Single-cell characterization of self-renewing primary trophoblast organoids as modeling of EVT differentiation and interactions with decidual natural killer cells.

BACKGROUND: Extravillous trophoblast cell (EVT) differentiation and its communication with maternal decidua especially the leading immune cell type natural killer (NK) cell are critical events for placentation. However, appropriate in vitro modelling system and regulatory programs of these two events are still lacking. Recent trophoblast organoid (TO) has advanced the molecular and mechanistic research in placentation. Here, we firstly generated the self-renewing TO from human placental villous and differentiated it into EVTs (EVT-TO) for investigating the differentiation events. We then co-cultured EVT-TO with freshly isolated decidual NKs for further study of cell communication. TO modelling of EVT differentiation as well as EVT interaction with dNK might cast new aspect for placentation research. RESULTS: Single-cell RNA sequencing (scRNA-seq) was applied for comprehensive characterization and molecular exploration of TOs modelling of EVT differentiation and interaction with dNKs. Multiple distinct trophoblast states and dNK subpopulations were identified, representing CTB, STB, EVT, dNK1/2/3 and dNKp. Lineage trajectory and Seurat mapping analysis identified the close resemblance of TO and EVT-TO with the human placenta characteristic. Transcription factors regulatory network analysis revealed the cell-type specific essential TFs for controlling EVT differentiation. CellphoneDB analysis predicted the ligand-receptor complexes in dNK-EVT-TO co-cultures, which relate to cytokines, immunomodulation and angiogenesis. EVT was known to affect the immune properties of dNK. Our study found out that on the other way around, dNKs could exert effects on EVT causing expression changes which are functionally important. CONCLUSION: Our study documented a single-cell atlas for TO and its applications on EVT differentiation and communications with dNKs, and thus provide methodology and novel research cues for future study of human placentation.

Chinese experts' consensus guideline on preimplantation genetic testing of monogenic disorders.

Recent developments in molecular biological technologies and genetic diagnostic methods, accompanying with updates of relevant terminologies, have enabled the improvements of new strategies of preimplantation genetic testing for monogenic (single gene) disorders (PGT-M) to prevent the transmission of inherited diseases. However, there has been much in the way of published consensus on PGT-M. To properly regulate the application of PGT-M, Chinese experts in reproductive medicine and genetics have jointly developed this consensus statement. The consensus includes indications for patient selection, genetic and reproductive counseling, informed consent, diagnostic strategies, report generation, interpretation of results and patient follow-ups. This consensus statement serves to assist in establishment of evidence-based clinical and laboratory practices for PGT-M.

Effect of noninvasive embryo viability testing versus conventional IVF on the live birth rate in IVF/ICSI patients: a study protocol for a double-blind, multicenter, randomized controlled trial.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) was demonstrated to be superior to conventional IVF in reducing the incidence of miscarriage and abnormal offspring after the first embryo transfer (ET). PGT-A requires several embryo trophectoderm cells, but its negative impacts on embryo development and long-term influence on the health conditions of conceived children have always been a concern. As an alternative, noninvasive PGT-A (niPGT-A) approaches using spent blastocyst culture medium (SBCM) achieved comparable accuracy with PGT-A in several pilot studies. The main objective of this study is to determine whether noninvasive embryo viability testing (niEVT) results in better clinical outcomes than conventional IVF after the first embryo transfer. Furthermore, we further investigated whether niEVT results in higher the live birth rate between women with advanced maternal age (AMA, > 35 years old) and young women or among patients for whom different fertilization protocols are adopted. METHODS: This study will be a double-blind, multicenter, randomized controlled trial (RCT) studying patients of different ages (20-43 years) undergoing different fertilization protocols (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]). We will enroll 1140 patients at eight reproductive medical centers over 24 months. Eligible patients should have at least two good-quality blastocysts (better than grade 4 CB). The primary outcome will be the live birth rate of the first embryo transfer (ET). Secondary outcomes will include the clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, cumulative live birth rate, ectopic pregnancy rate, and time to pregnancy. DISCUSSION: In this study, patients who undergo noninvasive embryo viability testing (niEVT) will be compared to women treated by conventional IVF. We will determine the effects on the pregnancy rate, miscarriage rate, and live birth rate and adverse events. We will also investigate whether there is any difference in clinical outcomes among patients with different ages and fertilization protocols (IVF/ICSI). This trial will provide clinical evidence of the effect of noninvasive embryo viability testing on the clinical outcomes of the first embryo transfer. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) Identifier: ChiCTR2100051408. 9 September 2021.

