Prostaglandins and Steroid Hormones in Follicular Fluid As Predictors of Outcome in In-Vitro Fertilisation

The evolution of in-vitro fertilisation over the last decade has produced major advances in ovarian hyperstimulation, cycle monitoring, oocyte retrieval and embryo transfer techniques. The methodology for culture of human oocytes and their fertilisation has been refined such that fertilisation rates of over 70% can now be obtained

Follicle selection dynamics in the mammalian ovary

The main objective of this thesis is to develop and analyse mathematical models of the regulation of the ovulation cycle in mammals. Specifically, we are interested in understanding the mechanisms that control the number of follicles ovulated in each cycle. In humans, a failure of such control mechanisms can lead to Polycystic Ovary Syndrome (PCOS), which accounts for a substantial fraction of all cases of anovulatory infertility found in women of reproductive age. Although treatment is available, it is highly desirable to improve it. Thus, a better understanding of the selection process of the ovulatory follicle is still required. The thesis begins with a biological description of the terminal phase of the ovarian cycle. This provides the necessary background for the understanding and formulation of the mathematical models presented in later chapters. Next, a review of existing models found in the literature is given and their relevance to the regulation process is analysed. Of these, the one due to Lacker (also referred as the symmetric model) is the best understood in terms of the control of ovulation and PCO. It is given by a system of non-linear differential equations and assumes the same growth rate for each follicle. This assumption is biologically implausible and leads the model to exhibit unrealistic behaviour in some cases. A non symmetric generalisation is therefore developed and Lacker's theoretical analysis of the symmetric model is extended to this case. The non-symmetric model exhibits behavior which more closely reflects that observed in PCO. The thesis then goes on to present a theoretical and numerical analysis of another version of the symmetric model which has been proposed by Mariana et al. This incorporates a variable representing the ageing of the follicle in the same framework as that of Lacker's original model. Finally, all of the above models use a somewhat arbitrary function to describe a follicle's sensitivity to hormonal stimulation. In order to provide a more biologically motivated basis for our analysis we therefore develop a model in terms of the gonadotropic receptors of follicular cells. It is believed that the degree of sensitivity of a follicle to pituitary hormones is one of the factors determining its selection. This model is studied using numerical techniques, since its mathematical structure is too complicated to allow a theoretical analysis. Tentative conclusions underlying the mechanisms that select the ovulatory follicle are given in terms of the different models described in this thesis. Some of these are rather speculative due to the greatly simplified nature of the models in comparison to the real biological system. Nevertheless, since the behaviour of the models is qualitatively consistent with the results obtained from experimental data, they provide useful insights into the mechanisms that control the ovulation number in mammals

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 metabolomic

Atresia of ovarian follicles in fishes, and implications and uses in aquaculture and fisheries

Atresia of ovarian follicles, that is the degenerative process of germ cells and their associated somatic cells, is a complex process involving apoptosis, autophagy and heterophagy. Follicular atresia is a normal component of fish oogenesis and it is observed throughout the ovarian cycle, although it is more frequent in regressing ovaries during the postspawning period. An increased occurrence of follicular atresia above physiological rates reduces fish fecundity and even causes reproductive failure in both wild and captive-reared fish stocks, and hence, this phenomenon has a wide range of implications in applied sciences such as fisheries and aquaculture. The present article reviews the available literature on both basic and applied traits of oocyte loss by atresia, including its morpho-physiological aspects and factors that cause a supraphysiological increase of follicular atresia. Finally, the review presents the use of early follicular atresia identification in the selection process of induced spawning in aquaculture and the implications of follicular atresia in fisheries management

Cell-Kinetics Based Calibration of a Multiscale Model of Structured Cell Populations in Ovarian Follicles

