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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 (UPRWomen 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 UPRWe found that several genes involved in UPROur findings suggest that the GCs of women with PCOS (with or without IR) are metabolically distressed and upregulate UPR

Caseinolytic peptidase P (CLPP) plays a central role in mitochondrial unfolded protein response (mtUPR) by promoting the breakdown of misfolded proteins and setting in motion a cascade of reactions to re-establish protein homeostasis. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Telomeres, on the other hand, are tandem repeats of 5’-TTAGGG-3’ sequences found at the ends of the chromosomes. Telomeres are essential for maintaining chromosome stability during somatic cell division and their shortening is associated with cellular senescence and aging. In this study, we asked whether the infertility and ovarian aging phenotype caused by global germline deletion of Clpp is associated with somatic aging, and tested telomere length in tissues of young and aging mice. We found that impaired mtUPR caused by the lack of CLPP is associated with accelerated telomere shortening in both oocytes and somatic cells of aging mice. In addition, expression of several genes that maintain telomere integrity were decreased, and double strand DNA breaks were increased in telomeric regions. Our results highlight how impaired mtUPR can affect telomere integrity and demonstrate a link between loss of mitochondrial protein hemostasis, infertility, and somatic aging.

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 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.

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

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.

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.

SummaryTelomere shortening during oocyte growth and development is related to reproductive ageing and infertility. The main mechanism involved in the maintenance of telomeres is based on telomerase activity, a specialized enzyme complex, which is capable of adding TTAGGG repeats at the ends of the chromosomes. Mitochondrial dysfunction may cause progressive shortening of telomeres by promoting the generation of reactive oxygen species. Mitofusin-1 is a protein required for mitochondrial fusion. Mice with the mitofusin-1 (Mfn1) deletion in the oocyte are characterized by accelerated follicular depletion and infertility, associated with defective oocyte maturation and follicular development. We hypothesized whether mitochondrial dysfunction in oocytes with targeted deletion of Mfn1 causes telomere shortening. We analyzed telomere length in oocyte and somatic cells in 3-, 6- and 9-month-old Mfn1−/− and wild-type mice. Immunofluorescence in oocyte mice of TRF1 and H2A.X was assessed to evaluate the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats. Mitochondrial dysfunction due to the deletion of Mfn1 does not seem to affect telomere length in mouse oocytes.

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.

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.

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 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

Purpose of review To discuss the utilization, performance, and interpretation of noninvasive prenatal testing (NIPT) results in women achieving pregnancy through in vitro fertilization (IVF) and preimplantation genetic testing for aneuploidy (PGT-A). Recent findings Although PGT-A is a highly accurate method for the selection of euploid embryos the possibility for error still exists. Many women pursue NIPT after conception via IVF with or without PGT-A, whereas some forgo prenatal screening all together. Recent evidence suggests that the prevalence of a positive NIPT following PGT-A is low, and the positive predictive value is altered in this population. Summary NIPT is a valuable prenatal screening tool that should be offered to pregnant women regardless of prior PGT. In women who conceive following IVF and PGT-A through the transfer of euploid embryos, positive test results should be interpreted with caution.

Intraovarian injection of platelet rich plasma (PRP) is a novel treatment for patients with poor ovarian response (POR) and primary ovarian insufficiency (POI). This article reviews the latest literature on the effect of PRP on markers of ovarian reserve, oocyte and embryo yield, and live birth for these poor prognosis patients.Several case series and one prospective trial have demonstrated improvements in markers of ovarian reserve in patients with POI and POR and improved oocyte and embryo yields in patients with POR. These studies report multiple live births in patients who had previously failed treatment. The positive effects of PRP persist throughout the literature despite the fact that multiple protocols for preparing and injecting PRP exist, with no consensus on the optimal protocol.Intra-ovarian injection of PRP is a promising new technology for poor prognosis patients. Rigorous and appropriately controlled clinical trials are warranted to confirm the utility of this treatment for improving patients' ability to successfully conceive.

