To compare obstetrical and neonatal outcomes of embryo transfer cycles using fresh vs. frozen-thawed testicular sperm derived from microTESE in non-obstructive azoospermia (NOA) patients. The retrospective cohort study included a total of 48 couples diagnosed with NOA who underwent 93 ET cycles, both fresh and frozen-thawed embryos, and resulted in pregnancy. ET cycles were divided into two groups according to sperm type, fresh (46 cycles, 49.5

Background: Does the Time-lapse Incubator (TLI) add value to reproductive outcomes when its two components, undisturbed culturing and morphokinetic embryo grading, are separated. Methods: A prospective pilot, randomized, controlled, double-blinded, single-center study was conducted during the years 2016–2020. In total, 102 patients were randomized into three groups: (1) conventional incubation with morphological evaluation only (n = 34), (2) TLI with both morphological and morphokinetic evaluations (n = 32), and (3) TLI with morphological evaluation only (n = 36). All arms were cultured in ESCO-MIRI® incubators. A total of 1061 injected mature oocytes were evaluated (420 in arm 1, 285 in arm 2, and 356 in arm 3). The primary outcome was live birth rates. Secondary outcomes included clinical and cumulative pregnancy rates as well as embryo quality. Embryos in arm 3 were retrospectively evaluated for their morphokinetic score. Results: No significant difference was found in the live birth rate for single embryo transfer cycles (SET) (35% vs. 31.6% vs. 24%, p = 0.708) or double embryo transfer (DET) cycles (41.7% vs. 38.5% vs. 36.4%, p = 0.966). Comparable pregnancy rates, clinical pregnancy rates, and cumulative pregnancy were found for similar top-quality embryos for days 2, 3, and blastocyst stages across groups. A similar number of embryos were suitable for either transfer or cryopreservation within the different groups. For 62.8% of the embryos in arm 3, the morphokinetic and morphologic evaluations were similar. In only 2/36 (5.6%) treatment cycles, the use of morphokinetic scoring may have helped the patient avoid undergoing an additional treatment cycle. In the other cases, morphokinetic scoring would not have changed the end point of pregnancy. Conclusions: The two components of the TLI system—undisturbed culturing and morphokinetic embryo grading—do not appear to have a significant additional value in reproductive outcome, although these results should be validated by an RCT.

OBJECTIVE:Pre-treatment (PT) therapies in IVF are known to be used as pre-stimulation modality to improve cycle outcomes. This study aims to assess whether PT in GnRH antagonist cycles triggered with GnRH-agonist impact oocyte maturation response. METHODS:Data were retrospectively collected for patients who underwent GnRH antagonist cycle with agonist triggering with and without PT. The patients were allocated to groups according to their PT status. The primary outcome evaluated was suboptimal maturation response. Suboptimal maturation to trigger was defined as no oocyte upon retrieval when adequate response was expected. RESULTS:The study population included 196 patients who underwent GnRH antagonist cycle with agonist triggering. The study group included 69 patients who received PT. The control group included 127 patients with no PT. In univariate analysis, the PT group significantly displayed suboptimal response compared to the controls (p = 0.008). All the patients in the study group with suboptimal response (with or without hCG re-triggering) were treated with GnRH-agonist as PT. Basal and pre-trigger LH values were significantly lower in the study group compared to controls (p < 0.001). Multivariate regression analysis revealed that PT with GnRH agonist was a significant predictor for suboptimal response. CONCLUSIONS:Pre-treatment, and particularly the use of GnRH-agonist as PT in antagonist cycles triggered with agonist, increases the risk of suboptimal response to GnRH-agonist trigger. This might be explained by prolonged pituitary suppression, which lasts beyond the PT cessation.