Human leukocyte antigen-G in gynaecological tumours.

Gynaecological tumours that threaten the health of women, especially when advanced and recurrent, have remained mostly intractable to existing treatments. Therefore, new therapeutic targets are urgently needed. Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex class I molecule typically expressed in foetuses for protection against destruction by the maternal immune system. HLA-G is also expressed under pathological conditions, such as in solid tumours, and may participate in tumour development and serve as a novel immune checkpoint in cancer. Furthermore, it is expressed in most gynaecological tumours. Therefore, inhibiting HLA-G and its receptors to block the immune escape pathway could represent a new strategy in cancer immunotherapy. To the best of our knowledge, this review is the first to summarize recent research findings on HLA-G in gynaecological oncology. We highlight the fact that HLA-G is expressed in gynaecological tumour tissues, wherein it inactivates immune effectors involved in tumour progression. Further studies on HLA-G in gynaecological oncology are needed to incorporate HLA-G into the design and evaluation of immunotherapy for malignant gynaecological diseases.

Preimplantation genetic testing for hereditary hearing loss in Chinese population.

PURPOSE: To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. METHODS: A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled. RESULTS: Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%. CONCLUSIONS AND RELEVANCE: In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.

Preimplantation genetic testing for aneuploidy helps to achieve a live birth with fewer transfer cycles for the blastocyst FET patients with unexplained recurrent implantation failure.

PURPOSE: This retrospective cohort study aimed to investigate the value of preimplantation genetic testing for aneuploidy (PGT-A) as a screening test for patients suffering from unexplained recurrent implantation failure (RIF). METHODS: After screening patients in one reproductive medicine center, twenty-nine, forty-nine and thirty-eight women (< 40 years old) who had suffered unexplained RIF with PGT-A, or RIF without PGT-A, or no RIF with PGT-A were included. The clinical pregnancy rate and live birth rate per transfer, the conservative and optimal cumulative clinical pregnancy rates (CCPR) and live birth rates (CLBR) after three blastocyst FETs were analyzed. RESULTS: The live birth rate per transfer was significantly higher in the RIF + PGT-A group than that in the RIF + NO PGT-A group (47.6% vs. 24.6%, p = 0.014). After 3 cycles of FET, RIF + PGT-A group had significantly higher conservative CLBR and optimal CLBR compared to the RIF + NO PGT-A group (69.0% vs. 32.7%, p = 0.002 and 73.7% vs. 57.5%, p = 0.016), but had similar conservative and optimal CLBRs compared to the NO RIF + PGT-A group. The number of FET cycles required when half women achieved a live birth was 1 in the PGT-A group and 3 in RIF + NO PGT-A group. The miscarriage rates were not different between the RIF + PGT-A and RIF + NO PGT-A, RIF + PGT-A and NO RIF + PGT-A groups. CONCLUSION: PGT-A did be superior in reducing the number of transfer cycles required to achieve a similar live birth rate. Further studies to identify the RIF patients who would benefit most from PGT-A are necessary.

Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.

During implantation, a symphony of interaction between the trophoblast originated from the trophectoderm of the implanting blastocyst and the endometrium leads to a successful pregnancy. Defective interaction between the trophoblast and endometrium often results in implantation failure, pregnancy loss, and a number of pregnancy complications. Owing to ethical concerns of using in vivo approaches to study human embryo implantation, various in vitro culture models of endometrium were established in the past decade ranging from two-dimensional cell-based to three-dimensional extracellular matrix (ECM)/tissue-based culture systems. Advanced organoid systems have also been established for recapitulation of different cellular components of the maternal-fetal interface, including the endometrial glandular organoids, trophoblast organoids and blastoids. However, there is no single ideal model to study the whole implantation process leaving more research to be done pursuing the establishment of a comprehensive in vitro model that can recapitulate the biology of trophoblast-endometrium interaction during early pregnancy. This would allow us to have better understanding of the physiological and pathological process of trophoblast-endometrium interaction during implantation.

Effect of long-duration oxygen vs room air during labor on umbilical cord venous partial pressure of oxygen: a randomized controlled trial.

