Risk of genetic and epigenetic alteration in children conceived following ART: Is it time to return to nature whenever possible?

Assisted reproductive technology may influence epigenetic signature as the procedures coincide with the extensive epigenetic modification occurring from fertilization to embryo implantation. However, it is still unclear to what extent ART alters the embryo epigenome. In vivo fertilization occurs in the fallopian tube, where a specific and natural environment enables the embryo's healthy development. During this dynamic period, major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Over the past decade, concerns relating to the raised incidence of epigenetic anomalies and imprinting following ART have been raised by several authors. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period; therefore, unphysiological conditions, including ovarian stimulation, in vitro fertilization, embryo culture, cryopreservation of gametes and embryos, parental lifestyle, and underlying infertility, have the potential to contribute to epigenetic dysregulation independently or collectively. This review critically appraises the evidence relating to the association between ART and genetic and epigenetic modifications that may be transmitted to the offspring.

Review of human oocyte cryopreservation in ART programs: Current challenges and opportunities.

Oocyte cryopreservation has notably increased in recent times, to become an essential part of clinical infertility treatment. Since the 1980s, many improvements in oocyte cryopreservation (OC) have been adopted, including the great advance with the application of vitrification. The commonly used vitrification protocol applies different cryoprotectants (Ethylene glycol and/or DMSO and/or PROH and sucrose and/or Trehalose) and two different steps: firstly, exposure in equilibration solution for 5-15 min, followed by a vitrification solution for 60-90 s at room temperature. The warming method includes a first step for 1 min at 37 °C and 3 subsequent steps at room temperature to remove the cryoprotectant for a total of 9-12 min. In addition, biosafety is a critical aspect to mention, and it is related to devices used during the vitrification, mainly in terms of whether the biological vitrified material comes in direct contact with liquid nitrogen (open vitrification) or not (closed vitrification), where LN2 may contain potentially contaminating viruses or pathogens. Furthermore, during early development major waves of epigenetic reprogramming take place. Recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, including osmotic shock, temperature, rapid changes of pH and toxicity of cryoprotectants. It is, therefore, important to better understand the potential perturbations of epigenetic modifications that may be associated with the globally used vitrification methods. Therefore, we here discuss the benefits and efficiency of human oocyte vitrification; we also review the evidence surrounding oocyte cryopreservation-related epigenetic modifications and potential epigenetic dysregulations, together with long-term consequences for offspring health.

The artificial uterus: on the way to ectogenesis.

The inability to support the growth and development of a mature fetus up to delivery results in significant human suffering. Current available solutions include adoption, surrogacy, and uterus transplantation. However, these options are subject to several ethical, religious, economic, social, and medical concerns. Ectogenesis is the process in which an embryo develops in an artificial uterus from implantation through to the delivery of a live infant. This current narrative review summarizes the state of recent research focused on human ectogenesis. First, a literature search was performed to identify published reports of previous experiments and devices used for embryo implantation in an extracorporeally perfused human uterus. Furthermore, studies fitting that aim were selected and critically evaluated. Results were synthesized, interpreted, and used to design a prospective strategy for future research. Therefore, this study suggests that full ectogenesis might be obtained using a computer-controlled system with extracorporeal blood perfusion provided by a digitally controlled heart-lung-kidney system. From a clinical perspective, patients who will derive significant benefits from this technology are mainly those women diagnosed with anatomical abnormalities of the uterus and those who have undergone previous hysterectomies, numerous abortions, and experienced premature birth. Ectogenesis is the complete development of an embryo in an artificial uterus. It represents the solutions for millions of women suffering from premature deliveries, and the inability to supply growth and development of embryos/fetuses in the womb. In the future, ectogenesis might replace uterine transplantation and surrogacy.

Real-time image and time-lapse technology to select the single blastocyst to transfer in assisted reproductive cycles.

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

Exploring the effect of cryopreservation in assisted reproductive technology and potential epigenetic risk.

Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.

Culture conditions in the IVF laboratory: state of the ART and possible new directions.

In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

The Polymorphism Asn680Ser on the FSH Receptor and Abnormal Ovarian Response in Patients with Normal Values of AMH and AFC.