Following administration of the SARS-CoV-2 vaccine, many women worldwide reported short-term menstrual irregularities. Although menstrual bleeding, "the fifth vital sign", is experienced by more than 300 million people on any given day worldwide, these changes were only partially studied. Irregular periods are important well beyond fertility and the discomfort they impose; they are associated with the risk of cardiovascular morbidity, chronic diseases, and premature mortality. Pre-clinical examination of the vaccine polymeric envelope indicates its accumulation in the ovaries. The somatic endocrine cells of the ovarian follicle - the granulosa cells (GCs)-participate in the strict hypothalamic-pituitary-ovarian (HPO) feedback loop that governs the menstrual cycle via endocrine and paracrine regulators, as AMH and Inhibins. We aimed to unravel the direct effect of the COVID-19 vaccine on GCs and link their post-vaccine activity to changes in menstrual patterns. Human primary GCs exposed in-vitro to the Pfizer COVID-19 vaccine BNT162b2, demonstrated no change in their viability but altered mRNA transcripts, specifically of the regulatory key factors: InhibinB was upregulated, whereas AMH was downregulated. We further examined pre- and post-vaccination blood samples from individual women and found a 2-3 folds change in the post-vaccination FSH/InhibinB protein level ratio, compared to their pre-vaccination values. This altered expression of InhibinB could significantly impact the HPO axis in vaccinated women and may ultimately influence the endometrium cyclicity, manifested clinically by the commonly reported changes in menstrual bleeding patterns.

BACKGROUND Awareness of the age-related decline in fertility potential has increased the popularity of planned oocyte cryopreservation (POC). However, data regarding outcomes of POC, including rates of women returning to thaw oocytes, as well as pregnancy and live birth rates, are scarce and based mostly on small case series.OBJECTIVE AND RATIONALE POC was defined as cryopreservation exclusively for prevention of future age-related fertility loss. The primary outcome was live birth rate per patient. The secondary outcomes included the return to thaw rate and laboratory outcomes. A meta-regression analysis examining the association between live birth and age above 40 or below 35 was conducted.SEARCH METHODS We conducted a systematic database search from inception to August 2022. The search included PubMed (MEDLINE) and EMBASE. Our search strategies employed a combination of index terms (Mesh) and free text words to compile relevant concepts. The systematic review and meta-regression were undertaken following registration of systematic review (PROSPERO registration number CRD42022361791) and were reported following guidelines of Preferred Reporting Items for Systematic Review and Meta-Analyses 2020 (PRISMA 2020).OUTCOMES The database search yielded 3847 records. After the selection process, 10 studies, conducted from 1999 to 2020, were included. Overall, 8750 women underwent POC, with a mean cryopreservation age of 37.2 (+/- 0.8). Of those, 1517 women returned to use their oocytes with a return rate of 11.1% (+/- 4.7%). The mean age at the time of cryopreservation for women who returned to use their oocytes was 38.1 (+/- 0.4), with an average of 12.6 (+/- 3.6) cryopreserved oocytes per woman. In a meta-analysis, the oocyte survival rate was 78.5% with a 95% CI of 0.74-0.83 (I2 = 93%). The live birth rate per patient was 28% with a 95% CI of 0.24-0.33 (I2 = 92%). Overall, 447 live births were reported. In a sub-group analysis, women who underwent cryopreservation at age >= 40 achieved a live birth rate per patient of 19% (95% CI 0.13-0.29, I2 = 6%), while women aged <= 35 years old or younger had a higher live birth rate per patient of 52% (95% CI 0.41-0.63, I2 = 7%).WIDER IMPLICATIONS POC emerges as a feasible option for women aiming to improve their chances of conceiving at a later reproductive age. Nonetheless, it must be acknowledged that the overall success rates of POC are limited and that the likelihood of successful live birth declines as the age at cryopreservation rises. With increasing interest in POC, the collation of comprehensive and high-quality data is imperative to clearly define the outcomes for various age groups.REGISTRATION NUMBER CRD42022361791. Graphical abstract In meta-analysis, Planned Oocyte Cryopreservation yielded 28% live birth rate, strongly correlated with age at cryopreservation.

Since the beginning of the SARS-CoV-2 pandemic, there have been rising concerns about the virus's possible ability to affect male and female fertility. Although effective vaccines were introduced and the vaccination rate of the general population is high, some reproductive-age individuals are still hesitant to receive the vaccine, because of an unestablished belief that the vaccine might impair fertility. In this single-center retrospective study, encompassing data from 387 medical files of in-vitro fertilization (IVF) patients we compared IVF cycle outcomes and sperm characteristics in vaccinated couples before and after vaccination, as well as between vaccinated patients and a control group of individuals who were neither vaccinated nor infected with COVID-19 before or during the cycles. We found no significant differences between vaccinated and non-vaccinated patients concerning the number of retrieved oocytes and the total motile sperm count (TMC). The mean number of retrieved oocytes showed a slight increase in the vaccinated group compared to the non-vaccinated control group (10.8 vs. 9.18, p = 0.14). Additionally, within the vaccinated group, no significant difference was observed in the mean number of oocytes before and after vaccination (9.7 and 10.8, p = 0.14). Other similar cycle outcomes between the groups were the rates of implantation, pregnancy, and ovarian hyperstimulation syndrome. This study emphasized that the mRNA anti-COVID-19 vaccination doesn't adversely affect ovarian response or sperm quality in IVF patients. These findings contribute valuable insights to the safety profile of anti-COVID-19 vaccines in the context of reproductive-aged populations, aiding decision-making during ongoing virus outbreaks and potential future scenarios.

