Evolution of biotechnological advances and regenerative therapies for endometrial disorders: a systematic review.

BACKGROUND: The establishment and maintenance of pregnancy depend on endometrial competence. Asherman syndrome (AS) and intrauterine adhesions (IUA), or endometrial atrophy (EA) and thin endometrium (TE), can either originate autonomously or arise as a result from conditions (i.e. endometritis or congenital hypoplasia), or medical interventions (e.g. surgeries, hormonal therapies, uterine curettage or radiotherapy). Affected patients may present an altered or inadequate endometrial lining that hinders embryo implantation and increases the risk of poor pregnancy outcomes and miscarriage. In humans, AS/IUA and EA/TE are mainly treated with surgeries or pharmacotherapy, however the reported efficacy of these therapeutic approaches remains unclear. Thus, novel regenerative techniques utilizing stem cells, growth factors, or tissue engineering have emerged to improve reproductive outcomes. OBJECTIVE AND RATIONALE: This review comprehensively summarizes the methodologies and outcomes of emerging biotechnologies (cellular, acellular, and bioengineering approaches) to treat human endometrial pathologies. Regenerative therapies derived from human tissues or blood which were studied in preclinical models (in vitro and in vivo) and clinical trials are discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase was conducted to identify original peer-reviewed studies published in English between January 2000 and September 2023. The search terms included: human, uterus, endometrium, Asherman syndrome, intrauterine adhesions, endometrial atrophy, thin endometrium, endometritis, congenital hypoplasia, curettage, radiotherapy, regenerative therapy, bioengineering, stem cells, vesicles, platelet-rich plasma, biomaterials, microfluidic, bioprinting, organoids, hydrogel, scaffold, sheet, miRNA, sildenafil, nitroglycerine, aspirin, growth hormone, progesterone, and estrogen. Preclinical and clinical studies on cellular, acellular, and bioengineering strategies to repair or regenerate the human endometrium were included. Additional studies were identified through manual searches. OUTCOMES: From a total of 4366 records identified, 164 studies (3.8%) were included for systematic review. Due to heterogeneity in the study design and measured outcome parameters in both preclinical and clinical studies, the findings were evaluated qualitatively and quantitatively without meta-analysis. Groups using stem cell-based treatments for endometrial pathologies commonly employed mesenchymal stem cells (MSCs) derived from the human bone marrow or umbilical cord. Alternatively, acellular therapies based on platelet-rich plasma (PRP) or extracellular vesicles are gaining popularity. These are accompanied by the emergence of bioengineering strategies based on extracellular matrix (ECM)-derived hydrogels or synthetic biosimilars that sustain local delivery of cells and growth factors, reporting promising results. Combined therapies that target multiple aspects of tissue repair and regeneration remain in preclinical testing but have shown translational value. This review highlights the myriad of therapeutic material sources, administration methods, and carriers that have been tested. WIDER IMPLICATIONS: Therapies that promote endometrial proliferation, vascular development, and tissue repair may help restore endometrial function and, ultimately, fertility. Based on the existing evidence, cost, accessibility, and availability of the therapies, we propose the development of triple-hit regenerative strategies, potentially combining high-yield MSCs (e.g. from bone marrow or umbilical cord) with acellular treatments (PRP), possibly integrated in ECM hydrogels. Advances in biotechnologies together with insights from preclinical models will pave the way for developing personalized treatment regimens for patients with infertility-causing endometrial disorders such as AS/IUA, EA/TE, and endometritis. REGISTRATION NUMBER: https://osf.io/th8yf/.

Effect of intraovarian platelet-rich plasma injection on IVF outcomes in women with poor ovarian response: the PROVA randomized controlled trial.