After the controlled ovarian stimulation (COS), the number of cumulus oocyte complexes collected is lower than predicted. The aim of this study is to understand if there is a possible reason for that deficient ovarian response. It was hypothesized that this is associated with the SNP (single-nucleotide polymorphism) of the FSH receptor (FSHr), specifically c.2039A > G, resulting in Asn680Ser. Two groups of patients were enrolled for this purpose: the normal (n = 36) and abnormal responses (n = 31). To predict the number of retrievable oocytes, according to the anti-Mullerian hormone (AMH) and the antral follicle count (AFC), the following formula was applied in a log scale: the number of oocytes retrieved = 2.584 − 0.015 × (age) − 0.035 × (FSH) + 0.038 × (AMH) + 0.026 × (AFC). Then, when the number of oocytes collected was less than 50% of the calculated value, it was proposed that the patients result in an abnormal response. DNA sample blood was collected from the women, and then the genetic assessment for the Asn680Ser of the FSHr was evaluated in both groups. The differences between the two categories were statistically analyzed with an independent samples t test, a Mann−Whitney U test and a Chi-squared test. In a patient with an abnormal response, a significant prevalence of the amino acid serine at position 680 of the FSHr compared to the counterpart group (p < 0.05) was detected. In conclusion, according to the results, the genetic evaluation of the FSHr could represent an accurate and predictive feature for patients undergoing assisted reproductive technology treatment.

Non-invasive oocyte quality assessment.

Oocyte quality is perhaps the most important limiting factor in female fertility; however, the current methods of determining oocyte competence are only marginally capable of predicting a successful pregnancy. We aim to review the predictive value of non-invasive techniques for the assessment of human oocytes and their related cells and biofluids that pertain to their developmental competence. Investigation of the proteome, transcriptome, and hormonal makeup of follicular fluid, as well as cumulus-oocyte complexes are currently underway; however, prospective randomized non-selection-controlled trials of the future are needed before determining their prognostic value. The biological significance of polar body morphology and genetics are still unknown and the subject of debate. The predictive utility of zygotic viscoelasticity for embryo development has been demonstrated, but similar studies performed on oocytes have yet to be conducted. Metabolic profiling of culture media using human oocytes are also limited and may require integration of automated, high-throughput targeted metabolomic assessments in real time with microfluidic platforms. Light exposure to oocytes can be detrimental to subsequent development and utilization of time-lapse imaging and morphometrics of oocytes is wanting. Polarized light, Raman microspectroscopy, and coherent anti-Stokes Raman scattering are a few novel imaging tools that may play a more important role in future oocyte assessment. Ultimately, the integration of chemistry, genomics, microfluidics, microscopy, physics, and other biomedical engineering technologies into the basic studies of oocyte biology, and in testing and perfecting practical solutions of oocyte evaluation, are the future for non-invasive assessment of oocytes.

Effects of mobile phone radiofrequency radiation on sperm quality.

In the last decades, the universal use of mobile phones has contributed to radiofrequency electromagnetic radiation environmental pollution. The steady growth in mobile phone usage has raised concerns about the effects of phone radiation on male reproductive health. Epidemiological studies report a sharp decline in sperm counts in developing countries, and worldwide with c. 14% of couples having difficulties to conceive, many of which are attributed to a male infertility factor. Environment and lifestyle factors are known to contribute to male infertility. Exposure to heat, radiation, or radioactivity might induce damage to biological tissue organs, including the testis. Given the ubiquitous use of mobile phones, the potential adverse effects of the resulting environmental radiation needs to be elucidated further. It seems to be an apparent relationship between the increased exposure to mobile phone radiofrequency and sperm quality decline, but the evidence is not conclusive. Our review summarizes the evidence concerning the possible adverse effects of cell phone radiation on the male reproductive system, with a focus on sperm quality. Also, we critically analyze the effects of elevated testicular temperature and oxidative stress on male fertility and how these factors could interfere with the physiological activities of the testis.

Impact of obesity on medically assisted reproductive treatments.

Increasing evidence has demonstrated that obesity impairs female fertility and negatively affects human reproductive outcome following medically assisted reproduction (MAR) treatment. In the United States, 36.5% of women of reproductive age are obese. Obesity results not only in metabolic disorders including type II diabetes and cardiovascular disease, but might also be responsible for chronic inflammation and oxidative stress. Several studies have demonstrated that inflammation and reactive oxygen species (ROS) in the ovary modify steroidogenesis and might induce anovulation, as well as affecting oocyte meiotic maturation, leading to impaired oocyte quality and embryo developmental competence. Although the adverse effect of female obesity on human reproduction has been an object of debate in the past, there is growing evidence showing a link between female obesity and increased risk of infertility. However, further studies need to clarify some gaps in knowledge. We reviewed the recent evidence on the association between female obesity and infertility. In particular, we highlight the association between fat distribution and reproductive outcome, and how the inflammation and oxidative stress mechanisms might reduce ovarian function and oocyte quality. Finally, we evaluate the connection between female obesity and endometrial receptivity.