RESEARCH QUESTION:What is the involvement of pigment epithelium-derived factor (PEDF), expressed in granulosa cells, in folliculogenesis? DESIGN:mRNA expression of PEDF and other key factors [Cyp19, anti-Müllerian hormone receptor (AMHR) and vascular endothelial growth factor (VEGF)] in mice follicles was examined in order to typify the expression of PEDF in growing follicles and in human primary granulosa cells (hpGC), and to follow the interplay between PEDF and the other main players in folliculogenesis: FSH and AMH. RESULTS:mRNA expression of PEDF increased through folliculogenesis, although the pattern differed from that of the other examined genes, affecting the follicular angiogenic and oxidative balance. In hpGC, prolonged exposure to FSH stimulated the up-regulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed: AMH stimulation reduced the expression of PEDF mRNA and PEDF stimulation reduced the expression of AMHR mRNA. CONCLUSIONS:Folliculogenesis, an intricate process that requires close dialogue between the oocyte and its supporting granulosa cells, is mediated by various endocrine and paracrine factors. The current findings suggest that PEDF, expressed in granulosa cells, is a pro-folliculogenesis player that interacts with FSH and AMH in the process of follicular growth. However, the mechanism of this process is yet to be determined.

Abstract Study question Does the TLI have an added value in reproductive outcome when separating the system’s two components, undisturbed culturing, and morphokinetic embryo grading? Summary answer The TLI system itself does not have a significant added value. More advanced algorithms should be applied to aid in embryo selection process. What is known already The TLI technology has been widely used for over two decades for embryo culturing. A Cochrane meta-analysis found no significant difference in clinical pregnancy rate using TLI compared to conventional incubators. Another RCT concluded that the addition of TLI morphokinetic data did not significantly improve clinical reproductive outcomes. On the other hand, the benefit of TLI is the identification of abnormal cleavage dynamics and analysis of other morphokinetic parameters although no proven increase in pregnancy rate was shown. Several Artificial intelligence algorithms use TLI data to increase the pregnancy rate and embryo selection process. Study design, size, duration A prospective, randomized, double-blinded, single-center, controlled study. After applying inclusion criteria, 102 women were included in the analysis, 34, 32, and 36 in arms 1,2, and 3 respectively, with a total of 1061 embryos evaluated, 420 in arm 1, 285 in arm 2, and 356 in arm 3. Patients were recruited between November 2017- July 2020. Participants/materials, setting, methods University-affiliated Medical Centre in vitro fertilization (IVF) clinic. Patients were randomized into 3 groups: 1) conventional incubator with morphological evaluation only; 2) TLI incubator with both morphological and morphokinetic evaluation; 3) TLI incubation with morphological evaluation only. All were cultured in MIRI ESCO incubators. The primary outcome was live birth rates. Secondary outcomes were clinical pregnancy rates and cumulative pregnancy rates. Embryo quality and morphokinetic scores. Main results and the role of chance No differences were found in the rated day 2 top quality embryos (TQE), day 3 TQE or blastocyst stage TQE, and a similar number of embryos were suitable for either transfer or cryopreservation between the groups. On the other hand, higher rates of multinucleation were found in group 1 (31.9±6 vs. 16.6±5 vs.14.9±5, p = 0.03 for groups 1,2 and 3 respectively). Regarding primary outcome, we did not find any significant difference in live birth rate between the groups, neither for single embryo transfer cycles (SET) (35% vs. 31.6% vs. 24%, p = 0.708) nor for double embryo transfer cycles (DET) (41.7% vs. 38.5% vs. 36.4%, p = 0.966 for groups 1,2 and 3 respectively). Also, no differences were noted between pregnancy rates and clinical pregnancy rates. After concluding the first part of the study, embryos in arm 3 were retrospectively reevaluated, gaining additional morphokinetic data for the embryo selection process. In more than half of the embryos selected for transfer, the morphokinetic and morphologic evaluations were similar. For the additional 32 embryos from a total of 12 cycles, there was a discrepancy between the grades. In the other cases, the use of morphokinetic scoring would not have changed significantly the end point of pregnancy. Limitations, reasons for caution The pandemic affected patient recruitment and smaller numbers than intended were achieved leaving the study sample underpowered for the primary endpoint. Both day 3 and day 5 transfers were performed, and cycles were not always restricted to single embryo transfer, although the study was designed as such. Wider implications of the findings This study underlines the maybe unjustified morphkinetic scoring grading system which requires much time and attention from the embryologist to view and annotate and should be reconsidered especially with the rise of substitute software-assisted assessment algorithms showing an additional advantage in embryo selection and implantation rate. Trial registration number ClinicalTrials.gov Identifier: NCT02657811.

