Markers of Oocyte Quality to Enhance Human IVF Outcomes: A Bibliographic Review

The markers of oocyte quality have remained a major controversy in the field of embryology due to the subjectivity of the different methods of oocyte assessment. Various scholars use oocyte quality and oocyte competence interchangeably. Oocyte quality can be defined as the overall health of an oocyte whereas oocyte competence refers to the ability of an oocyte to be fertilized and develop into a healthy embryo. Diminished oocyte quality is believed to be a result of alterations in oocyte growth and maturation processes that stem from several pelvic and systemic factors before and after oocyte retrieval. In this review, we focus on the morphological and nonmorphological markers of oocyte quality. Strict restrictions that limit the number of oocytes fertilized in various countries have triggered researchers around the world to come up with the most appropriate and noninvasive markers that enhance oocyte selection and optimize IVF outcomes. PubMed, Google Scholar, and the Cochrane Library were used to search for peer-reviewed, original articles about oocyte quality markers. The review was written in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Morphological markers are commonly used, but they are subjective, and no single marker can be used exclusively to predict oocyte competence and subsequent embryonic development potential. Furthermore, transcriptomics of differentially expressed genes in cumulus cells and assessment of metabolomics and other contents of follicular fluid have shown greater precision. However, their specificity to the different quality determinants needs further research.

Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids:the potential association with oocyte quality

Background: Ovarian follicular fluids(FFs) contain several kinds of regulatory factors that maintain a suitable micro-environment for oocyte development. Extracellular vesicles(EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs(miRNAs). This study aimed to investigate small-EV(s-EV) miRNAs in porcine FFs and their potential association with oocyte quality.Methods: Individual aspirated oocytes were stained with lissamine green B stain(LB), a vital stain for oocyte quality, and each oocyte was classified as high-quality(unstained;HQ) or low-quality(stained;LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina Nova Seq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation.Results: A total of 19 miRNAs(including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23(including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte developmental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution.Conclusion: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation.

Endogenous biosynthesis of docosahexaenoic acid(DHA)regulates fish oocyte maturation by promoting pregnenolone production

Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related to this phenomenon remains largely unknown.In this study,we employed two zebrafish genetic models,i.e.,elovl2^(-/-)mutant as an endogenous DHAdeficient model and fat1(omega-3 desaturase encoding gene)transgenic zebrafish as an endogenous DHA-rich model,to investigate the effects of DHA on oocyte maturation and quality.Results show that the elovl2^(-/-)mutants had much lower fecundity and poorer oocyte quality than the wild-type controls,while the fat1 zebrafish had higher fecundity and better oocyte quality than wildtype controls.DHA deficiency in elovl2^(-/-)embryos led to defects in egg activation,poor microtubule stability,and reduced pregnenolone levels.Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1,which encodes the cholesterol side-chain cleavage enzyme,thereby stabilizing microtubule assembly during oogenesis.In turn,the hypothalamic-pituitary-gonadal axis was enhanced by DHA.In conclusion,using two unique genetic models,our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.

Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress

Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are extremely sensitive to the heat stress.However,the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.Methods:Germinal vesicle(GV)porcine oocytes were cultured at 41.5℃ for 24 h to induce heat stress,and then cultured at 38.5℃ to the specific developmental stage for subsequent analysis.Nicotinamide mononucleotide(NMN)was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10μmol/L,20μmol/L,50μmol/L or 100μmol/L,respectively,during heat stress.Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.Results:Here,we report that NMN supplementation improves the quality of porcine oocytes under heat stress.Specifically,we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles,including the cytoskeleton assembly,cortical granule distribution and mitochondrial function.In addition,heat stress induced the production of excessive reactive oxygen species(ROS)and DNA damage,leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest.More importantly,we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.Conclusions:Taken together,our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.

Mitochondrial Dysfunction and Age-related Oocyte Quality

Fertility disorders have become a growing problem worldwide. It is well known that female fertility decreases with age, previous studies suggested that the age-related decline in female fertility potential was largely due to decrease in oocyte quality and mitochondrial dysfunction. Mitochondria play a crucial role during the process of oocyte maturation. Mitochondrial genetic, numerical and structural defects occur in oocyte aging process, mitochondrial abnormalities are believed to contribute to age-related infertility. Improvement of the mitochondrial function can lead to better fertility outcomes, and application of mitochondria replacement strategy or mitochondrial transfer to age-related infertility will be possible in the future. This review paper, we are trying to discuss current understanding about age-related changes in oocyte quality and mitochondrial dysfunction.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

Harmful Effects on African Clawed Frog （Xenopus laevis） Reproduction as Expression of High Water Phosphates Levels