International audienceIn this paper, we present a strategy for tuning the parameters of a multiscale model of structured cell populations in which physiological mechanisms are embedded into the cell scale. This strategy allows one to cope with the technical difficulties raised by such models, that arise from their anchorage in cell biology concepts: localized mitosis, progression within and out of the cell cycle driven by time-and possibly unknown-dependent, and nonsmooth velocity coefficients. We compute different mesoscopic and macroscopic quantities from the microscopic unknowns (cell densities) and relate them to experimental cell kinetic indexes. We study the expression of reaching times corresponding to characteristic cellular transitions in a particle-like reduction of the original model. We make use of this framework to obtain an appropriate initial guess for the parameters and then perform a sequence of optimization steps subject to quantitative specifications. We finally illustrate realistic simulations of the cell populations in cohorts of interacting ovarian follicles. Introduction. In this paper, we deal with the question of the numerical calibration of an existing multiscale model of cell-structured populations in the physiological context of ovulation. This model was formulated as a system of weakly coupled, non conservative transport equations with controlled velocities and sink terms, where the unknowns are the cell densities in each follicle [9, 8]. A number of theoretical studies have established the well-posedness of the model [19], examined optimal control problems related to the ovulatory trajectories in the framework of hybrid optimal control theory [6], and studied the reachability of final states corresponding to either ovulatory or atretic cases in the framework of backwards reachable sets [8]. Implementation of the model in an efficient and reliable computing environment has involved the design of a finite-volume scheme dealing with the discontinuous coefficients [3], embedding this scheme within a dedicated adaptive mesh based on a multi-resolution approach [4], and implementing it on parallel architecture [2]. This has left the question of model calibration to biological specifications to be resolved. We have to face a generic, yet unsolved issue in parameter fitting for physiologically-oriented multiscale mathematical models: although mechanistic knowledge in molecular and cell biology is available on the lower scales, quantitative experimental data are rather available on the higher scales. In our case, the question is how to infer the parameters entering the microscopic functions (on the level of the follicular cells) from mesoscopic (on the level of the individual follicles, i.e. the number of follicular cells) or macroscopic (on the level of the populations of follicles) information. In addition, even on the macroscopic level, data remain rather scarce and are rarely obtained directly a

From implantation to birth: insight into molecular melatonin functions

Melatonin is a lipophilic hormone synthesized and secreted mainly in the pineal gland, acting as a neuroendocrine transducer of photoperiodic information during the night. In addition to this activity, melatonin has shown an antioxidant function and a key role as regulator of physiological processes related to human reproduction. Melatonin is involved in the normal outcome of pregnancy, beginning with the oocyte quality, continuing with embryo implantation, and finishing with fetal development and parturition. Melatonin has been shown to act directly on several reproductive events, including folliculogenesis, oocyte maturation, and corpus luteum (CL) formation. The molecular mechanism of action has been investigated through several studies which provide solid evidence on the connections between maternal melatonin secretion and embryonic and fetal development. Melatonin administration, reducing oxidative stress and directly acting on its membrane receptors, melatonin thyroid hormone receptors (MT1 and MT2), displays effects on the earliest phases of pregnancy and during the whole gestational period. In addition, considering the reported positive effects on the outcomes of compromised pregnancies, melatonin supplementation should be considered as an important tool for supporting fetal development, opening new opportunities for the management of several reproductive and gestational pathologies

A SURVEY OF HETEROLOGOUS EXPRESSION SYSTEMS FOR THE PRODUCTION OF BOVINE FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE

Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are glycoprotein gonadotropins from the anterior pituitary gland that are involved in reproductive functions. They are heterodimeric proteins that share a common α-subunit that is highly conserved amongst mammalian species, and is also conserved across this family of proteins. The β-subunit confers receptor specificity and is distinct. Recombinant gonadotropin expression would be an important advance for the bovine superovulation industry, as there are a number of disadvantages associated with the currently used pituitary-derived gonadotropins. For example, these pituitary products are contaminated with other proteins including hormones, exhibit batch-to-batch inconsistencies, and harbor the potential to spread disease-causing agents. Recombinant preparations can overcome these negative issues because they are potentially free of contaminating proteins. Further, recombinant gonadotropins can be produced so they have no potential to transmit harmful agents. Here, two heterologous systems, bacteria and yeast, have been surveyed for their utility in the production of recombinant bovine FSH and LH (bFSH and bLH, respectively). bFSH was tested using a rat ovarian weight gain assay to determine biological activity and preliminary results indicate that the bacterial-derived bFSH had activity. The activity of yeast-derived bFSH could not be determined as a result of complications with the rat assay. bLH was assayed using bovine granulosa cells treated in vitro with LH for one day, followed by gene transcript profile analysis to characterize receptor binding and activation. Both recombinant preparations reduced expression of FSH receptor transcript levels similar to pituitary LH, as expected. Both the pituitary LH and yeast LH decreased transcript expression of LH receptor by 7.0-fold and 2.9 fold (respectively), suggesting an autocrine down-regulatory effect. Notably, the bacterial preparation failed to yield similar results (1.2-fold decrease), potentially because of the differences in post-translational modifications that have been suggested to be important for protein bioactivity. Recombinant bLH was also used in a rat ovarian ascorbic acid depletion bioassay to determine the impact of the compound at the whole animal level. Preliminary data suggests there was an ovarian ascorbic acid increase with both pituitary and recombinant LH treatments, rather than the expected decrease. Interestingly, the vehicle control caused an ascorbic acid increase for unknown reasons. While both the bacterial bLH and yeast bLH treatments resulted in an overall increase in relative ascorbic acid content post-LH treatment, the concentrations were less than the vehicle-only control, suggesting an overall reduction possibly as a result of activity maintained by the recombinant protein