PurposeTo compare pregnancy rates in GnRH-antagonist cycles triggered with hCG after luteal phase support with intranasal GnRH-agonist as sole luteal phase support versus standard vaginal progesterone preparation.MethodsProspective randomized controlled study of patients who underwent antagonist-based IVF cycles triggered with hCG at university-affiliated tertiary medical center between 2020 and 2022. Patients meeting the inclusion criteria were randomly assigned to either intranasal GnRH-agonist or vaginal progesterone for luteal phase support. Pregnancy rates were the main outcome compared between the two study groups.ResultsA total of 150 patients underwent 164 cycles, 127 cycles of which were included in the study cohort. Of them, 64 (50.4%) and 63 (49.6%) cycles were treated with GnRH-agonist or progesterone, respectively, as sole luteal phase support. A significantly higher pregnancy rate was demonstrated in the GnRH-agonist group compared with the progesterone group. After adjustment of several potential confounders such as age, body mass index, past obstetric history, number of IVF cycles, oocyte retrieved and embryos transferred, GnRH-agonist was still associated with a higher pregnancy rate (odds ratio 3.4, 95% confidence interval 1.4-8.3). Ovarian hyperstimulation syndrome rates were similar between the groups.ConclusionsThis prospective study suggests that nasal GnRH-agonist for luteal phase support is associated with higher pregnancy rates compared with standard progesterone support in an antagonist-based protocol triggered with hCG, while maintaining a similar safety profile.

Research question: Do preimplantation genetic testing (PGT) pregnancies have higher post-partum complications compared with naturally conceived pregnancies? Design: Retrospective cohort study conducted in 2008-2020 at the Shaare Zedek Medical Center (SZMC), including all patients aged 18-45 years old who conceived following PGT with a singleton live birth >24 weeks. Data were collected from computerized hospital databases and patient files. There were two control groups: (i) pregnancies following IVF-ICSI (intracytoplasmic sperm injection); (ii) four neighbourhood controls for each case delivery (two women delivered before and two after) of women with naturally conceived pregnancies. Results: Overall, 120 PGT, 779 IVF-ICSI and 3507 naturally conceived deliveries were included. Demographic variables were similar apart from slightly higher age in the PGT (P = 0.003) and ICSI (P = 0.002) groups (31.07 +/- 4.38 PGT, 31.66 +/- 5.03 ICSI, 28.77 +/- 5.72 naturally conceived). Composite post-partum placental-related complications (manual lysis of placenta, revision of uterine cavity, haemoglobin drop >= 3 g/dl, post-partum haemorrhage, need for blood transfusion) were more prevalent in both the PGT and IVF-ICSI groups as opposed to naturally conceived (20.0% versus 18.9% versus 10.3%, respectively, P < 0.001, P = 0.007). In a multivariate regression model PGT was not found to be independently associated with composite post-partum placental-related complications (adjusted odds ratio [aOR] 1.44, 95% confidence interval [CI] 0.83-2.15), while IVF-ICSI pregnancies had increased risk (aOR 1.52, 95% CI 1.20-1.97) compared with natural conception. No difference was found between fresh and frozen cycles or between day 3 and day 5 embryo transfer. Conclusions: PGT pregnancies have a comparable risk of post-partum placental-related complications to naturally conceived pregnancies, unlike IVF-ICSI pregnancies. It is possible that infertility itself is the main mediator for post-partum complications in IVF-ICSI pregnancies.