STUDY QUESTION: Does intraovarian platelet-rich plasma (PRP) injection increase the number of mature oocytes obtained after controlled ovarian stimulation (COS) in young women with poor ovarian response (POR) undergoing IVF? SUMMARY ANSWER: Intraovarian PRP injection procedure does not improve mature oocyte yield after COS in women less than 38 years old with an established IVF history of POR. WHAT IS KNOWN ALREADY: POR is frequently encountered among the infertile population and the number of women seeking infertility treatment related to POR is increasing. Effective treatment options for this patient population to conceive with autologous oocytes are lacking. Case series and cohort studies suggest that intraovarian PRP injection may improve follicular recruitment in women with premature ovarian insufficiency (POI) and POR, yet robust randomized studies have not been performed to date to determine the clinical utility of this intervention. STUDY DESIGN, SIZE, DURATION: This was a multi-center randomized controlled trial (RCT) conducted at university-affiliated reproductive centers in the USA and Turkey, between January 2020 and November 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients who met inclusion criteria (<38 years old, two or more prior cycles with <3 oocytes retrieved; and without single gene disorders, prior ovarian surgery, endometriomas, BMI >35 kg/m2, or severe male factor infertility) were randomized to either the PRP or control group. Patients in both groups subsequently underwent COS, oocyte retrieval, ICSI, preimplantation genetic testing for aneuploidy (PGT-A), and single euploid embryo transfer. Number of metaphase II (MII) oocytes obtained was the primary outcome. Secondary outcomes included ovarian reserve tests (antral follicle count [AFC] and anti-Müllerian hormone [AMH]), blastocyst and euploid blastocyst yields, and sustained implantation. The study was powered to detect a difference of one mature oocyte obtained at oocyte retrieval. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 83 patients met inclusion criteria and were randomized to receive autologous intraovarian PRP injection (n = 41) or to no intervention (n = 42). No significant differences were observed in number of MII oocytes retrieved per cycle (2.8 ± 2.4 vs 3.1 ± 3.3 in PRP vs control, respectively; P = 0.9), blastocysts (1.0 ± 1.3 vs 1.3 ± 2.1, P = 0.8), or euploid blastocysts (0.8 ± 1.1 vs 0.9 ± 1.6; P = 0.5). Similarly, no differences were observed in the likelihood of obtaining at least one euploid blastocyst (45% vs 37%, P = 0.4; relative risk [RR], 95% CI = 0.9, 0.6-1.2) or the rate of sustained implantation (31% vs 29%, P = 0.9; RR 1.0, 0.7-1.3). Posttreatment AFC (7.9 ± 4.5 vs 6.8 ± 4.8, P = 0.3) and AMH (0.99 ± 0.98 vs 0.7 ± 0.6, P = 0.2) were also not different between the groups. LIMITATIONS, REASONS FOR CAUTION: Results from this RCT may not be generalizable to other PRP preparations owing to heterogeneity and lack of standardization. The control groups did not undergo a sham ovarian injection, which would have been relevant had the results shown benefit of PRP injection. Only patients with POR were included in this study, and these results may not be generalizable to more severe diminution of ovarian reserve, as seen with POI. WIDER IMPLICATIONS OF THE FINDINGS: The intraovarian PRP injection procedure does not improve mature oocyte yield or other parameters of IVF outcome in women less than 38 years old with an established IVF history of POR. The results from this study do not support the use of intraovarian PRP injection in this population. STUDY FUNDING/COMPETING INTEREST(S): Departmental funds were used and no external funding was requested for this study. ES is a consultant for and receives grant funding from the Foundation for Embryonic Competence. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov Registry Identifier: NCT04163640. TRIAL REGISTRATION DATE: 15 November 2019. DATE OF FIRST PATIENT'S ENROLMENT: 24 February 2020.

Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123 987 patients.

STUDY QUESTION: What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? SUMMARY ANSWER: The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of 40% and 53.3%, respectively, culminating in a cumulative live birth rate of 98.1% (95% CI = 96.5-99.6%) after five euploid blastocyst transfers. WHAT IS KNOWN ALREADY: The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of 92.6%. STUDY DESIGN, SIZE, DURATION: An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123 987 patients with a total of 64 572 euploid blastocyst transfers were screened for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness <6 mm prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 (0.085% of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts (40% vs 53.3%, relative risk = 1.33, 95% CI = 0.93-1.9, P value = 0.14). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI >30 kg/m2, cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk = 0.84, 95% CI = 0.58-1.21, P = 0.29). The cumulative live birth rate after five euploid blastocyst transfers was 98.1% (95% CI = 96.5-99.6%). LIMITATIONS, REASONS FOR CAUTION: Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. WIDER IMPLICATION OF THE FINDINGS: Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of <2%. Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Treatment options for endometrial hypoproliferation.