Review of the impact of COVID-19 on male reproduction, and its implications on assisted reproductive technology services.

The announcement in 2019 of a new coronavirus disease that quickly became a major pandemic, is an exceptional challenge to healthcare systems never seen before. Such a public health emergency can largely influence various aspects of people's health as well as reproductive outcome. IVF specialists should be vigilant, monitoring the situation whilst contributing by sharing novel evidence to counsel patients, both pregnant women and would-be mothers. Coronavirus infection might adversely affect pregnant women and their offspring. Consequently, this review paper aims to analyse its potential risks for reproductive health, as well as potential effects of the virus on gamete function and embryo development. In addition, reopening fertility clinics poses several concerns that need immediate addressing, such as the effect of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) on reproductive cells and also the potential risk of cross-contamination and viral transmission. Therefore, this manuscript summarizes what is currently known about the effect of the SARS-CoV-2 infection on medically assisted reproductive treatments and its effect on reproductive health and pregnancy.

Progesterone: The Key Factor of the Beginning of Life.

Progesterone is the ovarian steroid produced by the granulosa cells of follicles after the LH peak at mid-cycle. Its role is to sustain embryo endometrial implantation and ongoing pregnancy. Other biological effects of progesterone may exert a protective function in supporting pregnancy up to birth. Luteal phase support (LPS) with progesterone is the standard of care for assisted reproductive technology. Progesterone vaginal administration is currently the most widely used treatment for LPS. Physicians and patients have been reluctant to change an administration route that has proven to be effective. However, some questions remain open, namely the need for LPS in fresh and frozen embryo transfer, the route of administration, the optimal duration of LPS, dosage, and the benefit of combination therapies. The aim of this review is to provide an overview of the uterine and extra-uterine effects of progesterone that may play a role in embryo implantation and pregnancy, and to discuss the advantages of the use of progesterone for LPS in the context of Good Medical Practice.

Focus on time-lapse analysis: blastocyst collapse and morphometric assessment as new features of embryo viability.

The main goal of assisted reproductive technology (ART) is to achieve a healthy singleton live birth after the transfer of one embryo. A major objective of IVF scientists has always been to use adequate criteria for selecting the embryo for transfer according to its implantation potential. Indeed, embryo quality is usually assessed by evaluating visual morphology, which relies on the removal of the embryo from the incubator and might include inter- and intra-evaluator variation among embryologists. Recently, an advancement in embryo culture has taken place with the introduction of a new type of incubator with an integrated time-lapse monitoring system, which enables embryologists to analyse the dynamic events of embryo development from fertilization to blastocyst formation. This novel practice is rapidly growing and has been used in many IVF centres worldwide. Therefore, the main aim of this review is to present the benefits of time-lapse monitoring in a modern embryology laboratory; in particular, we discuss blastocyst collapse and morphometric blastocyst assessment, and analyse their association with embryo viability and implantation potential. In addition, we highlight preliminary studies involving artificial intelligence and machine learning models as non-invasive markers of clinical pregnancy.

Use of time-lapse monitoring in medically assisted reproduction treatments: a mini-review.

During human in vitro culture, a morphological microscope analysis is normally performed to select the best embryo to transfer, with the hope of obtaining a successful pregnancy. The morphological evaluation may combine number and size of blastomeres, fragmentation, multinucleation, blastocyst expansion, inner-cell mass and trophectoderm appearance. However, standard microscopy evaluation involves the removal of the embryos from the incubator, exposing them to changes in pH, temperature, and oxygen level. Additionally, morphological assessments might include high inter-observer variability. Recently, continuous embryo culture using time-lapse monitoring (TLM) has allowed embryologists to analyse the dynamic and morphokinetic events of embryo development and, based on that, the embryologist is able to scrutinize the complete sequence of embryonic evolution, from fertilization to the blastocyst formation. Therefore, TLM allows an uninterrupted culture condition, reducing the need to remove embryos from the incubator. The monitoring system is normally composed of a standard incubator with an integrated microscope coupled to a digital camera, which is able to collect images at regular times, and subsequently processed into video. These data can be annotated and analyzed using an integrated software, therefore this allows embryologists to facilitate the process of embryo selection for transfer. The main aim of this paper is to discuss the potential benefits and uses of the TLM in the embryology laboratory.