Mitochondria are dynamic organelles that regulate their size, shape, and morphology through mechanisms called fusion and fission, to continually adapt themselves to their bioenergetic environment. These mechanisms play a critical role to maintain the mitochondrial function under metabolic and environmental stress. Mitofusin 1 (MFN1) and mitofusin 2 (MFN2) are transmembrane GTPases that regulate mitochondrial fusion mechanism and are required for the maintenance of cellular homeostasis. In this study, we aimed to determine the role of mitofusins in female reproductive competence and senescence using a mouse model with oocyte-specific double deletion of Mfn1 and Mfn2, eliminating the potential functional redundancy of these two proteins. Oocyte-specific targeted double deletion of Mfn1 and Mfn2 in mice resulted in female infertility associated with impaired follicular development and oocyte maturation. It also resulted in altered mitochondrial dynamics and mitochondrial dysfunction. Lack of Mfn1 and Mfn2 in oocytes resulted in accelerated follicular depletion and impaired oocyte quality which are consistent with phenotype of reproductive aging.

STUDY QUESTION Is poor ovarian response associated with a change in predicted age based on a DNA methylation-derived age prediction model (the Horvath algorithm) in white blood cells (WBCs) or cumulus cells (CCs)? SUMMARY ANSWER In young women, poor ovarian response is associated with epigenetic age acceleration within WBC samples but is not associated with age-related changes in CC. WHAT IS KNOWN ALREADY The majority of human tissues follow predictable patterns of methylation which can be assessed throughout a person’s lifetime. DNA methylation patterns may serve as informative biomarkers of aging within various tissues. Horvath’s ‘epigenetic clock’, which is a DNA methylation-derived age prediction model, accurately predicts a subject’s true chronologic age when applied to WBC but not to CC. STUDY DESIGN, SIZE, DURATION A prospective cohort study was carried out involving 175 women undergoing ovarian stimulation between February 2017 and December 2018. Women were grouped according to a poor (≤5 oocytes retrieved) or good (>5 oocytes) response to ovarian stimulation. Those with polycystic ovary syndrome (PCOS) (n = 35) were placed in the good responder group. PARTICIPANTS/MATERIALS, SETTING, METHODS DNA methylation patterns from WBC and CC were assessed for infertile patients undergoing ovarian stimulation at a university-affiliated private practice. DNA was isolated from peripheral blood samples and CC. Bisulfite conversion was then performed and a DNA methylation array was utilized to measure DNA methylation levels throughout the genome. Likelihood ratio tests were utilized to assess the relationship between predicted age, chronologic age and ovarian response. MAIN RESULTS AND THE ROLE OF CHANCE The Horvath-predicted age for WBC samples was consistent with patients’ chronologic age. However, predicted age from analysis of CC was younger than chronologic age. In subgroup analysis of women less than 38 years of age, poor ovarian response was associated with an accelerated predicted age in WBC (P = 0.017). Poor ovarian response did not affect the Horvath-predicted age based on CC samples (P = 0.502). No alternative methylation-based calculation was identified to be predictive of age for CC. LIMITATIONS, REASONS FOR CAUTION To date, analyses of CC have failed to identify epigenetic changes that are predictive of the aging process within the ovary. Despite the poor predictive nature of both the Horvath model and the novel methylation-based age prediction model described here, it is possible that our efforts failed to identify appropriate sites which would result in a successful age-prediction model derived from the CC epigenome. Additionally, lower DNA input for CC samples compared to WBC samples was a methodological limitation. We acknowledge that a universally accepted definition of poor ovarian response is lacking. Furthermore, women with PCOS were included and therefore the group of good responders in the current study may not represent a population with entirely normal methylation profiles. WIDER IMPLICATIONS OF THE FINDINGS The process of ovarian and CC aging continues to be poorly understood. Women who demonstrate poor ovarian response to stimulation represent a common clinical challenge, so clarifying the exact biological changes that occur within the ovary over time is a worthwhile endeavor. The data from CC support a view that hormonally responsive tissues may possess distinct epigenetic aging patterns when compared with other tissue types. Future studies may be able to determine whether alternative DNA methylation sites can accurately predict chronologic age or ovarian response to stimulation from CC samples. Going forward, associations between epigenetic age acceleration and reproductive and general health consequences must also be clearly defined. STUDY FUNDING/COMPETING INTEREST(S) No external funding was obtained for the study and there are no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Funder: Weston Havens Foundation; doi: https://doi.org/10.13039/100011223, 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.