PURPOSE OF REVIEW: Endometrial hypoproliferation refers to the failure of the endometrium to reach optimal thickness during fresh or frozen embryo transfer cycles in women undergoing infertility treatment with in-vitro fertilization (IVF). This review discusses the treatment options for endometrial hypoproliferation. RECENT FINDINGS: Apart from factors related to the embryo quality, ultrasonographic findings associated with the endometrium, such as endometrial thickness, endometrial pattern and subendometrial blood flow, are considered key factors associated with the outcome of assisted reproductive treatment. To date, a consensus has not been reached regarding the definition of thin endometrium, while thresholds of 6, 7 or 8 mm have been used in the literature. Strategies to increase endometrial thickness can be reviewed in three groups: endocrine approaches, vitamins & supplements, and new experimental therapeutic interventions. Some of the recently introduced experimental therapeutic interventions such as platelet-rich plasma injection, stem cell treatment and tissue bioengineering are exciting potential therapies that need to be further studied. SUMMARY: Despite a large number of publications on the topic, diagnosing and treating endometrial hypoproliferation remains a challenge. Well designed studies are needed to establish a widely accepted endometrial thickness cut-off value below which endometrial hypoproliferation is diagnosed and to generate meaningful data that would allow an evidence-based discussion of available therapeutic options with patients.

Distress response in granulosa cells of women affected by PCOS with or without insulin resistance.

PURPOSE: In this study, we investigated whether metabolic dysfunction in women with Polycystic ovarian syndrome (PCOS) induces granulosa cell (GC) stress and activates in the endoplamatic reticulum and the mitochondria (UPRer and UPRmt, respectively). METHODS: Women who were diagnosed with PCOS (based on the Rotterdam criteria), were divided into two groups, PCOS with insulin resistance (PCOS-IR; n = 20) and PCOS with no insulin resistance (PCOS-nIR; n = 20), and compared to healthy oocyte donors (CONT; n = 20). Insulin resistance (IR) was assessed on the results of homeostasis model assessment (HOMA) that determines IR using the concentration of fasting plasma glucose and fasting insuline. Expression of UPRer genes (i.e., IRE1, ATF4, ATF6, XBP1, BIP, and CHOP), and UPRmt genes (i.e., HSP60, HSP10, CLPP, and HSP40) was assessed in cumulus GCs by qRT-PCR. RESULTS: We found that several genes involved in UPRer and UPRmt were overexpressed in the GCs of PCOS-IR and PCOS-nIR compared to CONT. IRE1, ATF4 and XBP1, that are activated by ER stress, were significantly overexpressed in PCOS-IR compared to CONT. BIP and CHOP were overexpressed in PCOS groups compared to CONT. HSP10 and HSP40 were upregulated in PCOS-IR and PCOS-nIR groups compared to the CONT. HSP60 and CLPP showed no statistical different expression in PCOS-IR and PCOS-nIR compared to CONT group. CONCLUSION: Our findings suggest that the GCs of women with PCOS (with or without IR) are metabolically distressed and upregulate UPRer and UPRmt genes. Our study contributes to the understanding of the molecular mechanisms underlying the pathological changes that occur in the follicular microenvironment of women with PCOS.

Human Umbilical Cord-Based Therapeutics: Stem Cells and Blood Derivatives for Female Reproductive Medicine.

There are several conditions that lead to female infertility, where traditional or conventional treatments have limited efficacy. In these challenging scenarios, stem cell (SC) therapies have been investigated as alternative treatment strategies. Human umbilical cord (hUC) mesenchymal stem cells (hUC-MSC), along with their secreted paracrine factors, extracts, and biomolecules, have emerged as promising therapeutic alternatives in regenerative medicine, due to their remarkable potential to promote anti-inflammatory and regenerative processes more efficiently than other autologous treatments. Similarly, hUC blood derivatives, such as platelet-rich plasma (PRP), or isolated plasma elements, such as growth factors, have also demonstrated potential. This literature review aims to summarize the recent therapeutic advances based on hUC-MSCs, hUC blood, and/or other plasma derivatives (e.g., extracellular vesicles, hUC-PRP, and growth factors) in the context of female reproductive medicine. We present an in-depth analysis of the principal molecules mediating tissue regeneration, compiling the application of these therapies in preclinical and clinical studies, within the context of the human reproductive tract. Despite the recent advances in bioengineering strategies that sustain delivery and amplify the scope of the therapeutic benefits, further clinical trials are required prior to the wide implementation of these alternative therapies in reproductive medicine.

The chances of obtaining a euploid embryo and subsequent live birth remain consistent with national age-based rates after an in vitro fertilization cycle that produced only aneuploid embryos.