Live birth and clinical outcome of vitrification-warming donor oocyte programme: an experience of a single IVF unit.

Medically assisted reproductive (MAR) treatments using donated oocytes are commonly applied in several countries to treat women who cannot conceive with their own gametes. Historically, in Italy, gamete donation has been prohibited but, in 2014, the law changed and gamete donation became allowed for couples undergoing MAR treatments. Consequently, in the last decade, there has been an increase in application of the oocyte donation programme. This study reports an egg-donation programme's clinical efficacy, based on importing donated vitrified oocytes from cryo-banks located in a foreign country. For this, we conducted a retrospective analysis of data from a single reproductive unit located in Italy (Donna Salus Women's Health and Fertility, Bozen). The study group consisted of 681 vitrified oocytes, which were warmed and culture to be replaced in 100 recipients. The survival rate after warming was 79.1% (n = 539/681), whereas the fertilization and blastulation rates were 90.2% (n = 486/539) and 47.9% (n = 233/486), respectively. Positive pregnancy test, clinical pregnancy rates, and live-birth rates per embryo transfer were 37.8%, 31.1% and 28.4%, respectively. The multiple pregnancy rate was 0.7%. This study is one of the first to report on the efficacy of a donor oocyte programme in Italy using imported vitrified oocytes. The above data may reassure women who are undertaking donation programmes using vitrified oocytes imported from commercial egg banks.

Clinical pregnancy is significantly associated with the blastocyst width and area: a time-lapse study.

In order to maintain pregnancy rates following single embryo transfer, optimisation of embryo culture and selection is vital. Time-lapse monitoring (TLM) has the potential to play a crucial role by providing sequential images of embryo development and minimal disturbance. Therefore, in this study morphometric assessment of blastocyst area and maximum width was performed in order to evaluate if these parameters are associated with pregnancy outcomes in IVF/ICSI cycles. This is a retrospective study of 664 patients who had elective single blastocyst transfer (eSBT). The EmbryoScope drawing tools were used to measure specific variables such as the maximum blastocyst width and blastocyst area. Our results show that women who were pregnant had significantly (P < 0.01) larger blastocyst width [median (range) µm] 184 (125-239) versus non-pregnant, 160 (120-230)] and area [median (range) µm2] 26099 (12101-45,280) versus non-pregnant women, 22,251 (10992-37,931)]. A univariate logistic regression performed showed that blastocyst width [(OR = 1.026, 95% CI = (1.019, 1.033)] was significant (P < 0.01) and for every µm increase of blastocyst width, the odds of clinical pregnancy increase by 2.6%. A univariate logistic regression performed showed that blastocyst area [(OR = 1.00008, 95% CI = (1.00006, 1.00011)] was significant with P < 0.01. For every µm2 increase of blastocyst area, our data showed the odds of clinical pregnancy increase by 0.008%. Hosmer-Lemeshow tests of calibrations were performed to verify calibration. Although our findings show a clear correlation between blastocyst dimensions and the clinical pregnancy rate, further studies are necessary to confirm these observations.

PGT-A preimplantation genetic testing for aneuploidies and embryo selection in routine ART cycles: Time to step back?

Embryo aneuploidies may be responsible for implantation failures, miscarriages and affects IVF outcomes. A variety of technologies have been implemented to individuate euploid embryos in IVF treatments, which is named preimplantation genetic testing for aneuploidies (PGT-A). According to this strategy, a better embryo selection should increase IVF results. In reality, several issues remain unaddressed including the sampling strategy, involving the test outcomes, and the frequent occurrence of embryo mosaicism, affecting the criteria for selection of supposed viable embryos and possibly posing an ethical dilemma. Safety issues are in place, including perinatal and postnatal consequences of embryo sampling and the epigenetic weaknesses from a prolonged in vitro culture, necessary for trophectoderm biopsy. On the other side, chromosome number mistakes are progressively recognized as physiologic events in the early pre-implantation embryo with many corrective mechanisms in place and their destiny in the post-implantation development is unclear. Accordingly, the increasing precision of the diagnostic tools should be used to investigate the effect of such interventions within rigorous research programs in the sake of improved clinical outcomes. Meantime the diagnosis of embryo aneuploidies in IVF cycles should be considered as a research tool and systematic implementation in clinical practice may appear unjustified.