ABSTRACT: this paper proposes that early modern Ottoman intellectuals familiar with a medieval cosmographic theory of wonder formed a large emotional community whose members valued and practiced this emotion towards imperial Ottoman mosques. To achieve this objective, I draw upon a variety of sources from the early modern period, including chronicles, treatises on architecture, and travelogues, created by individuals who belonged to this emotional community. Sources such as these contain words, symbols, and gestures, that aided the whole group in recognizing this desirable emotion in the context of imperial architecture. Through their writing, these individuals sought to instill cosmographic wonder among their readers, while at the same time conveying the message that imperial mosques represent the creation of God. Such a narrative was employed by the Ottomans as long as cosmographies were in demand. From the beginning of the seventeenth century, when updated studies on geography gradually replaced cosmographies, there were those within this emotional community who underwent an epistemological change. From this point on, wonder at Ottoman architecture was generated by different stimuli which were not necessarily related to the cosmographic theory of wonder.

This multi-center study evaluated a novel microscope system capable of quantitative phase microscopy (QPM) for label-free sperm-cell selection for intracytoplasmic sperm injection (ICSI). Seventy-three patients were enrolled in four in vitro fertilization (IVF) units, where senior embryologists were asked to select 11 apparently normal and 11 overtly abnormal sperm cells, in accordance with current clinical practice, using a micromanipulator and 60× bright field microscopy. Following sperm selection and imaging via QPM, the individual sperm cell was chemically stained per World Health Organization (WHO) 2021 protocols and imaged via bright field microscopy for subsequent manual measurements by embryologists who were blinded to the QPM measurements. A comparison of the two modalities resulted in mean differences of 0.18 µm (CI −0.442–0.808 µm, 95%, STD—0.32 µm) for head length, −0.26 µm (CI −0.86–0.33 µm, 95%, STD—0.29 µm) for head width, 0.17 (CI −0.12–0.478, 95%, STD—0.15) for length–width ratio and 5.7 for acrosome–head area ratio (CI −12.81–24.33, 95%, STD—9.6). The repeatability of the measurements was significantly higher in the QPM modality. Surprisingly, only 19% of the subjectively pre-selected normal cells were found to be normal according to the WHO2021 criteria. The measurements of cells imaged stain-free through QPM were found to be in good agreement with the measurements performed on the reference method of stained cells imaged through bright field microscopy. QPM is non-toxic and non-invasive and can improve the clinical effectiveness of ICSI by choosing sperm cells that meet the strict criteria of the WHO2021.

Abstract Study question What are the chances of achieving a live birth after planned oocyte cryopreservation (POC)? Summary answer The mean thawing rate is 11.9% (± 4.9). Among women who thawed their oocytes, the mean live birth per patient is 28.1% (± 13.0). What is known already Trends of delayed childbearing have become increasingly common. The age-related decline in fertility potential increased the popularity of planned oocyte cryopreservation. However, Data on the outcomes of planned oocyte cryopreservation including a return for a thaw, pregnancy, and live-birth rates are scarce, being based mostly on small case series. Study design, size, duration A systematic review and meta-regression followed the PRISMA and MOOSE guidelines. A systematic search was conducted in Medline, Embase, and the Cochrane Library. The search strategies incorporated index terms (Mesh) and free text words for the search concepts. The first domain contained terms on indication the second domain related to oocytes cryopreservation. The detailed protocol is documented online in the International Prospective Register of Systematic Reviews registry. Participants/materials, setting, methods POC was defined as cryopreservation for future age-related fertility loss exclusively. All studies that reported primary data on POC were considered eligible for screening. Observational and non-observational studies were included. The primary outcome was the live birth rate per woman. The secondary outcomes included the thawing rate and other laboratory outcomes. Meta-regression analyses regarding the association between oocyte survival after thawing and live birth and age above 40 or method of freezing were conducted. Main results and the role of chance A systematic search from inception to October 2022 yielded 3847 citations. After the selection process, 9 studies, conducted from 1999 to 2020, were included. In total, 8059 women underwent POC, and 1463 returned to use their oocytes. The mean thawing rate was 11.9% (±4.9). The mean age at thawing was 41.9 years (±0.8) with a mean time from freezing to thawing of 4.0 years (±- 0.4). 68.3% (± 18.4 ) of patients returned with a partner while the others used donor sperm. Five studies used vitrification exclusively while the others included both slow-freezing and vitrification. The oocytes survival rate after thawing was 79.0% (±5.4 ) and the mean live birth per patient was 28.1% (± 13.0). A meta-regression analysis revealed an association between age above 40 years and decreased live birth (R2=0.08) and oocyte survival (R2=0.16). No statistically significant association was found between the method of cryopreservation to oocyte survival rate or live-birth Limitations, reasons for caution Variations in expertise and technical experience between different centers may yield different results. In addition, most data reports outcomes for women who underwent POC aged 35-40, while information on women who had this procedure at younger or older ages is scarce. Wider implications of the findings Data presented here may be valuable for consultation and informed decision-making of women considering POC Trial registration number CRD42022361791 02/10/2022