OBJECTIVE: To determine the prognosis of patients who were only able to obtain aneuploid embryos in their first in vitro fertilization (IVF) cycle if they attempted a second cycle. DESIGN: Case series and retrospective cohort study. SETTING: A single, large fertility center. PATIENT(S): All patients who obtained only aneuploid embryos after IVF with preimplantation genetic testing for aneuploidy during the initial cycle and returned for a second cycle. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The percentage of patients who obtained a euploid embryo and live birth rates in the second cycle, stratified by Society for Assisted Reproductive Technology-defined age groups, was compared with that of controls from the same period. RESULT(S): A total of 538 patients with only aneuploid embryos in their first cycle were included. Three hundred (56%) patients obtained euploid blastocysts in the second cycle, with younger women having a higher chance of obtaining at least 1 euploid embryo (81% in women aged <35 years vs. 25% in women aged >42 years). The cumulative live birth rates were 71%, 62%, 46%, 27%, and 13% for the age groups <35, 35-37, 38-40, 41-42, and >42 years, respectively. The live birth rates per first embryo transfer were >57% across all the age groups and similar to those of the controls in the same age groups. CONCLUSION(S): Patients who obtained only aneuploid embryos during their initial IVF cycle retained favorable prognosis in their second cycle, with outcomes comparable with the national age-based standards. Younger women and those who had more embryos available for biopsy had the highest chance of success. These women should receive age-appropriate counseling and should not be discouraged from a second IVF attempt based on the results of their first cycle.

Transcriptomic landscape of granulosa cells and peripheral blood mononuclear cells in women with PCOS compared to young poor responders and women with normal response.

STUDY QUESTION: Are transcriptomic profiles altered in ovarian granulosa cells (GCs) and peripheral blood mononuclear cells (PBMNCs) of women with polycystic ovary syndrome (PCOS) compared to young poor responders (YPR) and women with normal response to ovarian stimulation? SUMMARY ANSWER: RNA expression profiles in ovarian GCs and PBMNCs were significantly altered in patients with PCOS compared with normoresponder controls (CONT) and YPR. WHAT IS KNOWN ALREADY: PCOS is characterised by a higher number of follicles at all developmental stages. During controlled ovarian hyperstimulation, PCOS women develop a larger number of follicles as a result of an exacerbated response, with an increased risk of ovarian hyperstimulation syndrome. Despite the number of developing follicles, they are often heterogeneous in both size and maturation stage, with compromised quality and retrieval of immature oocytes. Women with PCOS appear to have a longer reproductive lifespan, with a slightly higher menopausal age than the general population, in addition to having a higher antral follicular count. As a result, the ovarian follicular dynamics appear to differ significantly from those observed in women with poor ovarian response (POR) or diminished ovarian reserve. STUDY DESIGN, SIZE, DURATION: Transcriptomic profiling with RNA-sequencing and validation using quantitative reverse transcription PCR (qRT-PCR). Women with PCOS (N = 20), YPR (N = 20) and CONT (N = 20). Five patients for each group were used for sequencing and 15 samples per group were used for validation. PARTICIPANTS/MATERIALS, SETTING, METHODS: PCOS was defined using the revised Rotterdam diagnostic criteria for PCOS. The YPR group included women <35 years old with <4 mature follicles (at least 15 mm) on the day of the trigger. According to internal data, this group represented the bottom 15th percentile of patients' responses in this age group. It was consistent with Patient-Oriented Strategies Encompassing Individualize D Oocyte Number (POSEIDON) criteria for POR (Group 3). The young CONT group included women <35 years without PCOS or anovulation, who developed >14 mature follicles (at least 15 mm on transvaginal ultrasound). According to internal data, a threshold of >14 mature follicles was established to represent the top 25% of patients in this age group in this clinic.Overall, n = 60 GCs and PBMNCs samples were collected and processed for total RNA extraction. To define the transcriptomic cargo of GCs and PBMNCs, RNA-seq libraries were successfully prepared from samples and analysed by RNA-seq analysis. Differential gene expression analysis was used to compare RNA-seq results between different groups of samples. Ingenuity pathway analysis was used to perform Gene Ontology and pathways analyses. MAIN RESULTS AND THE ROLE OF CHANCE: In PBMNCs of PCOS, there were 65 differentially expressed genes (DEGs) compared to CONT, and 16 compared to YPR. In GCs of PCOS, 4 genes showed decreased expression compared to CONT, while 58 genes were differentially expressed compared to YPR. qRT-PCR analysis confirmed the findings of the RNA-seq. The functional enrichment analysis performed revealed that DEGs in GCs of PCOS compared to CONT and YPR were prevalently involved in protein ubiquitination, oxidative phosphorylation, mitochondrial dysfunction and sirtuin signaling pathways. LARGE SCALE DATA: The data used in this study is partially available at Gene Ontology database. LIMITATIONS, REASONS FOR CAUTION: The analysis in PBMNCs could be uninformative due to inter-individual variability among patients in the same study groups. Despite the fact that we considered this was the best approach for our study's novel, exploratory nature. WIDER IMPLICATIONS OF THE FINDINGS: RNA expression profiles in ovarian GCs and PBMNCs were altered in patients with PCOS compared with CONT and YPR. GCs of PCOS patients showed altered expression of several genes involved in oxidative phosphorylation, mitochondrial function and sirtuin signaling pathways. This is the first study to show that the transcriptomic landscape in GCs is altered in PCOS compared to CONT and YPR. STUDY FUNDING/COMPETING INTEREST(S): This study was partially supported by grant PI18/00322 from Instituto de Salud Carlos III, and European Regional Development Fund (FEDER), 'A way to make Europe' awarded to S.H. M.C., S.H., S.T., L.R., M.R., I.R., A.P. and R.C. declare no conflict of interests concerning this research. E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence. TRIAL REGISTRATION NUMBER: N/A.

