Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

Morphological assessment of oocyte quality during assisted reproductive technology cycle.

Following the advancement of medically assisted reproduction (MAR) technology, and the rationale to extend the culture to the blastocyst stage, performing elective single embryo transfer (eSET), gamete quality and assessment have acquired large relevance in ART. Embryo quality is strictly correlated with gametes quality and culture conditions. Oocyte maturity assessment is therefore imperative for fertilization and embryo evolution. Mature oocytes at the metaphase II stage result in a higher fertilization rate compared to immature oocytes. Indeed, oocyte morphology evaluation represents an important and challenging task that may serve as a valuable prognostic tool for future embryo development and implantation potential. Different grading systems have been reported to assess human embryos, however, in many cases, it is still a major challenge to select the single embryo to transfer with the highest implantation potential. Further, eSET has conferred a challenge to embryologists, who must try to enhance embryo culture and selection to provide an adequate success rate, whilst reducing the overall number of embryos transferred. Above the standard morphological assessment, there are several invasive or non-invasive approaches for embryo selection such as preimplantation genetic testing, time-lapse technology, proteomics and metabolomics, as well as oxygen utilization and analysis of oxidative stress in culture medium. This short review is not designed to be a comprehensive review of all possible features that may influence oocyte quality. It does give, however, a brief overview and describes the prognostic value of the morphological characteristics of human oocytes on their developmental capacity following ART treatments.

Decrease in Sperm Parameters in the 21st Century: Obesity, Lifestyle, or Environmental Factors? An Updated Narrative Review.

Semen quality represents a compelling factor for fertility, and delineating the normal values has proven difficult. In the last four decades, several authors have reported a noticeable decline in sperm parameters. Also, studies investigating 'time to pregnancy' have shown that fecundity begins to be reduced when sperm numbers decrease below 30 million, even though according to the 6th edition of the WHO manual, the normal value is currently 16 million/mL or 39 million per ejaculate. There exists sufficient data to suggest a decline in sperm counts over time, even though the clear reason for this adverse trend is not well established, but some associations have been hypothesised, such as maternal smoking during pregnancy. Additional potential factors have yet to be fully illustrated but involve poor diet, increased obesity, and exposure to environmental toxins. Moreover, the change in environmental conditions and more common exposure to endocrine-disrupting chemicals (EDCs), such as pesticides and herbicides, as well as bisphenol A, phthalates, polychlorinated biphenyls, and heavy metals, starting from prenatal life and continuing into adulthood, may exhibit probable features explaining the reduction in sperm parameters. Therefore, the main goal of this narrative review is to furnish an overview of the possible effects of exposure to EDCs on testicular function and spermatogenesis and, also, to summarise the evidence regarding a decrease in sperm quality and examine its potential consequences.

Intracytoplasmic sperm injection vs. in-vitro fertilization in couples in whom the male partners had a semen analysis within normal reference ranges: An open debate.

During recent decades, the application of intracytoplasmic sperm injection has increased considerably worldwide, especially in couples with non-male factor infertility. However, several studies analyzing the broad use of intracytoplasmic sperm injection, even in cases with a normal semen analysis, have collectively demonstrated no benefits compared to conventional in-vitro fertilization. Currently, there is insufficient evidence to support the intracytoplasmic sperm injection technique vs. in-vitro fertilization in cases of poor ovarian response or a low number of oocytes collected, or in patients with advanced maternal age. Since the intracytoplasmic sperm injection technique is more operator-dependent and invasive, its use should only be recommended in cases of male-factor infertility. There is some evidence showing that intracytoplasmic sperm injection is linked with an increased risk of birth defects. Albeit this evidence is limited, and currently it is not possible to draw a firm conclusion on these concerns, we do believe that these risks should be rigorously investigated. Thus, this review aims to clarify the debate on the application of the intracytoplasmic sperm injection procedure, as compared to standard in-vitro fertilization, in those assisted reproductive technology cycles without a clear male factor infertility. Furthermore, we try to clarify whether intracytoplasmic sperm injection would result in a higher live birth rate than in-vitro fertilization, in couples with non-male factor infertility.

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.

Exploring the Impact of Controlled Ovarian Stimulation and Non-Invasive Oocyte Assessment in ART Treatments.

Invasive and noninvasive features are normally applied to select developmentally competent oocytes and embryos that can increase the take-home baby rates in assisted reproductive technology. The noninvasive approach mainly applied to determine oocyte and embryo competence has been, since the early days of IVF, the morphological evaluation of the mature cumulus-oocyte complex at the time of pickup, first polar body, zona pellucida thickness, perivitelline space and cytoplasm appearance. Morphological evaluation of oocyte quality is one of the options used to predict successful fertilization, early embryo development, uterine implantation and the capacity of an embryo to generate a healthy pregnancy to term. Thus, this paper aims to provide an analytical revision of the current literature relating to the correlation between ovarian stimulation procedures and oocyte/embryo quality. In detail, several aspects of oocyte quality such as morphological features, oocyte competence and its surrounding environment will be discussed. In addition, the main noninvasive features as well as novel approaches to biomechanical parameters of oocytes that might be correlated with the competence of embryos to produce a healthy pregnancy and live birth will be illustrated.