Amphibians are considered as reliable indicators of environmental quality. In Europe, a general decline of amphibians population parallels a worldwide decline, and some of the factors which are thought to be responsible to this decline are habitat loss, introduction of exotic species, disease and decreasing water quality. Recent investigations suggest that these eutrophic conditions may be associated with frog reproduction problems. Water quality criteria settled for the different species of amphibians do not currently exist, but in the present paper, the authors report their observation about water phosphate concentrations in association with hipofertility in African clawed frogs （Xenopus laevis）. The study considered two different groups of African clawed frog showing hypofertility （22 patients） hosted in captivity. The animals were visited, and the filtration systems functionality and the water quality were checked, i.e., temperature, pH, ammonia, nitrite, nitrate, phosphates, hardness and alkalinity. In the first group, it was decided to perform ultrasound scans, parasitological tests on fecal samples, and microscopical/macroscopical examination, as well as microbiological analysis on collected oocytes. In the second group, only fecal samples were collected in order to perform parasitologiacal exams. No pathological findings were showed by veterinary tests. Water changes were carried out in both facilities and after two months, the fertility in African clawed frogs improved, evidencing the involvement of phosphates values in the onset of the problem and even more in its resolution. More studies are needed to further define this correlation.

Expression of Target Genes in Cumulus Cells Derived from Human Oocytes with and without Blastocyst Formation

Objective:The transcriptional profile of cumulus cells(CCs)during oocyte maturation provides information for predicting oocyte developmental competence.Our previous study using a mouse model indicated that there were nine different genes related to oocyte development potential expressed in CCs during oocyte maturation.The purpose of this study was to elucidate whether gene expression levels of CCs during oocyte maturation are associated with oocyte developmental competence.Methods:The human CCs collected from each oocyte were divided into two groups after tracking depending on whether or not they developed to the blastocyst stage:(1)the oocytes were developed to blastocyst stage after fertilization(B+)and(2)the oocytes were not developed to blastocyst stage after fertilization(B−).The expression levels of the nine selected genes(ARRB1,ATP2C1,CDH5,CNTNAP1,LGR4,MKLN1,RHOBTB1,SIX2,and SMC2)were examined.CCs were obtained from 29 women who were undergoing intracytoplasmic sperm injection treatment cycles.Quantitative reverse transcriptase-polymerase chain reaction analysis was performed on cumulus masses collected before insemination.Each sample was run three times.Statistically significant differences in mRNA expression of the target genes in independent samples were evaluated by two-tailed Student’s t-test,and P<0.05 was considered significantly different.Results:There were significant differences in the mRNA expression levels of ARRB1(P=0.016),LGR4(P=0.025),and SMC2(P=0.013)between the groups B+and B−.Gene expression of ARRB1,LGR4,and SMC2 in CCs is related to blastocyst development.Conclusions:Analysis of expression of ARRB1,LGR4,and SMC2 genes in CCs as biomarkers may provide predictive information on oocyte developmental competence before insemination and fertilization.

Ovarian aging:mechanisms and intervention strategies

Ovarian reserve is essential for fertility and influences healthy aging in women.Advanced maternal age correlates with the progressive loss of both the quantity and quality of oocytes.The molecular mechanisms and various contributing factors underlying ovarian aging have been uncovered.In this review,we highlight some of critical factors that impact oocyte quantity and quality during aging.Germ cell and follicle reserve at birth de-termines reproductive lifespan and timing the menopause in female mammals.Accelerated diminishing ovarian reserve leads to premature ovarian aging or insufficiency.Poor oocyte quality with increasing age could result from chromosomal cohesion deterioration and misaligned chromosomes,telomere shortening,DNA damage and associated genetic mutations,oxidative stress,mitochondrial dysfunction and epigenetic alteration.We also discuss the intervention strategies to delay ovarian aging.Both the efficacy of senotherapies by antioxidants against reproductive aging and mitochondrial therapy are discussed.Functional oocytes and ovarioids could be rejuvenated from pluripotent stem cells or somatic cells.We propose directions for future interventions.As couples increasingly begin delaying parenthood in life worldwide,understanding the molecular mechanisms during female reproductive aging and potential intervention strategies could benefit women in making earlier choices about their reproductive health.

Recent progress in the treatment of women with diminished ovarian reserve

Diminished ovarian reserve(DOR)refers to a decrease in the number and/or quality of oocytes in the ovary,accompanied by a decline in reproductive potential,which is generally related to advanced age or ovarian disease.In in vitro fertilization(IVF)clinical practice,managing patients with DOR remains one of the most challenging tasks.In recent years,increased research on improving ovarian function has provided us with new insights into treating patients with DOR.Many therapeutic options have been proposed to improve the ovarian function of patients with DOR,yet they are not widely utilized in clinical practice because of limited evidence of safety and effectiveness.In this review,we focus on the mechanisms from animal models and clinical trials that have been applied to the treatment of DOR in recent years,intending to improve IVF outcomes in patients with DOR.Furthermore,new insights and perspectives on the molecular and cellular regulation of follicular development and ovarian reserve are emphasized to provide more clues for research on the treatment of DOR.