Reproductive aging is characterized by a decline in ovarian function and in oocytes’ quantity and quality. Pigment epithelium-derived factor (PEDF), a pivotal player in ovarian angiogenic and oxidative balance, was evaluated for its involvement in reproductive aging. Our work examines the initial stage of reproductive aging in women and mice, and the involvement of PEDF in the process. Granulosa cells from reproductively-aged (RA) women and mice (36–44 years old and 9–10 months old, respectively) indicated an increase in the level of PEDF mRNA (qPCR), with yet unchanged levels of AMH and FSHR mRNAs. However, the PEDF protein level in individual women showed an intra-cellular decrease (ELISA), along with a decrease in the corresponding follicular fluid, which reflects the secreted fraction of the protein. The in vitro maturation (IVM) rate in the oocytes of RA mice was lower compared with the oocytes of young mice, demonstrated by a reduced polar body extrusion (PBE) rate. The supplementation of PEDF improved the hampered PBE rate, manifested by a higher number of energetically-competent oocytes (ATP concentration and mtDNA copy number of individual oocytes). Our findings propose PEDF as an early marker of reproductive aging, and a possible therapeutic in vitro agent that could enhance the number of good-quality oocytes in older IVF patients.

Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I-arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse-transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT-1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin-associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.

To report outcome of planned oocyte cryopreservation (POC) in the first 8 years of this treatment in our center. A retrospective study in a university-affiliated medical center. A total of 446 women underwent POC during 2011–2018. Fifty-seven (13

As we age, shifts in heterochromatin levels change the epigenetic landscape in our cells. One of the consequences of heterochromatin de-regulation may be the de-repression of transposable elements (TE). In this work we examine the epigenetic changes in aged oocytes, and their impact on oocyte quality. Importantly, we examined this effect before the onset of significant aneuploidy. Epigenetic status was investigated by immunofluorescence (IF) in mouse and human oocytes. Young and older mouse oocytes were stained in situ or by chromosome spread. Retrotransposon activity and processing was explored using RT-PCR and smRNA sequencing and confirmed by IF. IF was performed to examine RNA processing machinery components, and DNA damage markers. In vitro maturation assays in the presence of heterochromatin modifying chemicals were used to confirm the casual relationship between heterochromatin loss, retrotransposon activity and maturation potential. The loss of heterochromatin in aged oocytes was demonstrated by decreased immuno-staining signal for heterochromatin markers H3K9me2, HP1γ and H3K27me3. Staining for H3K9me2 in human oocytes showed decrease as well. The de-regulation on TE transcription were observed by increased RNA expression. LINE-1 overexpression was confirmed by high presence of L1-ORF1p in IF staining of old oocytes. In accordance with increased activity of these TE, older oocytes show increased recruitment of DNA repair proteins indicative of DNA damage. This was shown by elevated Rad51 in older oocytes, and of γH2Ax. Inhibiting retrotransposon reverse-transcriptase through Azidothymidine (AZT) treatment in older oocytes partially rescued maturation rates , and lowered their DNA damage. TEs were intensively processed by the oocyte. Staining for Dicer and dsRNA showed an increase with age. Specific TE types showed higher processing in older oocytes as evident by smRNA-seq. In order to show a causal link between epigenetic deterioration and maturation defects, we treated young mouse oocytes with heterochromatin damaging compounds (e.g. Chaetocin, TSA). Treated oocytes presented loss of maturation, accompanied with major loss of H3K9me2, and increased L1-ORF1p activity. Our work demonstrates an epigenetic aspect of oocyte aging, which occurs before the onset of aneuploidy. This aspect is characterized by the loss of heterochromatin associated chromatin marks and activation of retrotransposons which cause DNA damage and impairs oocyte maturation.