BACKGROUND: There are limited data to guide the duration and dose of oxygen supplementation for pregnant women undergoing labor. OBJECTIVE: To assess the effect of maternal long-duration high-concentration oxygen administration during labor on umbilical cord venous partial pressure of oxygen. STUDY DESIGN: This randomized clinical trial was conducted between January and October of 2021 in the obstetrics wards of 3 tertiary teaching hospitals in Beijing, China. Women undergoing the latent phase of labor with no existing medical conditions or obstetrical complications who were admitted for delivery were eligible. The women who met inclusion criteria with category I fetal heart rate tracings in labor were randomized in a 1:1 ratio to oxygen or room air. The oxygen group received 10 L of oxygen per minute by simple, tight-fitting face mask until delivery. The room-air group received room air only, without a face mask. The primary outcome was the umbilical cord venous partial pressure of oxygen. RESULTS: A total of 661 women were screened, and 521 were excluded; 140 participants with category I fetal heart rate tracings were enrolled and randomized to oxygen (N=70) or room air (N=70). A total of 135 women with valid paired umbilical cord venous and arterial gas values were included in the umbilical cord venous partial pressure of oxygen and arterial pH analyses. All 140 women were included in the fetal heart rate tracings analysis. Baseline characteristics were similar between the oxygen and room-air groups. The duration of oxygen exposure was approximately 322±147 minutes. There were no differences between the oxygen and room-air groups in the umbilical cord venous partial pressure of oxygen (mean difference, 1.1 mm Hg; 95% confidence interval, -1.0 to 3.2; P=.318) or the proportion of participants with category II fetal heart rate tracings (81.4% vs 78.6%; relative risk, 1.04; 95% confidence interval, 0.88-1.22; P=.672). However, the umbilical cord arterial pH was significantly lower in the oxygen group than in the room-air group (median, 7.23; interquartile range, 7.20-7.27 vs median 7.27; interquartile range, 7.20-7.30; P=.005). CONCLUSION: Maternal long-duration high-concentration oxygen administration during labor did not affect either the umbilical cord venous partial pressure of oxygen or fetal heart rate pattern distribution but resulted in a deterioration of the umbilical cord arterial pH at birth.

PGT-A: The biology and hidden failures of randomized control trials.

OBJECTIVE: Preimplantation Genetic Testing - Aneuploidy (PGT-A) for embryo selection has undergone significant advancements in the last 2 decades and yet many studies still fail to demonstrate any clinical benefits over traditional embryo morphology selection (Mo-S). To understand this conundrum, we performed a multi-center clinical study of PGT-A patients, where Mo-S and euploid selection (Eu-S) outcomes were directly compared. METHOD: All suitable blastocysts were biopsied and analyzed for chromosome copy number. Outcomes (positive beta hCG, implantation, ongoing pregnancy, and live birth rates) for Eu-S were compared to Mo-S using single embryo transfers. RESULTS: Compared to Eu-S embryos, Mo-S embryos resulted in significant reduction of outcomes for positive beta hCG (p = 0.0005), implantation (p = 0.0008), ongoing pregnancy (p = 0.0046), livebirth (p = 0.0112), babies per transfer (p = 0.0112), and babies per embryo transferred (p = 0.0112). Morphology selection resulted in patients of all age groups having non-euploid embryos chosen for transfer. Post-hoc evaluation of individual clinic performances showed variable transfer outcomes that could potentially confound the true benefits of PGT-A. CONCLUSION: Embryo chromosome status is central to improved embryo transfer outcomes and sole reliance on current morphology-based selection practices, without Eu-S, will always compromise outcomes. Often overlooked but a major effector of successful PGT-A outcomes are individual clinic performances.

A pathogenic DMC1 frameshift mutation causes nonobstructive azoospermia but not primary ovarian insufficiency in humans.

Nonobstructive azoospermia (NOA) and diminished ovarian reserve (DOR) are two disorders that can lead to infertility in males and females. Genetic factors have been identified to contribute to NOA and DOR. However, the same genetic factor that can cause both NOA and DOR remains largely unknown. To explore the candidate pathogenic gene that causes both NOA and DOR, we conducted whole-exome sequencing (WES) in a non-consanguineous family with two daughters with DOR and a son with NOA. We detected one pathogenic frameshift variant (NM_007068:c.28delG, p. Glu10Asnfs*31) following a recessive inheritance mode in a meiosis gene DMC1 (DNA meiotic recombinase 1). Clinical analysis showed reduced antral follicle number in both daughters with DOR, but metaphase II oocytes could be retrieved from one of them. For the son with NOA, no spermatozoa were found after microsurgical testicular sperm extraction. A further homozygous Dmc1 knockout mice study demonstrated total failure of follicle development and spermatogenesis. These results revealed a discrepancy of DMC1 action between mice and humans. In humans, DMC1 is required for spermatogenesis but is dispensable for oogenesis, although the loss of function of this gene may lead to DOR. To our knowledge, this is the first report on the homozygous frameshift mutation as causative for both NOA and DOR and demonstrating that DMC1 is dispensable in human oogenesis.