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (

Embryo diagnostics are somewhat controversial in clinical assisted reproduction technology (ART) practice and remain an active area of investigation. Application of embryo diagnostics holds great potential to raise the standard of clinical care by eliminating futile transfers, allowing highly effective single-embryo transfer, and reducing the probability of clinical loss and ongoing abnormal gestations. These advantages are accompanied by risks, principally the chance that a reproductively competent embryo will be mislabeled and discarded. This would lower the ultimate probability that one or more of the embryos might implant and lead to delivery of a healthy infant. Rigorous validation should be required for embryo diagnostics. Metrics for validation can be divided into three simple areas: analytical validation, determination of clinical predictive values for normal and abnormal test results, and a randomized clinical trial to demonstrate that the selection advantage gained through the diagnostic improves clinical outcomes.

Purpose of review To discuss the recent applications of electrophysiological principles to the optimization and automation of the IVF laboratory. Recent findings There is growing evidence showing improvement of live birth rates following oocyte electro-activation. Novel applications using electrophysiological techniques are now employed to determine oocyte penetration and viability in real-time. Summary In this short review, we summarize the recent advances in the integration of electrophysiological techniques into the assisted reproductive technology laboratories. We describe the potential clinical applications and their advantages such as creation of reliable automated cell injection systems and novel manual intracytoplasmic sperm injection (ICSI) training platforms. We also discuss theoretical adverse effects and ways to mitigate them.

We aimed to determine whether intraovarian injection of autologous platelet rich plasma (PRP) improves response to ovarian stimulation and in vitro fertilization (IVF) outcome in women with primary ovarian insufficiency (POI). Women (N=311; age 24-40) diagnosed with POI based on ESHRE criteria underwent intraovarian PRP injection. Markers of ovarian reserve, and IVF outcome parameters were followed. PRP treatment resulted in increased antral follicle count (AFC) and serum antimullerian hormone (AMH), while serum follicle stimulating hormone (FSH) did not change significantly. After PRP injection, 23 women (7.4%) conceived spontaneously, 201 (64.8%) developed antral follicle(s) and attempted IVF, and 87 (27.8%) had no antral follicles and therefore did not receive additional treatment. Among the 201 women who attempted IVF, 82 (26.4% of total) developed embryos; 25 of these women preferred to cryopreserve embryos for transfer at a later stage, while 57 underwent embryo transfer resulting in 13 pregnancies (22.8% per transfer, 4% of total). In total, of the 311 women treated with PRP, 25 (8.0%) achieved livebirth/sustained implantation (spontaneously or after IVF), while another 25 (8.0%) cryopreserved embryos. Our findings suggest that in women with POI, intraovarian injection of autologous PRP might be considered as an alternative experimental treatment option.

Caseinolytic peptidase P (CLPP) plays a central role in mitochondrial unfolded protein response (mtUPR) and is required for maintaining protein homeostasis in the mitochondria. Global germline Clpp deletion causes female infertility and accelerated follicular depletion. In the current study, we aimed to characterize the role of CLPP in cumulus cell function, gene expression, and mitochondrial ultrastructure. We found that mitochondria in Clpp-deficient cumulus cells have a smaller aspect ratio (length/width) and have a larger coverage area (mitochondrial area/cytoplasmic area) under electron microscopy. These ultrastructural changes were accompanied with diminished expression of mitochondrial dynamics genes. RNA sequencing analysis revealed a significant change in genes related to cellular metabolism in Clpp-deficient cumulus cells compared to wild type. In addition, apoptosis and phagosome pathways were significantly affected. Immunofluorescence assessment confirmed increased apoptotic activity and decreased cell proliferation in cumulus oophorus complexes (COCs) of Clpp-deficient mice. Our findings demonstrate that deletion of CLPP results in significant structural and functional changes in cumulus cells and suggests that mtUPR is required for cumulus cell function.

