Single-cell analysis of [Ca²⁺]i signalling in sub-fertile men:characteristics and relation to fertilization outcome

STUDY QUESTIONWhat are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+]i signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability?SUMMARY ANSWERSingle cell analysis of progesterone-induced (CatSper-mediated) [Ca2+]i showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate.WHAT IS KNOWN ALREADYStimulation with progesterone is a widely used method for assessing [Ca2+]i mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability.STUDY DESIGN, SIZE, DURATIONThis was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA).MAIN RESULTS AND THE ROLE OF CHANCEFor analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve).LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study and caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies

IVF, same-sex couples and the value of biological ties

Ought parents, in general, to value being biologically tied to their children? Is it important, in particular, that both parents be biologically tied to their children? I will address these fundamental questions by looking at a fairly new practice within IVF treatments, so-called IVF-with-ROPA ( Reception of Oocytes from Partner ), which allows lesbian couples to „share motherhood‟ with one partner providing the eggs while the other becomes pregnant. I believe that IVF-with-ROPA is, just like other IVF treatments, morally permissible; but here I argue that the increased biological ties which IVF-with-ROPA allows for do not have any particular value beside the satisfaction of a legitimate wish, because there is no intrinsic value in a biological tie between parents and children; further, I argue that equality within parental projects cannot be achieved by redistributing biological ties

Impact of contraception and IVF hormones on metabolic, endocrine, and inflammatory status

Assisted reproductive technologies (ART) represent commonly utilized management strategies for infertility with multifactorial causes (including genetically predisposed diseases). Amongst ART, in vitro fertilization (IVF) is the most popular. IVF treatment may predispose the mother to increased risks and complications during pregnancy, and there may be adverse fetal outcomes. Hormonal therapies, including oral contraceptives, may impair glucose and lipid metabolism, and promote insulin resistance and inflammation. IVF treatment involves administration of reproductive hormones, similar in composition but in much higher doses than those used for oral contraception. The provision of IVF reproductive hormones to mice associates with glucose intolerance. In addition, the physiological and hormonal changes of pregnancy can trigger an inflammatory response, and metabolic and endocrine changes. There is controversy regarding the potential effects of IVF hormonal therapies in the promotion of diabetogenic and inflammatory states, additional to those that occur during pregnancy, and which may therefore predispose women with IVF-conceived pregnancies to adverse obstetric outcomes compared with women with spontaneously conceived pregnancies. This review summarizes the limited published evidence regarding the effect of IVF-based fertility therapies on glucose homeostasis, insulin resistance, cardio-metabolic profile, and markers of inflammation

Breast Cancer Risk After Ovarian Stimulation for In Vitro Fertilization

The study by Dr van den Belt-Dusebout and colleagues1 investigated a debated aspect of reproductive medicine: breast cancer risk following ovarian stimulation for in vitro fertilization (IVF).2-5 The authors concluded that “these findings are consistent with absence of a significant increase in long-term risk of breast cancer among IVF-treated women”.1 However, some important points should be discussed. For about 23 % of women, sub-fertility diagnosis and number of IVF cycles were collected using a questionnaire since medical records were not available. This high rate threatens the reliability of results. It is not possible to compare a detailed report of official medical records with data deriving from subjective memory of treatments received many years before. This may be a strong bias, because reproductive medicine, IVF strategies and the pharmacological protocols have changed rapidly in the last decades. Dates of diagnosis and histology were reported but unfortunately not disease staging. It would be interesting to investigate if ovarian stimulation with the use of IVF techniques can promote the occurrence of biologically different types of breast cancer, as in the case of tamoxifen-related endometrial cancer, a neoplasia with better prognostic profile and outcome. Also, the authors reported that breast cancer risk decreased with more IVF cycles (7 or more compared with 1-2). They suggested as potential explanations that women treated with more IVF cycles received more hCG or had longer periods of down-regulation with low estradiol and progesterone levels, or the women requiring more IVF cycles were inherently different. It is difficult to provide a definitive conclusion since the clinical outcomes of IVF cycles were not reported. The decreased risk in women treated with many IVF cycles also could be related to the improvement of ovarian function after repeated endocrine stimulations. Infertility and infertility-related nulliparity must be considered as risk factors for breast cancer, and prolonged treatment of anovulatory or poor ovulatory cycles could be one approach for restoring normal ovarian activity and reducing breast cancer risk

Pre-implantation mouse embryos cultured In vitro under different oxygen concentrations show altered ultrastructures

Abstract Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2

Understanding the ethical concerns that have shaped European regulation of human embryonic stem cell research

Human embryonic stem cell research has generated much hope, but also fear and repulsion. National legislators, as well as the European Parliament, the European Patent Office and the European Court of Justice have had to make decisions relating to what is or is not allowed in the field of hESC research and patenting, and their decisions are often difficult to reconcile. In order to understand this divergence and the specific restrictions that different regulators impose, insight is needed into the different opinions regarding the moral status of the pre-implantation embryo (blastocyst), into the moral distinction between using IVF embryos donated for research versus creating embryos for research purposes, and into the moral distinction between producing and using hESC lines for non-commercial research and allowing such production and research in a commercial or industrial setting. While one need not agree that all of these perceived differences are in fact morally relevant, knowing that many people perceive them as being relevant is in itself valuable for understanding the debate and the decisions that different regulators make

IVF Battles: Legal Categories and Comparative Tales

Coupled with modern reproductive technologies, the ancient desire for parenthood has led to novel legal challenges. This essay discusses landmark cases addressing those challenges. At the outset, it distinguishes between two litigation paradigms in this area—termed “horizontal” and “vertical.” Horizontal controversies involve private parties who have different aspirations regarding a joint parenthood project (e.g., between two partners who began an IVF procedure and later disagree whether to complete the process). In contrast, vertical controversies concern clashes between an individual (or individuals) and the state, such as when the state or one of its authorities does not allow the individual to move forward with technologies that may lead to parenthood (e.g., new surrogacy procedures), though all affected individuals consent. The essay then focuses on horizontal litigation, and examines the ways in which various legal systems draw on, and sometimes adjust to the particular circumstances of the case, traditional concepts such as contract, reliance, property, and more to resolve such disputes

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations