Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination.

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Endometrial stromal cells from women with repeated implantation failure display impaired invasion towards trophoblastic spheroids.

In brief: Implantation failure can occur even after the transfer of good-quality embryos. This study showed that the migration of human endometrial stromal cells towards embryonic trophoblasts is higher in women with live births in the first in vitro fertilization cycle than those with repeated implantation failure, suggesting that the chemotactic response of stroma cells is associated with successful pregnancy. Abstract: The success rate of in vitro fertilization (IVF) remains limited in some women despite transfers of good-quality embryos in repeated attempts. There is no reliable tool for assessing endometrial receptivity. This study aimed to assess the interaction between decidualized human primary endometrial stromal cells (1°-EnSC) and human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) and to compare the invasion ability of decidualized 1°-EnSC towards BAP-EB between women attaining live birth in the first IVF cycle and those with repeated implantation failure (RIF). The invasion of the decidualized human endometrial cell line (T-HESC) and 1°-EnSC towards BAP-EB was studied. Real-time quantitative PCR and immunocytochemistry were employed to determine the expression of decidualization markers at mRNA and protein levels, respectively. Trophoblast-like BAP-EB-96h, instead of early trophectoderm (TE)-like BAP-EB-48h, facilitated the invasion ability of decidualized T-HESC and decidualized 1°-EnSC. Human chorionic gonadotropin at supra-physiological levels promoted the invasiveness of decidualized 1°-EnSC. The extent of BAP-EB-96h-induced invasion was significantly stronger in decidualized 1°-EnSC from women who had a live birth in the first IVF cycle when compared to those with RIF. While no difference was found in the expression of decidualization markers, PRL and IGFBP1 among two groups of women, significantly lower HLA-B was detected in the non-decidualized and decidualized 1°-EnSC from women with RIF. Collectively, the findings suggested that the invasion of decidualized 1°-EnSC towards trophoblast-like BAP-EB-96h was higher in women who had a live birth in the first IVF cycle than those with RIF.

Non-Coding RNAs as Biomarkers for Embryo Quality and Pregnancy Outcomes: A Systematic Review and Meta-Analysis.

Despite advances in in vitro fertilization (IVF), there is still a lack of non-invasive and reliable biomarkers for selecting embryos with the highest developmental and implantation potential. Recently, small non-coding RNAs (sncRNAs) have been identified in biological fluids, and extracellular sncRNAs are explored as diagnostic biomarkers in the prediction of IVF outcomes. To determine the predictive role of sncRNAs in embryo quality and IVF outcomes, a systematic review and meta-analysis was performed. Articles were retrieved from PubMed, EMBASE, and Web of Science from 1990 to 31 July 2022. Eighteen studies that met the selection criteria were analyzed. In total, 22 and 47 different sncRNAs were found to be dysregulated in follicular fluid (FF) and embryo spent culture medium (SCM), respectively. MiR-663b, miR-454 and miR-320a in FF and miR-20a in SCM showed consistent dysregulation in two different studies. The meta-analysis indicated the potential predictive performance of sncRNAs as non-invasive biomarkers, with a pooled area under curve (AUC) value of 0.81 (95% CI 0.78, 0.844), a sensitivity of 0.79 (95% CI 0.72, 0.85), a specificity of 0.67 (95% CI 0.52, 0.79) and a diagnostic odds ratio (DOR) of 8 (95% CI 5, 12). Significant heterogeneity was identified among studies in sensitivity (I2 = 46.11%) and specificity (I2 = 89.73%). This study demonstrates that sncRNAs may distinguish embryos with higher developmental and implantation potentials. They can be promising non-invasive biomarkers for embryo selection in ART. However, the significant heterogeneity among studies highlights the demand for prospective multicenter studies with optimized methods and adequate sample sizes in the future.

Myd88 Signaling Is Involved in the Inflammatory Response in LPS-Induced Mouse Epididymitis and Bone-Marrow-Derived Dendritic Cells.