Re-analysis of whole blastocysts after trophectoderm biopsy indicated chromosome aneuploidy.

BACKGROUND: To compare the concordance between trophectoderm (TE) analysis and whole blastocyst analysis of embryos from chromosomal structural rearrangement (SR) carriers. METHOD: Sixty-three abnormal blastocysts identified by preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) were included. The whole blastocysts were processed through multiple displacement amplification cycle and sequenced for 24-chromosome aneuploidy screening by next-generation sequencing (NGS). The sequencing results were compared with those of TE biopsy from the same blastocysts and the primary chromosomal rearrangement of the couples. RESULTS: Analysis of the 63 blastocysts showed 68% (43/63) complete concordance between TE sequencing analysis and whole blastocyst results. Approximately one third (20/63, 32%) of the sequencing results showed some level of discordance between the two samples. Of these, 14% (9/63) of the embryos were identified as euploid after whole blastocyst sequencing. Among them, seven blastocysts were classified as chromosome mosaicism (five whole chromosomes, two segmental) after TE analysis, while two displayed non-SR related segmental changes in the TE biopsy. Of the original analyses, 70% (44/63) of findings were associated with the primary parental chromosomal rearrangement, while 30% (19/63) had no association. CONCLUSIONS: TE biopsy with NGS for PGT-SR is an efficient strategy to identify embryos suitable for transfer. While there was a high concordance between TE and whole blastocyst chromosome results, some embryos classified as mosaic in the original analysis and therefore unsuitable for transfer were reclassified as chromosomally balanced. To maximize the number of embryos available for PGT-SR patients, we suggest that embryos with mosaic non-SR chromosomal rearrangement should be stored and considered for transfer after appropriate counseling.

IVF embryo choices and pregnancy outcomes.

OBJECTIVE: Investigate the chromosome status and transfer outcomes of embryos selected using routine "best morphology" IVF practices. METHOD: A prospective multi-center, non-selection cohort study involving patients undertaking IVF treatment. Study entry conditions were blastocyst biopsy, >1 embryo with chromosome analysis and frozen transfer of the best morphology embryo. Primary analyses were ßhCG positive, implantation, ongoing pregnancy and birth rates and pregnancy-stage progression failures. RESULTS: After transfer, embryo chromosome status was assigned and outcomes divided into two primary groups - euploids (n = 135) and aneuploids (n = 53). Compared to euploid embryo transfers, aneuploid embryos had significantly lower primary outcomes (+ßhCG: 67% vs. 30%, p < 0.0001; IR: 56% vs. 19%, p < 0.0001; ongoing week 12: 51% vs. 9%, p < 0.0001; and livebirths: 50% vs. 8%, p < 0.0001, respectively). Transfers were further subdivided into smaller groups according to their main chromosomal feature. Stage analysis showed higher failure rates for aneuploids to initiate a pregnancy (p < 0.0001), higher subclinical miscarriage rate (p = 0.0402) and higher clinical miscarriage rate (p = 0.0038). CONCLUSION: Routine morphology-based embryo selection resulted in a high euploid selection rate but a significant number of aneuploid embryos were still inadvertently selected for transfer (28%) with the subsequent high failure rates for pregnancy initiation and progression having implications for appropriate patient management.

Third-generation sequencing: any future opportunities for PGT?

PURPOSE: To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT). METHODS: Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore. RESULTS: High-depth sequencing identified the deletion interval for the relatively large HBA1/2--SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies. CONCLUSION: TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT.

The Effect of Sacubitril-Valsartan in Heart Failure Patients With Mid-Range and Preserved Ejection Fraction: A Meta-Analysis.