Embryology outcomes after oocyte vitrification with super-cooled slush nitrogen are similar to outcomes with conventional liquid nitrogen: a randomized controlled trial.

OBJECTIVE: To determine whether the use of slush nitrogen (SN), a super-cooled form of nitrogen with a temperature from -207 to -210 °C, can improve oocyte survival after vitrification and warming compared with conventional liquid nitrogen (LN). DESIGN: Randomized controlled trial. SETTING: Academic-affiliated private practice. PATIENT(S): A total of 556 metaphase II oocytes from 32 oocyte donor cycles were included. INTERVENTION(S): Donor oocytes were block randomized to undergo vitrification with either SN or LN. Vitrification was followed by warming, fertilization with donor sperm, embryo culture to the blastocyst stage, and preimplantation genetic testing for aneuploidy via trophectoderm biopsy with targeted next-generation sequencing. MAIN OUTCOME MEASURE(S): The primary outcome was oocyte survival after vitrification and warming. Secondary outcomes included rates of fertilization, usable blastocyst formation, and whole chromosome aneuploidy. RESULT(S): Half of the metaphase II oocytes (n = 278) were randomized to undergo vitrification with SN, whereas the other half (n = 278) were randomized to undergo vitrification with LN. There were no statistically significant differences noted in oocyte survival rate (85.3% vs. 86.3%), fertilization rate (84.0% vs. 80.0%), rate of usable blastocyst formation (54.3% vs. 55.7%), or rate of whole chromosome aneuploidy (9.4% vs. 11.7%) between the SN and LN arms, respectively. CONCLUSION(S): The implementation of an SN oocyte vitrification protocol resulted in similar embryology outcomes compared with LN. The use of SN did not lead to any demonstrable improvement in oocyte survival after vitrification and warming. CLINICAL TRIAL REGISTRATION NUMBER: NCT04342364.

B-cell lymphoma 6 expression is not associated with live birth in a normal responder in vitro fertilization population.

OBJECTIVE: To determine whether increased endometrial B-cell lymphoma 6 (BCL6) expression is associated with live birth in a normal responder in vitro fertilization (IVF) population. DESIGN: Case-control study. SETTING: University-affiliated infertility center. PATIENT(S): Two groups of women undergoing IVF with preimplantation genetic testing for aneuploidy followed by warmed, single, euploid embryo transfer. Group 1 consisted of women who failed to achieve live birth, and group 2 consisted of women who achieved live birth. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Endometrial BCL6 expression measured by immunohistochemistry in endometrial tissue samples. Overexpression was defined by mean HSCORE with a cutoff of positivity of >1.4, as previously described in the literature. RESULT(S): Twenty-seven patients who achieved live birth and 23 patients who failed to achieve live birth were included. B-cell lymphoma 6 expression/HSCORE and live birth rate were not associated (Odds ratio [OR], 0.78 [0.24-2.55]). Using a cutoff of >1.4 for positivity, 8 of 23 samples were positive for BCL6 in the no live birth group, whereas 7 of 27 were positive in the live birth group. There was no significant association between BCL6 positivity and live birth (OR, 0.66 [0.19-2.21]). CONCLUSION(S): The proportion of patients with BCL6 positivity did not significantly differ between those who achieved live birth and those who did not. In the population of patients at our center, who compromise of women who respond normally to IVF stimulation, BCL6 overexpression was not associated with IVF success. Physicians implementing BCL6 testing as a diagnostic tool for clinical decision making should counsel patients that results may have limited utility in predicting IVF outcomes in this population.