Can 1-day old human unfertilized oocytes activate and blastulate after exposure to calcium ionophore (Ca.I) A23187?Prospective randomized trial analysis of sibling oocytes. Seventy unfertilized sibling oocytes from 24 couples were randomly split into two groups. In the treatment group, 35 oocytes were cultured with 5-µM Ca.I A23187 for 10 min, washed and cultured until day 6 of development (D+6). The remaining 35 oocytes (control group) were similarly cultured until D+6. Activation, cleavage and blastulation rates were compared between the two groups.Comparable activation rates were observed in the oocytes incubated with Ca.I A23187 and in the control group (11.4% versus 17.1%; P = 0.49). The cleavage rate observed was 45.7% in both groups. None of the embryos reached blastocyst stage.Activation and cleavage can occur in unfertilized oocytes after the diagnosis of failure to fertilize. Unfortunately, the prevalence of activation is not affected by exposure to Ca.I A23187. Additionally, these embryos have no tangible reproductive potential as they arrest before reaching the blastocyst stage.

Mitochondrial unfolded protein response (UPRmt) is a highly conserved mechanism, which is activated upon cellular or metabolic stress and aims to help cells maintain homeostasis. CLPP (caseinolytic peptidase P) plays a crucial factor for UPRmt, it promotes the degradation of unfolded mitochondrial proteins. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Here, we asked whether CLPP is necessary for granulosa/cumulus cell function. Clppflox/flox mice were generated and crossbred with Cyp19a1-Cre mice to generate mice with granulosa/cumulus cell-specific Clpp deletion (Clpp&minus, /&minus, ). Mature (8-week-old) Clpp&minus, female mice (8-week-old) were compared to same age wild type (WT) mice. We found that mature Clpp&minus, female mice were fertile and produced a similar number of pups per litter compared to WT. Folliculogenesis was not affected by the loss of CLPP in granulosa/cumulus cells as Clpp&minus, and WT mice had a similar number of primordial, primary, secondary, early antral, and antral follicles. The number of germinal vesicles (GV) and MII oocytes collected from Clpp&minus, and WT female mice were also similar. Our findings demonstrate that fertility in female mice is not affected by granulosa/cumulus cell-specific UPRmt disruption through CLPP deletion.

The aim of the current study was to characterize ovarian reserve parameters and IVF outcomes in women with a history of poor ovarian response (POR) treated with intraovarian injection of autologous platelet rich plasma (PRP). Reproductive age women (N=510; age range 30-45yo) diagnosed with POR based on Poseidon criteria were included in the study. PRP treatment resulted in higher AFC, higher serum AMH, lower serum FSH, and a higher number of mature oocytes and cleavage and blastocyst stage embryos. After PRP injection, 22 women (4.3%) conceived spontaneously, 14 (2.7%) were lost to follow up, and 474 (92.9%) attempted IVF. Among women who attempted IVF, 312 (65.8%) generated embryos and underwent embryo transfer, 83 (17.5%) achieved a pregnancy, and 54 (11.4%) achieved sustained implantation/live birth (SI/LB). In total, of the 510 women with POR and mean age of 40.3, PRP resulted in improvement of ovarian reserve parameters, a pregnancy rate of 20.5% and SI/LB rate of 12.9%. Our findings suggest that PRP treatment may be considered in women with POR. For wider clinical application, its clinical efficacy will need to be demonstrated in prospective randomized clinical trials.