BACKGROUND: The effect of sacubitril-valsartan in heart failure patients with mid-range (HFmEF) and preserved (HFpEF) ejection fractions remains unclear. This study aimed to investigate the clinical benefits of sacubitril-valsartan in HFmEF and HFpEF patients. METHODS: PubMed, EMBASE, Cochrane Library, and China National Knowledge Infrastructure were searched from inception to 29 February 2020 to identify pertinent articles. Studies meeting the inclusion criteria were included and analysed. RESULTS: Six (6) studies, with a total of 5,503 patients, were included. Compared with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, sacubitril-valsartan significantly reduced the rate of HF hospitalisation (risk ratios, 0.84; 95% CI, 0.77-0.91; p<0.001) and improved the New York Heart Association class (risk ratios, 1.25; 95% CI, 1.10-1.43; p=0.001) in HFmEF and HFpEF patients. Both the cardiovascular mortality and all-cause mortality were not significantly decreased by sacubitril-valsartan. In addition, there were no significant between-group differences in the N-terminal pro-B-type natriuretic peptide and left ventricular ejection fraction changes. Regarding safety, sacubitril-valsartan was likely to increase the risk of hypotension, but the incidence of serum creatinine elevation was significantly lower in the sacubitril-valsartan group than in the angiotensin-converting enzyme inhibitors and angiotensin receptor blockers group. CONCLUSIONS: This meta-analysis suggests that sacubitril-valsartan may be an effective and safe strategy with which to improve the clinical symptoms and reduce HF hospitalisation in HFmEF and HFpEF patients.

Maternal oxygen exposure may not change umbilical cord venous partial pressure of oxygen: non-random, paired venous and arterial samples from a randomised controlled trial.

BACKGROUND: Despite the widespread use of oxygen (O2) in intrauterine resuscitation, the obstetric scientists' understanding of O2 therapy is full of contradictions. We tested the hypothesis that higher maternal arterial partial pressure of oxygen (PO2) is associated with higher umbilical cord venous PO2 (UvPO2). METHODS: This is a planned secondary analysis of a randomised controlled trial (RCT), 443 normal women were 1:1 randomly allocated to receive 2 L/min O2 or room air from the onset of second stage to delivery. We reported that maternal 2 L/min O2 exposure cannot affect the umbilical cord arterial pH or the fetal heart rate (FHR) pattern. In 217 non-random samples, we found 2 L/min O2 exposure increased the maternal arterial PO2 to the median 150 mmHg (hemoglobin would be saturated). The primary outcome for this analysis was UvPO2 in these non-random samples. RESULTS: There were no significant differences between the O2 group (N = 107) and the control group (N = 110) in the UvPO2 (median 30.2, interquartile 25.4-35.2 versus median 28.3, interquartile 23.4-35.3, mmHg, P = 0.379). There were also no significant differences between room air and different percentiles of O2 exposure duration (< 25th, ≧ 25th < 50th, ≧ 50th < 75th, ≧ 75th percentile) in the UvPO2. CONCLUSIONS: Maternal O2 exposure at super-physiological levels (median arterial blood PO2 150 mmHg) in normal labor may not change the UvPO2. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02221440 , first posted in 20 August 2014.

Analysis of balanced reciprocal translocations in patients with subfertility using single-molecule optical mapping.

PURPOSE: Approximately 1% of individuals who carry a balanced reciprocal translocation (BRT) are subfertile. Current karyotyping does not have the resolution to determine whether the breakpoints of the involved chromosomes perturb genes important for fertility. The aim of this study was to apply single-molecule optical mapping (SMOM) to patients presenting for IVF (in vitro fertilization) to ascertain whether the BRT disrupted any genes associated with normal fertility. METHODS: Nine subfertile patients with different BRTs were recruited for the study. Methyltransferase enzyme DLE1 was used to fluorescently label their genomic DNA samples at the recognition motif CTTAAG. The SMOM was performed on the Bionano platform, and long molecules aligned against the reference genome hg19 to identify the breakpoint regions. Mate-pair and PCR-Sanger sequencing were used to confirm the precise breakpoint sequences. RESULTS: Both breakpoint regions in each of the nine BRTs were finely mapped to small regions of approximately 10 Kb, and their positions were consistent with original cytogenetic banding patterns determined by karyotyping. In three BRTs, breakpoints disrupted genes known to be associated with male infertility, namely NUP155 and FNDC3A [46,XY,t(5;13)(p15;q22)], DPY19L1 [46,XY,t(1;7)(p36.3;p15), and BAI3 [46,XY,t(3;6)(p21;q16)]. CONCLUSIONS: The SMOM has potential clinical application as a rapid tool to screen patients with BRTs for underlying genetic causes of infertility and other diseases.

SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and Small interfering RNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a Sma- and Mad-related protein (SMAD)-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.

Chromosome constitution of equal-sized three-cell embryos using next-generation sequencing technology.