A review of the pathophysiology of recurrent implantation failure.

Implantation is a critical step in human reproduction. The success of this step is dependent on a competent blastocyst, receptive endometrium, and successful cross talk between the embryonic and maternal interfaces. Recurrent implantation failure is the lack of implantation after the transfer of several embryo transfers. As the success of in vitro fertilization has increased and failures have become more unacceptable for patients and providers, the literature on recurrent implantation failure has increased. While this clinical phenomenon is often encountered, there is not a universally agreed-on definition-something addressed in an earlier portion of this Views and Reviews. Implantation failure can result from several different factors. In this review, we discuss factors including the maternal immune system, genetics of the embryo and parents, anatomic factors, hematologic factors, reproductive tract microbiome, and endocrine milieu, which factors into embryo and endometrial synchrony. These potential causes are at various stages of research and not all have clear implications or immediately apparent treatment.

Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes.

RESEARCH QUESTION: Can cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DESIGN: Cumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. RESULTS: A total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell-cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. CONCLUSIONS: These findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

Individual culture leads to decreased blastocyst formation but does not affect pregnancy outcomes in the setting of a single, vitrified-warmed euploid blastocyst transfer.

PURPOSE: To evaluate embryology and pregnancy outcomes following individual and group embryo culture in the setting of contemporary laboratory practices and freeze-all cycles. METHODS: Patients underwent ovarian stimulation followed by intracytoplasmic sperm injection (ICSI). Embryos proceeded through individual culture and then underwent preimplantation genetic testing for aneuploidy (PGT-A) via trophectoderm biopsy. In a subsequent cycle, participants underwent single embryo transfer of a vitrified-warmed, euploid embryo. Outcomes were compared to controls undergoing group culture during the same time frame. The Mann-Whitney U test and logistic regression models were utilized. RESULTS: Outcomes were assessed for 144 patients whose embryos underwent individual culture and 449 controls whose embryos underwent group culture. There were no significant differences in fertilization rates between groups (81.7% for individual culture vs. 84.1% for group culture, p = 0.22). However, individual culture was associated with a decreased rate of blastocyst formation compared to group culture (43.5% vs. 48.5%, p < 0.01). Following single, vitrified-warmed euploid blastocyst transfer, there were no significant differences between individual culture and group culture, respectively, in rates of positive ßhCG (81.9% vs. 81.5%, p = 0.91), sustained implantation (63.9% vs. 65.0%, p = 0.80), biochemical miscarriage (16.7% vs. 12.3%, p = 0.18), or clinical miscarriage (1.4% vs. 4.2%, p = 0.13). CONCLUSION: While individual culture appears to negatively impact the rate of usable blastocyst formation compared to group culture, there were no significant differences in pregnancy outcomes following transfer of a single, vitrified-warmed euploid blastocyst.

Emerging follicular activation strategies to treat women with poor ovarian response and primary ovarian insufficiency.

PURPOSE OF THE REVIEW: Female reproductive aging remains one of the key unsolved challenges in the field of reproductive medicine. This article reviews three of the most recent and cutting-edge strategies that are currently being investigated to address the issues of poor ovarian response (POR) and primary ovarian insufficiency (POI). RECENT FINDINGS: Publications revealing the mechanism of mechanical disruption of the Hippo signaling pathway paved the way to studies on its potential application for fertility treatments. This, in combination with Akt stimulation, resulted in live births and ongoing pregnancies in women with POI. Building on previous reports on the effects of bone marrow transplants on fertility after chemotherapy, another approach involved autologous stem cell ovarian transplantation (ASCOT). The method proved effective in achieving live births in women previously diagnosed with POR. A third approach, intraovarian injection of autologous platelet-rich plasma, resulted in live births and ongoing pregnancies both spontaneously and via in vitro fertilization (IVF) in women with POI and POR. SUMMARY: New paths are being charted to address the issues of POI and POR. Although these are preliminary studies that should be interpreted with caution, they represent great promise for the women affected by these conditions and the physicians treating them.

Evaluation of genome-wide DNA methylation profile of human embryos with different developmental competences.