Epididymitis is an epididymal inflammation that may lead to male infertility. Dendritic cells (DCs) and myeloid differentiation primary response gene 88 (Myd88) were associated with epididymitis in rodents. However, the functions of Myd88 on epididymal DCs remain unclear. This study investigated the role of Myd88 in DCs for epididymitis. The Myd88 signaling pathway, phenotypes of DC subsets, and cytokines were investigated in lipopolysaccharide (LPS)-induced epididymitis in mice. CRISPR-Cas9 was used to knockout Myd88 in bone-marrow-derived dendritic cells (BMDCs) and immortalized mouse epididymal (DC2) cell line. In the vivo experiments, levels of the proinflammatory cytokines IL-1α, IL-6, IL-17A, TNF-α, IL-1ß, MCP-1, and GM-CSF, mRNA for MyD88 related genes, and the percentages of monocyte-derived DCs (Mo-DCs) were significantly elevated in mice with epididymitis. In the vitro experiments, LPS significantly promoted the apoptosis of BMDCs. In addition, the concentration of inflammatory cytokines in BMDCs and DC2s were increased in the LPS group, while decreasing after the knockout of Myd88. These findings indicate that Myd88 on DCs is involved in the inflammation of epididymitis in mice, which may be a potential target for better strategies regarding the treatment of immunological male infertility.

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation.

Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not ß-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception.

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.

Hyperglycemia Altered DNA Methylation Status and Impaired Pancreatic Differentiation from Embryonic Stem Cells.

The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1, NKX6-1 and NKX6-2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6-2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6-1. Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.

Connexin 43 is involved in early differentiation of human embryonic stem cells.

Gap junctional intercellular communication (GJIC) is important for maintaining the pluripotency of mouse embryonic stem cells (mESC). However, human ESC (hESC) have a high level of connexin (Cx) molecules with unknown function. In this study, we found that the major Cx molecule, Cx43, was highly expressed in undifferentiated hESC. It was down-regulated upon spontaneously differentiation by embryoid body formation and induced differentiation along ectoderm, mesoderm and extraembryonic lineages, but up-regulated along endoderm differentiation. The knockdown of Cx43 and GJIC had no effect on the maintenance of hESC, as demonstrated by no morphological changes and similar expression levels of pluripotent markers (OCT4, NANOG, SSEA-3 and SSEA-4) and early differentiation markers (KRT8 and KRT18). Meanwhile, Cx43 knock down had no effect on endodermal markers (SOX17, FOXA2 and CXCR4) expression when hESC were differentiating into definitive endoderm lineage. On the contrary, it led to lower levels of mesodermal markers (CD56, CD34 and PDGFR-α) when cells were undergoing mesoderm differentiation. When compared to control, Cx43 knockdown led to higher attachment rate, HCG secretion and cell invasion of the hESC derived trophoblastic cells. Cx43 knockdown also resulted in up-regulated expressions of placental hormone (ß-hCG) and implantation related genes (LIFR, CDH5, LEP, PGF, TGFBR2). Our study suggested that Cx43 and GJIC had no effect on the undifferentiated growth of hESC but affected specific lineage differentiation.

Independent association of serum vitamin D with anti-Mullerian hormone levels in women with polycystic ovary syndrome.

OBJECTIVE: This study aimed at investigating the association of serum vitamin D (25(OH)D) and anti-Mullerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) as well as non-PCOS healthy ovulatory women and the possible confounding effects of adiposity and androgen. METHOD: This was a cross-sectional study conducted on serum samples collected from 451 women diagnosed with PCOS as well as 244 age-matched healthy ovulatory women in a tertiary gynaecology out-patient clinic and a family planning clinic. RESULTS: Serum 25(OH)D level was significantly higher in women recruited during summer and autumn than those recruited in winter and spring. Both serum 25(OH)D and AMH levels peaked during summer in women with PCOS. In ovulatory women, only serum 25(OH)D but not AMH level showed such seasonal variation. Serum 25(OH)D level in women with PCOS significantly correlated positively with AMH, AMH/antral follicle count (AFC) ratio, serum total testosterone, sex-hormone-binding globulin and quantitative insulin-sensitivity check index and inversely with body mass index (BMI), insulin, triglycerides and homeostatic model assessment of insulin resistance. After controlling for BMI, 25(OH)D level remained significantly correlated positively with serum AMH, AMH/AFC and total testosterone, and inversely with triglycerides. 25(OH)D level was an independent predictor of serum AMH level after controlling for age, BMI and free androgen index in women with PCOS. CONCLUSION: Serum 25(OH)D level is an independent factor significantly associated with AMH level in women with PCOS but not in ovulatory women.

Effect of ulipristal acetate and mifepristone at emergency contraception dose on the embryo-endometrial attachment using an in vitro human trophoblastic spheroid and endometrial cell co-culture model.

STUDY QUESTION: Do both ulipristal acetate (UPA) and mifepristone inhibit embryo-endometrial attachment at concentrations corresponding to the emergency contraception (EC) dose? SUMMARY ANSWER: Both UPA and mifepristone at concentrations corresponding to the EC dose do not have an inhibitory effect on embryo implantation, although mifepristone at a higher concentration appeared to have such an effect. WHAT IS KNOWN ALREADY: Levonorgestrel is commonly used for EC, but it only acts through inhibition of ovulation. UPA and mifepristone have higher efficacy as EC compared to levonorgestrel; while there is some suggestion that mifepristone may interfere with implantation, whether UPA has post-ovulatory action in inhibiting implantation is yet to be confirmed. STUDY DESIGN, SIZE, DURATION: An in vitro experimental study using trophoblastic spheroids made from JAr cell line as the embryo surrogate, and the Ishikawa cell line and primary human endometrial cells cultured to monolayer as the endometrial surrogate. The primary endometrial cells were collected from nine volunteer women in the mid-luteal phase with consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a university gynaecology unit. The JAr and Ishikawa cell lines (or primary endometrial cells) were treated with graded concentrations of UPA (0, 0.04, 0.4 and 4 µM) or mifepristone (0, 0.1, 1 and 10 µM) for 24 h. Embryo-endometrial attachment was studied using an in vitro JAr spheroid-endometrial co-culture model. Expressions of progesterone receptor, ß-catenin and glycogen synthase kinase 3 ß (GSK-3ß) were studied with real-time RT-PCR and Western blotting, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In the Ishikawa experiments, there was no significant difference in the JAr spheroid attachment rate after treatment with UPA at 0 (93.0%), 0.04 (93.6%), 0.4 (93.4%) and 4 (91.4%) µM concentrations (P > 0.05); the attachment rate was reduced after treatment with mifepristone only at 10 µM (79.8%, P < 0.0001) but not at 0.1 (92.1%) or 1.0 (95.2%) µM concentrations. In the primary endometrial cell experiments, again no significant difference was observed in the JAr spheroid attachment rate after treatment with UPA 4 µM (42.6%) compared to control (46.5%, P > 0.05). Both UPA and mifepristone could significantly up-regulate progesterone receptor expression. There was no significant alteration in expression of ß-catenin and GSK-3ß after treatment with UPA 4 µM or mifepristone 10 µM (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The co-culture model is only a surrogate which may not fully represent the complicated process of embryo implantation in vivo, although there is no existing perfect model for studying implantation in vitro which fully resembles the latter. WIDER IMPLICATIONS OF THE FINDINGS: The lack of inhibitory effect on embryo implantation by UPA and possibly mifepristone at concentrations corresponding to the EC dose is an important information for contraceptive counseling. STUDY FUNDING/COMPETING INTEREST(S): We had free supply of the UPA compound used in this study from Laboratoire HRA Pharma. This work was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, The University of Hong Kong, Hong Kong.

A randomized double-blinded controlled trial of hCG as luteal phase support in natural cycle frozen embryo transfer.

STUDY QUESTION: Does the use of hCG as luteal phase support in natural cycle frozen embryo transfer (FET) increase the ongoing pregnancy rate? STUDY ANSWER: The use of hCG in natural cycle FET did not improve the ongoing pregnancy rate. WHAT IS KNOWN ALREADY: The use of luteal phase support in stimulated cycles has been associated with higher live-birth rates and the results are similar when using hCG or progesterone. STUDY DESIGN SIZE, DURATION: This is a randomized double-blinded controlled trial of 450 women recruited between August 2013 and October 2015. PARTICIPANTS/MATERIALS SETTING METHODS: Women with regular cycles undergoing natural cycle FET were recruited. Serial serum hormonal concentrations were used to time natural ovulation and at least Day 2 cleavage embryos were replaced. Patients were randomized into either: (i) the treatment group, receiving 1500 IU hCG on the day of FET and 6 days after FET, or (ii) the control group, receiving normal saline on these 2 days. MAIN RESULTS AND THE ROLE OF CHANCE: The ongoing pregnancy rate [60/225 (26.7%) in the treatment group vs 70/225 (31.3%) in the control group, odds ratio 1.242 (95% CI 0.825-1.869)], implantation rate and miscarriage rate were comparable between the two groups. In the treatment group, there were significantly more cycles with top quality embryos transferred and a significantly higher serum oestradiol level, but a comparable serum progesterone level, 6 days after FET. However, no significant differences were observed in serum oestradiol and progesterone levels 6 days after FET between the pregnant and non-pregnant women. In the multivariate logistic regression, the number of embryos transferred was the only significant factor predictive of the ongoing pregnancy rate after natural cycle FET. LIMITATIONS REASON FOR CAUTION: This study only included FET with cleavage stage embryos and only hCG, not vaginal progesterone, was used as luteal phase support. WIDER IMPLANTATIONS OF THE FINDINGS: The findings in this study do not support the use of hCG for luteal phase support in natural cycle FET. STUDY FUNDING/COMPETING INTERESTS: No external funding was used and there were no competing interests. TRIAL REGISTRATION NUMBER: clinicaltrial.gov identifier: NCT01931384. TRIAL REGISTRATION DATE: 23/8/2013. DATE OF FIRST PATIENT'S ENROLMENT: 30/8/2013.

Effects of adrenomedullin on the expression of inflammatory cytokines and chemokines in oviducts from women with tubal ectopic pregnancy: an in-vitro experimental study.

BACKGROUND: The occurrence of tubal ectopic pregnancy (tEP) is related to the inflammation of the oviduct. Recently, Adrenomedullin (ADM) was found highly expression in human oviduct. The current study is to investigate whether ADM have a modulatory action on inflammatory cytokines and chemokines in oviductal tissue from women with tubal ectopic pregnancy (tEP). METHODS: Oviductal isthmus samples were collected from women with tEP undergoing salpingectomy, and women undergoing hysterectomy for benign gynaecological conditions. The mRNA and protein levels of inflammatory cytokines/chemokines were assayed by PCR (n = 6 for tEP, n = 5 for controls) and protein microarray methods (n = 5 for both tEP and controls) respectively. RESULTS: Some of the inflammatory cytokines/chemokines were upregulated by ADM in oviducts from tEP patients at both mRNA and protein levels. Incubation of oviduct from tEP patients with ADM for 24 h down-regulated some of these cytokines/chemokines. CONCLUSION: Our results suggest an additional mechanism whereby ADM insufficiency may increase the susceptibility to tEP through diminished anti-inflammatory activity. The actual impact of the relationship between ADM and inflammatory process on tubal implantation needs further exploration.

Assessment of adulteration of soybean proteins in dairy products by 2D microchip-CE device.

To determine the adulteration of soybean proteins in dairy product, a microchip-CE device was developed to isolate selected fraction of soybean and milk proteins in pI range from 5.5 ∼ 7.0 by 1D IEF, followed by ITP/CZE in the embedded capillary for preconcentration, separation and UV detection at 280 nm. Compared to IEF-CZE without ITP preconcentration, the enhancement factor (EF) in detection of soybean proteins was 20 times. Adulteration of 0.1% soybean protein in total dairy proteins can be detected in less than 10 min.