Research question: What are the reproductive choices and retrospective reflections of women at least 4 years after planned oocyte cryopreservation (POC)? Design: This was an internet survey, using the REDCap application, of women who underwent POC, at a single-centre university affiliated IVF unit, 4-8 years before the survey. The questionnaire addressed reproductive choices and outcomes following POC. Results: Seventy-nine women who underwent POC during 2011-2014 were invited to participate, and 70 (89%) responded. Mean age at cryopreservation was 37.1 +/- 2.4 (range 30-41) years, mean age at study participation 42.6 +/- 2.6 (range 35- 48) years, and mean time from first cryopreservation cycle to study participation 5.5 +/- 1.3 (range 4-8) years. The main retrospectively reported reason for POC was not wanting to become pregnant without a partner (59, 84%). During the followup period, 44 women (63%) attempted to conceive either naturally or by assisted reproductive technology using fresh or cryopreserved oocytes. Of those, 28 women achieved a live birth (64% of those who tried to conceive). Fourteen respondents (20% of all respondents) reported using their cryopreserved oocytes, and three (21%) achieved a birth using those oocytes. Fifteen women (34%) of those who tried to conceive used donor spermatozoa. Conclusions: The most common reasons for not using frozen oocytes were achieving pregnancy without frozen oocytes or preferring not to have a child without a partner. A considerable proportion of women who had POC and were not interested in being a single parent by choice eventually try to conceive using donor spermatozoa several years later.

PURPOSE:We previously developed Haploseek, a method for comprehensive preimplantation genetic testing (PGT). However, some key features were missing, and the method has not yet been systematically validated.METHODS:We extended Haploseek to incorporate DNA from embryo grandparents and to allow testing of variants on chromosome X or in regions where parents share common haplotypes. We then validated Haploseek on 151 embryo biopsies from 27 clinical PGT cases. We sequenced all biopsies to low coverage (0.2×), and performed single-nucleotide polymorphism (SNP) microarray genotyping on the embryos' parents and siblings/grandparents. We used the extended Haploseek to predict chromosome copy-number variants (CNVs) and relevant variant-flanking haplotypes in each embryo. We validated haplotype predictions for each clinical sample against polymerase chain reaction (PCR)-based PGT case results, and CNV predictions against established commercial kits.RESULTS:For each of the 151 embryo biopsies, all Haploseek-derived haplotypes and CNVs were concordant with clinical PGT results. The cases included 17 autosomal dominant, 5 autosomal recessive, and 3 X-linked monogenic disorders. In addition, we evaluated 1 Robertsonian and 2 reciprocal translocations, and 17 cases of chromosome copy-number counting were performed.CONCLUSION:Our results demonstrate that Haploseek is clinically accurate and fit for all standard clinical PGT applications.

Molecular changes, caused by various environmental factors, affect the quality and developmental potential of oocytes. Oxidative stress (OS) is a major factor involved in various gynecologic disorders and/or in aging. Recent studies suggest that elevated reactive oxygen species (ROS) hamper oocyte quality and future embryonic development. Pigment epithelium-derived factor (PEDF) is a pleiotropic protein, known for its antiangiogenic, anti-inflammatory, and antioxidative properties. Our previous findings demonstrate the antioxidative role of rPEDF in maintaining granulosa cell viability. In the current study, we examined the ability of PEDF to negate the adverse impact of OS on oocytes. Maturation rate of oocytes exposed to OS was significantly lower than that of control oocytes. The number of mtDNA copies in OS-exposed oocytes was significantly higher than in control oocytes (>3 times), whereas ATP concentration was significantly lower. Oocytes exposed to OS demonstrated impaired chromosome arrangement at the metaphase plate. PEDF significantly improved maturation rate of untreated OS-exposed oocytes. Moreover, mtDNA copy number, ATP concentration, and chromosome arrangement at the metaphase plate in rPEDF-treated OS-exposed oocytes were restored to the level of control oocytes. Our findings demonstrate that OS hampers the ability of oocytes to undergo proper in vitro maturation. The energetic balance of OS-exposed oocyte is characterized by excessive mtDNA replication and reduced ATP concentration; it hampers the ability of oocytes to perform high fidelity chromosome segregation. PEDF alleviates this damage, improves the rate of oocyte maturation, and preserves mtDNA level and ATP content, thus enabling oocytes to form proper metaphase plate and improve oocyte competence.