Cytokines and Ovulation in the Mouse Ovary

Ovulation has been hypothesized as an inflammatory process. Interleukin(IL)-1$\alpha$, IL-1$\beta$ and tumor necrosis factor(TNF)-$\alpha$ are potent cytokines produced from macrophages and various other cell types, and are pivotal components of inflammation. Although previous studies have investigated cytokine activities in the reproductive system, there is little information on their precise localization and activities during the periovulatory period. To investigate the role of cytokines in ovulation, experiments were designed to determine the immunohistochemical localization and time specific production of cytokines IL-1 and TNF-$\alpha$ using a mouse model at 36h, 12h, 6h, 2h before ovulation, and at 6h and 18h after ovulation in vivo. Isolated individual follicles in vitro were used to determine more precise roles of cytokines on follicular development, ovulation and steroidogenesis. From these studies it was found that (1) granulosa cells were the primary sites of IL-1$\alpha$ and TNF-$\alpha$ production from large antral follicles and preovulatory follicles in vivo, (2) production of IL-1$\alpha$ and TNF-$\alpha$ increased as ovulation neared, first appearing in the cumulus cells and expanding to antral and mural granulosa cells, (3) less intense staining of these cytokines in the theca layer of smaller follicles suggests that theca cells may contribute to the production of these cytokines to some extent, (4) but there was no IL-1$\beta$ production, (5) localized and temporal production of cytokines during the periovulatory period suggests precise regulation, (6) decrease of IL-1$\alpha$ in the ovary after gonadotropin injection determined by enzyme linked immunoadsorbent assay suggests that IL-1$\alpha$ production may be under the control of gonadotropins, (7) in follicle culture without bone marrow derived cells, granulosa cells were confirmed as the main source of cytokine production, (8) addition of IL-1$\alpha$ and TNF-$\alpha$ to follicles in culture tend to decrease estradiol production. In conclusion, immunoreactive cytokine production correlated positively with the periovulatory follicular development suggesting their role as ovulatory mediators. It requires further studies on what are the signals for the initiation and termination of cytokine production, how transcription and translation of these cytokines are regulated during the periovulatory period, and how they contribute to the ovulation

Cumulus cells and their extracellular matrix affect the quality of the spermatozoa penetrating the cumulus mass

Objective: To investigate the role of the cumulus cells and the cumulus matrix in affecting the penetrability, morphology, acrosome reaction, and motility of human spermatozoa penetrating the cumulus oophorus. Design: Controlled experimental laboratory study. Setting: University gynecology unit. Patient(s): Women undergoing assisted reproduction treatment and men visiting the subfertility clinics. Intervention(s): Human spermatozoa were allowed to penetrate through the cumulus oophorus and cell-depleted cumulus matrix in a capillary, and were treated with cumulus cell extract or hyaluronic acid. Main Outcome Measure(s): The morphology, acrosomal status, and motility of human spermatozoa were determined. Result(s): Fewer spermatozoa could penetrate the fresh cell-depleted matrix compared with intact cumulus oophorus. Spermatozoa that penetrated through the cumulus oophorus had higher percentages of normal morphology and acrosome reaction and had specific motility pattern. These effects were lost or reduced in the cell-depleted matrix that had been stored overnight. Hyaluronic acid, a main component of the cumulus matrix at concentration found in the cumulus oophorus, modulated sperm motility but did not affect spontaneous acrosome reaction. Cumulus cell extract did not affect sperm motility, but induced acrosome reaction. Conclusion(s): Both the cumulus matrix and the cumulus cells contribute to the effect of cumulus oophorus on spermatozoa penetrating through it. © 2009 American Society for Reproductive Medicine.postprin