Clinical utility of freeze-all approach in ART treatment: A mini-review.

A significant proportion of couples at reproductive age rely on assisted reproductive technology to overcome infertility. In vitro fertilisation (IVF) involves typically the use of exogenous gonadotropins to stimulate the ovary to produce oocytes, which are collected surgically. After fertilization by conventional IVF or intracytoplasmic sperm injection (ICSI), embryos are cultured in the embryology laboratory for a few days before being replaced into the uterus (fresh embryo transfer). Spare embryos can be vitrified and stored in liquid nitrogen to be transferred in a subsequent cycle. Over the years, concerns have arisen about possible adverse outcomes of transferring embryos back to the uterus immediately after controlled ovarian stimulation (COS) as regards to obstetrical and perinatal outcomes. It has been suggested that high hormonal levels during COS could create a relatively hostile environment for embryo implantation whilst increasing the risk of ovarian hyperstimulation syndrome (OHSS). With the remarkable improvement of vitrification as an alternative to the slow-freezing technique for human embryos, a new strategy the so-called "freeze-all" (FA) or "elective frozen embryo transfer" (eFET) was introduced. This approach involves COS, followed by the elective cryopreservation of the entire cohort of viable embryos to be transferred to the uterus in subsequent cycles in a possibly more physiological environment, thus avoiding the supra-physiologic hormonal levels observed during COS. The initial reports suggested that this policy could lead to improved pregnancy rates and reduced perinatal complications, which resulted in a steady increase and widespread use of FA globally. However, as data accumulated, it became clear that the use of FA to unselected couples undergoing ART offered no additional benefits over the conventional approach. Nonetheless, current evidence based on randomized controlled trials and observational studies indicates that FA might be justified in selected clinical scenarios, such as those involving the risk of OHSS. By contrast, there is a lack of evidence to support the FA policy for other indications, such as implantation failure or high progesterone levels on the trigger day. This review summarizes the clinical effectiveness of FA with the main focus on the health of offspring.

Fertility preservation and preimplantation genetic assessment for women with breast cancer.

Breast cancer is the most common cancer diagnosed among reproductive aged women, and its treatment can compromise future fertility. Options for fertility preservation include oocyte or embryo cryopreservation after ovarian stimulation (OS), which are the most established choices and are applicable for adult women with cancer. Ovarian tissue freezing may also be appropriate, as it offers potentially the least delay. The recognisation of the role of BRCA1 and BRCA2 mutations in some women has led to the involvement of preimplantation genetic diagnosis (PGD), recently renamed preimplantation genetic testing for monogenic disorder (PGT-M), whereby embryos are created by IVF and cell(s) are removed and genetically analyzed for specific disease-related mutations. PGT-M offers a valid option for women wishing to avoid transmission of the predisposition for hereditary breast cancer to their offspring. The aim of this paper is to provide an overview of the factors that influence fertility preservation in newly diagnosed breast cancer patients, and to illustrate the option of PGT-M to enable conception of an unaffected child.

Cryopreservation of human embryos and oocytes for fertility preservation in cancer and non cancer patients: a mini review.

The term 'cryopreservation' illustrates the process of freezing cells and storing at very low temperature in liquid nitrogen (-196 °C). Cooling is not a physiological condition for human cells especially due to the high concentration of water in the living matter, whose conversion to ice crystals may be associated with cell death. Human oocytes are particularly sensitive to the freezing process, primarily because of their large size and the presence of the meiotic spindle, which at low temperature can degenerate. In the last decade, the cryopreservation technology has become highly important as an option for fertility preservation (FP) in women with cancer. Anticancer therapy might promote premature ovarian failure and negatively affects the reproductive outcome. Over the years, scientists have proposed different cryopreservation strategies in the effort to maintain the physiological functions of oocytes and embryo. However, despite the first success obtained in the 1980s with frozen oocytes, it was not until recently that a new approach has been proposed: the 'Vitrification' which allowed a breakthrough in this procedure. FP is a major determinant for cancer survivor women in the reproductive age. This article describes the FP options currently available, focusing mainly on oocyte and embryo cryopreservation.