STUDY QUESTION: Do embryos with different developmental competence exhibit different DNA methylation profiles at the blastocyst stage? SUMMARY ANSWER: We established genome-wide DNA methylome analysis for embryo trophectoderm (TE) biopsy samples and our findings demonstrated correlation of methylation profile of trophectoderm with euploidy status and with maternal age, indicating that genome-wide methylation level might be negatively correlated with embryo quality. WHAT IS KNOWN ALREADY: DNA methylation is a fundamental epigenetic regulatory mechanism that affects differentiation of cells into their future lineages during pre-implantation embryo development. Currently there is no established approach available to assess the epigenetic status of the human preimplantation embryo during routine IVF treatment. STUDY DESIGN, SIZE, DURATION: In total, we collected trophectoderm biopsy samples from 30 randomly selected human blastocysts and conducted whole-genome bisulfite sequencing (WGBS) to evaluate their DNA methylation profile. Nested linear models were used to assess association between DNA methylation level and ploidy status (aneuploidy [n = 20] vs. euploidy [n = 10]), maternal age (29.4-42.5 years old), and time of blastulation (day 5 [n = 16] vs. day 6 [n = 14]), using embryo identity as a covariate. PARTICIPANTS/MATERIALS, SETTING, METHODS: TE biopsy samples were obtained and submitted to bisulfite conversion. For WGBS, whole-genome sequencing libraries were then generated from the converted genome. An average of 75 million reads were obtained for each sample, and about 63% of the reads aligned to human reference. An average of 40 million reads used for the final analysis after the unconverted reads were filtered out. MAIN RESULTS AND THE ROLE OF CHANCE: We revealed an increase of genome-wide DNA methylation level in aneuploid embryo TE biopsies compared to euploid embryos (25.4% ± 3.2% vs. 24.7% ± 3.2%, P < 0.005). We also found genome-wide DNA methylation level to be increased with the maternal age (P < 0.005). On a chromosomal scale, we found monosomic embryos have lower methylation levels on the involved chromosome while no drastic change was observed for the involved chromosome in trisomies. Additionally, we revealed that WGBS data precisely revealed the chromosome copy number variance. LIMITATIONS, REASONS FOR CAUTION: Though our results demonstrated a negative correlation of genome-wide methylation level and embryo quality, further WGBS analysis on a greater number of embryos and specific investigation of its correlation with implantation and live birth are needed before any practical use of this approach for evaluation of embryo competence. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed a change in genome-wide DNA methylation profile among embryos with different developmental potentials, reinforcing the critical role of DNA methylation in early development. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this study. Intramural funding was provided by the Foundation for Embryonic Competence (FEC). E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence; he is also co-founder and a shareholder of ACIS LLC and coholds patent US2019/055906 issued for utilizing electrical resistance measurement for assessing cell viability and cell membrane piercing. TRIAL REGISTRATION NUMBER: N/A.

Noninvasive preimplantation genetic testing for aneuploidy exhibits high rates of deoxyribonucleic acid amplification failure and poor correlation with results obtained using trophectoderm biopsy.

OBJECTIVE: To validate a commercially available noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) assay by investigating the following: prevalence of deoxyribonucleic acid (DNA) amplification failure with niPGT-A; factors affecting amplification failure with niPGT-A; and frequency of discordant results between niPGT-A and traditional preimplantation genetic testing for aneuploidy. DESIGN: Prospective cohort study SETTING: Academic-affiliated private practice PATIENT(S): One hundred sixty-six blastocysts and their surrounding culture media from couples undergoing in vitro fertilization between July 2019 and May 2020 were analyzed. INTERVENTION(S): Blastocyst-stage spent culture media samples underwent niPGT-A using a commercially available kit that used whole-genome amplification with a modified multiple annealing and looping-based amplification cycle protocol followed by next-generation sequencing. Preimplantation genetic testing for aneuploidy of trophectoderm (TE) biopsies was performed using targeted next-generation sequencing. MAIN OUTCOME MEASURE(S): The primary outcome was failure to achieve an interpretable result with niPGT-A. Factors affecting DNA amplification were also assessed. Discrepancies between niPGT-A and TE biopsy results were analyzed, and clinical outcomes were evaluated. RESULT(S): Deoxyribonucleic acid amplification failures with niPGT-A were observed in 37.3% (62/166) of the samples. With TE biopsy, no embryos exhibited DNA amplification failure. Embryos with a shorter duration of exposure to the culture media and no evidence of whole-chromosome aneuploidy on the TE biopsy displayed high rates of DNA amplification failure with niPGT-A. Of 104 embryos with both niPGT-A and TE biopsy results available, whole-chromosome discordance was noted in 42 cases (40.4%). Three embryos classified as aneuploid based on the niPGT-A result progressed to successful delivery. CONCLUSION(S): The rates of DNA amplification failure were high among the niPGT-A samples, virtually precluding the clinical applicability of niPGT-A in its current form.