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.

Inadvertent Administration of 72 µg of Follitropin-Δ for Three Consecutive Days Does Not Appear to Be Dangerous for Poor Responders: A Case Series.

Follitropin delta (Δ) is a recombinant human follicle-stimulating hormone (rFSH), like natural human FSH, that can stimulate the development and growth of multiple follicles. Treatment with Follitropin-Δ may cause mild to severe adverse reactions, such as the risk of developing ovarian hyperstimulation syndrome, resulting in nausea, vomiting and diarrhea, weight loss, respiratory difficulty, stomach swelling and discomfort of the pelvic area, headaches, and fatigue. To date, the effects of a Follitropin-Δ overdosage are unknown, and no data are reported in the scientific literature or in the drug data sheet. Therefore, this study aimed to describe the effects of Follitropin-Δ overdosages in poorly responding women who underwent IVF cycles. This is a descriptive case series of four nulligravid, poorly responding patients, two of whom made requests for fertility preservation. Four poorly responding patients who were prescribed 20.0 µg/day of Follitropin-Δ for three consecutive days wrongly injected the total cartridge of 72 µg Follitropin-Δ every day. After the incorrect injection of Follitropin-Δ, the patients continued their controlled ovarian stimulation and underwent vaginal ovarian pick up. The analyzed patients had no side effects or adverse reactions. The evaluations reported in this case series showed that the accidental use of 72 µg/day of Follitropin-Δ for three days did not cause side effects or adverse reactions in poor responders.

Can Cryopreservation in Assisted Reproductive Technology (ART) Induce Epigenetic Changes to Gametes and Embryos?

Since the birth of Louise Brown in 1978, more than nine million children have been conceived using assisted reproductive technologies (ARTs). While the great majority of children are healthy, there are concerns about the potential epigenetic consequences of gametes and embryo manipulation. In fact, during the preimplantation period, major waves of epigenetic reprogramming occur. Epigenetic reprogramming is susceptible to environmental changes induced by ovarian stimulation, in-vitro fertilization, and embryo culture, as well as cryopreservation procedures. This review summarizes the evidence relating to oocytes and embryo cryopreservation and potential epigenetic regulation. Overall, it appears that the stress induced by vitrification, including osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, might induce epigenetic and transcriptomic changes in oocytes and embryos. It is currently unclear if these changes will have potential consequences for the health of future offspring.

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.

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.

A Multi-Institutional Informed Consent Proposal as a Prevention Tool for Combined Oral Contraceptive Intake and Thrombotic Risk.

Combined oral contraceptives (COC), are among the most widely used contraceptive methods in the world today. Despite the different changes in terms of estrogen/progestogen combinations and dosages, the thromboembolic risk for a woman who takes combined oral contraceptives persists to date. METHODS: The review of relevant literature and international guidelines on prescription of combined oral contraceptives made it possible to create a proposal for informed consent to be used for prescribing. RESULTS: The several sections of our consent proposal were designed according to a rationale in order to cover all the aspects presented by worldwide guidelines: how to take, adverse effects, advertisements, extra-contraceptive benefits and effects, a checklist for condition at risk of thromboembolism, the signature of the woman. CONCLUSIONS: An informed consent to standardize combined oral contraceptives prescription can improve women's eligibility, mitigate thromboembolic risk, and assure legal protection to healthcare providers. In this systematic review in particular, we refer to the Italian medical-legal scenario, to which our group of researchers belongs. However, the model proposed was designed in the respect of main healthcare organization guidelines, and it could be easily used by any center in the world.

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 potential impact of human papillomavirus on infertility and assisted reproductive technology outcomes.

Human papillomavirus (HPV) is a common sexually transmitted disease that has been linked to both cancer and reproductive health issues. While its impact on fertility and pregnancy success has been studied, there is still too little evidence about the influence of HPV on assisted reproductive technology (ART). Therefore, there exists a need for HPV testing in couples undergoing infertility treatments. Infertile men have been found to have a higher prevalence of seminal HPV infection, which can compromise sperm quality and reproductive function. As such, it could be important to investigate the correlation between HPV and ART outcomes in order to improve the quality of evidence. Understanding the potentially detrimental effects of HPV on ART outcomes may have promising important implications for the management of infertility. This minireview summarizes the so far limited developments in this area and highlights the major need for further well-designed studies to address this issue.

Total fertilization failure after ICSI: insights into pathophysiology, diagnosis, and management through artificial oocyte activation.

