors that regulate specific genes involved in steroidogenesis. Using RNA interference (RNAi)-mediated reduction of GATA4 and/or GATA6 with microarray analysis, we aimed to identify novel GATA target genes in luteinizing porcine granulosa cells under vehicle- and cAMP-treated conditions. Microarray analysis identified IGF1 mRNA to be cAMP- and GATA-responsive, and real-time PCR demonstrated that the cAMP-induced increase in IGF1 mRNA was reduced under conditions of GATA6 depletion and GATA4 plus GATA6 depletion, but not GATA4 depletion. Insulin-like growth factor 1 protein levels in media were also decreased by GATA6 or GATA4 plus GATA6 reduction. IGFBP2 and IGFBP4 mRNAs were increased and IGFBP5 mRNA decreased with vehicle and cAMP treatment under GATA4 plus GATA6 RNAi conditions. GATA6 reduction alone increased basal IGFBP4 and decreased IGFBP5 with both vehicle and cAMP, and GATA4 reduction alone lowered cAMP IGFBP5 levels with cAMP. No changes in IGFBP3 mRNA were observed with GATA reduction relative to the control RNAi condition. Levels of insulin-like growth factor binding proteins 2-5 in media as assessed by Western ligand blotting were not altered by GATA reduction. Electromobility gel shift assays with two GATA-containing oligonucleotides of the IGF1 5'-regulatory region showed GATA4 and GATA6 could bind the more proximal GATA-B site. These studies indicate that although GATA4 and GATA6 can bind the porcine IGF1 5'-region, GATA6 is functionally most important for cAMP-stimulated mRNA levels. Using microarray analysis, we identified other mRNAs that were altered by GATA-reduced conditions, including ALDH1, DIO2, and EDNRB. Our findings further support GATA as a coordinator of endocrine/paracrine/autocrine signals in the ovary.

Purpose To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes. Methods This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes. Results PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy. Conclusions Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.

Purpose To utilize a novel mitochondrial function assay with pooled granulosa cells to determine whether mitochondrial function would differ by patient demographics and embryo development. Methods This was a prospective pilot study in a hospital-based assisted reproductive program and public university. Mitochondrial metabolic substrate utilization was assessed in pooled granulosa cells from 40 women undergoing in vitro fertilization during 2018 and 2019. Results Assessment of mitochondrial substrate metabolism in pooled granulosa cells revealed higher citric acid, L-malic acid, and octanoyl-L-carnitine utilization with higher body mass index (BMI). Utilization of citric acid, cis-aconitic acid, D-alphaketo-glutaric acid, L-glutamine, and alanine plus glycine was significantly lower as total dosage of FSH administered increased. Utilization of glycogen was significantly higher in patients with a higher percentage of fertilized oocytes. D-alpha-keto-glutaric acid utilization was significantly lower in patients with a higher percentage of good 8-cell embryos. L-glutamine utilization was significantly lower, with a higher percentage of blastocyst formation. Mitochondrial metabolic scores (MMS), which reflect overall mitochondrial activity of the granulosa pool, were significantly higher in patients with higher BMI and with greater numbers of mature oocytes retrieved. MMS in granulosa decreased as total FSH dose administered increased. Conclusions Granulosa cell utilization of substrates feeding into the citric acid cycle changed with total FSH dosage and BMI. Fertilization rate, 8-cell embryo quality, and blastocyst formation also associated with different energy substrate usage. Mitochondrial substrate utilization by granulosa cells from individual follicles could be further developed into a useful diagnostic tool.

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