PURPOSE: To study the chromosome constitution of equal-sized three-cell embryo. METHODS: We determined the chromosome constitution of 105 blastomeres from 35 embryos using multiple annealing and looping-based amplification cycles (MALBAC) together with NGS sequencing technology. Chromosomal copy number variation (CNV) analysis was successfully performed in 27 embryos. We also analyzed radius, perimeter, area, and volume of each blastomere to explore the possibility of selecting the normal embryos. RESULTS: Majority of the embryos (77.8%, 21/27) studied were mosaic or aneuploid, and only 22.2% (6/27) had normal chromosome numbers. The aneuploid chromosomes spread across all chromosomes and the most frequent aneuploidies were for chromosomes 1, 16, and 18 followed by 13, 19, and 21. Statistical analyses showed no significant difference between euploid and aneuploid embryos regarding radius, perimeter, area, and volume of their blastomeres. CONCLUSIONS: Our results showed that majority of the equal-sized three-cell embryos were chromosomally abnormal and could not be distinguished by morphology observation, so they should be given lower priority at selection for transfer.

Niacin Inhibits Apoptosis and Rescues Premature Ovarian Failure.

BACKGROUND/AIMS: Over 99% of mouse and human ovarian follicles will undergo specialized cell death including atresia and apoptosis. Reduction of apoptosis may help reduce infertility and maintain the reproductive ability in women. METHODS: 3-day B6D2F1 mice were used to culture small follicle and ovary tissue with niacin and 18-day mice were intraperitoneal injected with niacin to determine its effect on follicle development. Then establish 8-weeks POF animal model with cytoxan (CTX) or radiation. Treatment group was given 0.1 mL of 100 mM niacin by an intraperitoneal injection twice before ovulation. The ovaries were collected and the follicles were counted and categorized, and ovarian histologic sections were stained for TUNEL. Ovarian function was then evaluated by monitoring ovulation. Microarray analyses, Western blot, immunofluorescence and real-time quantitative PCR were used to assess the mechanism of ovarian injury and repair. RESULTS: We found that niacin promotes follicle growth in the immature oocyte and it increased the levels of a germ-line cell marker DDX4, and a cell proliferation marker PCNA in the ovary. Addition of niacin to the cell culture reduced oocyte apoptosis in vitro. Administration of niacin to treat premature ovarian failure (POF) in mouse models showed inhibition of follicular apoptosis under harmful conditions, such as radiation and chemotherapy damage, by markedly reducing cumulus cell apoptosis. Additionally, the number of developing follicles increased after administration of niacin. CONCLUSION: Niacin may have an important function in treating POF by reducing apoptosis in clinical applications.

Interleukin-6 regulates expression of Fos and Jun genes to affect the development of mouse preimplantation embryos.

AIM: We investigated whether recombinant mouse interleukin-6 (IL-6) affects the development of preimplantation embryos and induces the -signal transducers and activators of transcription (JAK-STAT) signaling pathway by binding IL-6 signal transducer (IL-6st) and regulating Fos and Jun gene expression, thereby accounting for the negative effect of superovulation on embryo development. METHODS: We compared rates of blastocyst formation from embryos cultured with different concentrations of IL-6 or/and anti-interleukin 6 receptor antibody (anti-IL-6RAb) in superovulated experimental and normal control groups. IL-6 expression in preimplantation embryos was determined by immunofluorescence identification. Fos, Jun and IL-6st messenger RNA expression was detected by PCR and microarray experiments. RESULTS: Rates of blastocyst formation significantly decreased in superovulated embryos, whether or not they were incubated in 0.1, 1, 25 or 50 pg/mL IL-6, (P < 0.01) compared to embryos from naturally ovulated controls, whereas incubation in 5 and 10 pg/mL IL-6 reversed the negative effects of superovulation. The addition of anti-IL-6RAb to naturally ovulated embryos reduced blastocyst rates to those of superovulated embryos. Gene chip analysis indicated that the JAK-STAT signaling pathway contained differentially expressed IL-6, IL-6st, Jun and Fos genes. Both anti-IL-6RAb or ovarian stimulation downregulated IL-6st, Jun, and Fos messenger RNA expression, but expression of the same three genes increased in 5 pg/mL IL-6. CONCLUSION: Ovarian stimulation negatively impacts the development of preimplantation embryos by reducing IL-6 release. IL-6 affects the rate of development of zygotes to blastocyst by regulating IL-6st, Fos and Jun expression in the JAK-STAT signaling pathway.