A multicenter, prospective, blinded, nonselection study evaluating the predictive value of an aneuploid diagnosis using a targeted next-generation sequencing-based preimplantation genetic testing for aneuploidy assay and impact of biopsy.

OBJECTIVE: To determine the predictive value of an aneuploid diagnosis with a targeted next-generation sequencing-based preimplantation genetic testing for aneuploidy (PGT-A) assay in prognosticating the failure of a successful delivery. DESIGN: Prospective, blinded, multicenter, nonselection study. All usable blastocysts were biopsied, and the single best morphologic blastocyst was transferred before genetic analysis. Preimplantation genetic testing for aneuploidy was performed after clinical outcome was determined. Clinical outcomes were compared to PGT-A results to calculate the predictive value of a PGT-A aneuploid diagnosis. SETTING: Fertility centers. PATIENT(S): Couples undergoing their first in vitro fertilization cycle without recurrent pregnancy loss, antral follicle count < 8, or body mass index ≥ 35 kg/m2. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcome was the ability of the analytical result of aneuploid to predict failure to deliver (clinical result). A secondary outcome was the impact of the trophectoderm biopsy on sustained implantation. RESULT(S): Four hundred two patients underwent 484 single, frozen, blastocyst transfers. The PGT-A aneuploid diagnosis clinical error rate was 0%. There was no difference in sustained implantation between the study group and an age-matched control group, where biopsy was not performed (47.9% vs. 45.8). CONCLUSION(S): The PGT-A assay evaluated was highly prognostic of failure to deliver when an aneuploid result was obtained. Additionally, the trophectoderm biopsy had no detectable adverse impact on sustained implantation. CLINICAL TRIAL REGISTRATION NUMBERS: NCT02032264 and NCT03604107.

Developmental potential of aneuploid human embryos cultured beyond implantation.

Aneuploidy, the presence of an abnormal number of chromosomes, is a major cause of early pregnancy loss in humans. Yet, the developmental consequences of specific aneuploidies remain unexplored. Here, we determine the extent of post-implantation development of human embryos bearing common aneuploidies using a recently established culture platform. We show that while trisomy 15 and trisomy 21 embryos develop similarly to euploid embryos, monosomy 21 embryos exhibit high rates of developmental arrest, and trisomy 16 embryos display a hypo-proliferation of the trophoblast, the tissue that forms the placenta. Using human trophoblast stem cells, we show that this phenotype can be mechanistically ascribed to increased levels of the cell adhesion protein E-CADHERIN, which lead to premature differentiation and cell cycle arrest. We identify three cases of mosaicism in embryos diagnosed as full aneuploid by pre-implantation genetic testing. Our results present the first detailed analysis of post-implantation development of aneuploid human embryos.

Mitochondrial function in women with polycystic ovary syndrome.

PURPOSE OF REVIEW: To provide an overview of mitochondrial functional alterations in women with polycystic ovary syndrome (PCOS). RECENT FINDINGS: Although numerous studies have focused on PCOS, the pathophysiological mechanisms that cause this common disease remain unclear. Mitochondria play a central role in energy production, and mitochondrial dysfunction may underlie several abnormalities observed in women with PCOS. Recent studies associated mtDNA mutations and low mtDNA copy number with PCOS, and set out to characterize the potential protective role of mitochondrial and endoplasmic reticulum unfolded protein responses (UPR and UPR). SUMMARY: Mitochondrial dysfunction likely plays a role in the pathogenesis of PCOS by increasing reactive oxygen (ROS) and oxidative stress. This occurs in a metabolic milieu often affected by insulin resistance, which is a common finding in women with PCOS, especially in those who are overweight or obese. Mutations in mtDNA and low mtDNA copy number are found in these patients and may have potential as diagnostic modalities for specific PCOS phenotypes. More recently, UPR and UPR are being investigated as potential cellular rescue mechanisms in PCOS, the failure of which may lead to apoptosis, and contribute to decreased reproductive potential.