BACKGROUND: Total fertilization failure (TFF) is the failure of all metaphase II oocytes to fertilize in ART cycles. The phenomenon represents a known cause of infertility, affecting 1-3% of ICSI cycles. Oocyte activation deficiency (OAD) is the leading cause of fertilization failure, attributed to sperm- or oocyte-related issues, although until recently little attention has been given to oocyte-related deficiencies. Different strategies for overcoming TFF have been proposed in clinical settings, mainly using artificial oocyte activation (AOA) by calcium ionophores. Typically, AOA has been blindly applied with no previous diagnosis testing and, therefore, not considering the origin of the deficiency. The scarcity of data available and the heterogeneous population subjected to AOA make it challenging to draw firm conclusions about the efficacy and safety of AOA treatments. OBJECTIVE AND RATIONALE: TFF leads to an unexpected, premature termination of ART, which inflicts a substantial psychological and financial burden on patients. This review aims to provide a substantial update on: the pathophysiology of fertilization failure, focusing both on sperm- and oocyte-related factors; the relevance of diagnostic testing to determine the cause of OAD; and the effectiveness and safety of AOA treatments to overcome fertilization failure. SEARCH METHODS: Relevant studies were identified in the English-language literature using PubMed search terms, including fertilization failure, AOA, phospholipase C zeta (PLCζ), PLCZ1 mutations, oocyte-related factors, wee1-like protein kinase 2 (WEE2) mutations, PAT1 homolog 2 (PATL2) mutations, tubulin beta-8 chain (TUBB8) mutations, and transducin-like enhancer protein 6 (TLE6) mutations. All relevant publications until November 2022 were critically evaluated and discussed. OUTCOMES: Fertilization failure after ART has been predominantly associated with PLCζ deficiencies in sperm. The reason relates to the well-established inability of defective PLCζ to trigger the characteristic pattern of intracellular Ca2+ oscillations responsible for activating specific molecular pathways in the oocyte that lead to meiosis resumption and completion. However, oocyte deficiencies have recently emerged to play critical roles in fertilization failure. Specifically, mutations have been identified in genes such as WEE2, PATL2, TUBB8, and TLE6. Such mutations translate into altered protein synthesis that results in defective transduction of the physiological Ca2+ signal needed for maturation-promoting factor (MPF) inactivation, which is indispensable for oocyte activation. The effectiveness of AOA treatments is closely related to identifying the causal factor of fertilization failure. Various diagnostic tests have been developed to determine the cause of OAD, including heterologous and homologous tests, particle image velocimetry, immunostaining, and genetic tests. On this basis, it has been shown that conventional AOA strategies, based on inducing the calcium oscillations, are highly effective in overcoming fertilization failure caused by PLCζ-sperm deficiencies. In contrast, oocyte-related deficiencies might be successfully managed using alternative AOA promoters that induce MPF inactivation and meiosis resumption. Such agents include cycloheximide, N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), roscovitine, and WEE2 complementary RNA. In addition, when OAD is caused by oocyte dysmaturity, applying a modified ovarian stimulation protocol and trigger could improve fertilization. WIDER IMPLICATIONS: AOA treatments represent a promising therapy to overcome fertilization failure caused by sperm- and oocyte-related factors. Diagnosing the cause of fertilization failure will be essential to improve the effectiveness and safe utilization of AOA treatments. Even though most data have not shown adverse effects of AOA on pre- and post-implantation embryo development, the literature is scarce on the matter concerned and recent studies, mainly using mice, suggest that AOA might cause epigenetic alterations in the resulting embryos and offspring. Until more robust data are available, and despite the encouraging results obtained, AOA should be applied clinically judiciously and only after appropriate patient counseling. Currently, AOA should be considered an innovative treatment, not an established one.

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.

Considerations on staffing levels for a modern assisted reproductive laboratory.

The duties recently performed in the embryology laboratory have deeply increased compared to those realized a couple of decades ago. Currently, procedures include conventional in vitro fertilization (IVF) and ICSI techniques, or processing of surgically retrieved sperm, embryo culture and time-lapse monitoring, blastocyst culture, as well as trophectoderm biopsy for preimplantation genetic testing and cryopreservation. These techniques require not only time, but also high knowledge level and acutely concentration by the embryologist team. The existing data indicate that an IVF laboratory need to have adequate staffing levels to perform the required daily duties, and to work in optimal conditions that are critical to assure a high quality service, as well as avoiding incidents and to provide the best outcomes. As a result, IVF clinics have invested in human resources, but there is still a large discrepancy between IVF centres on the number of embryologists employed. Currently there is no golden standard on the human resource requirements for assisted reproductive technology procedures; therefore, in this review paper we aim to provide arguments to take into account to determine the embryology staffing requirements in an embryology laboratory to assure optimal safety and efficiency of operations.

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.

Healthy Live Births after the Transfer of Mosaic Embryos: Self-Correction or PGT-A Overestimation?

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of "self-correction" mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as "mosaic" those embryos that are uniformly euploid or aneuploid. NGS assumption of "intermediate copy number profiles" as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate.

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.