Ulipristal acetate resembles mifepristone in modulating human fallopian tube function.

STUDY QUESTION: Do ulipristal acetate (UPA) and mifepristone have an effect on ciliary beat frequency and muscular contractions in the human Fallopian tube? SUMMARY ANSWER: UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube, probably through an agonistic effect on the tubal progesterone receptor. WHAT IS KNOWN ALREADY: UPA, like mifepristone, acts as an emergency contraceptive mainly by inhibiting ovulation. Little is known about its effects on tubal function. STUDY DESIGN, SIZE, DURATION: This was an in vitro experimental study using Fallopian tube samples collected from 11 women undergoing hysterectomy for benign non-tubal gynaecological conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The tubal epithelium and longitudinal smooth muscle fibres were isolated, cultured and treated with UPA at graded concentrations of 0, 20, 200 and 2000 ng/ml, and mifepristone at graded concentrations of 0, 300, 3000 and 30 000 ng/ml, respectively. After treatment, ciliary beat frequency was determined using a photometric method. Basal tone, amplitude and frequency of muscular contraction were recorded through a force transducer. The mRNA expression of progesterone receptor (total and PR-B isoform), glycodelin and adrenomedullin were determined by real-time quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE: There was an overall dose-dependent suppressive effect on ciliary beat frequency (P < 0.0001) after treatment with UPA at all concentrations and with mifepristone at 3000 ng/ml or above. The basal tone, amplitude and frequency of muscular contractions were significantly reduced (P < 0.05) after treatment with UPA at 200 ng/ml or above, and with mifepristone at 3000 ng/ml or above. UPA treatment at 200 ng/ml or above significantly up-regulated the mRNA expression of progesterone receptor and glycodelin and down-regulated the mRNA expression of adrenomedullin in Fallopian tube tissue (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Whether or not the tubal effect may translate into additional mechanisms for contraceptive action in vivo is uncertain. WIDER IMPLICATIONS OF THE FINDINGS: The clinical relevance of UPA with regard to contraceptive activity is worthy of further exploration. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong. All authors have no competing interest to declare.

Cumulative live-birth rate in women with polycystic ovary syndrome or isolated polycystic ovaries undergoing in-vitro fertilisation treatment.

PURPOSE: This retrospective cohort study evaluated the cumulative live birth rate in women with polycystic ovary syndrome (PCOS) and isolated polycystic ovaries (PCO) undergoing in-vitro fertilisation (IVF) treatment. METHODS: We studied 104 women with PCOS, 184 with PCO and 576 age-matched controls undergoing the first IVF treatment cycle between 2002 and 2009. The main outcome measure was cumulative live birth in the fresh plus all the frozen embryo transfers combined after the same stimulation cycle. RESULTS: Women in both the PCOS (n = 104) and isolated PCO groups (n = 184) had higher ovarian response parameters compared to age-matched controls (n = 576), and higher rates of withholding fresh embryo transfer for risk of ovarian hyperstimulation syndrome (OHSS). The actual incidence of moderate to severe OHSS was significantly higher in the PCOS (11.5 %) but not the isolated PCO group (8.2%) compared to controls (4.9%). The live birth rates in the fresh cycle were comparable among the 3 groups, but the PCOS group had a significantly higher miscarriage rate compared to the other 2 groups. Cumulative live birth rate was significantly higher in the isolated PCO group (60.3%), but not the PCOS group (50.0%), compared to controls (47.5%). CONCLUSIONS: Women in the isolated PCO group, but not the PCOS group, had a significantly higher cumulative live birth rate compared to controls. This could be explained by the quantitative effect of the higher number of transferable embryos obtained per stimulation cycle, which is uncompromised by the unfavourable embryo competence otherwise observed in PCOS.

The effect of letrozole with misoprostol for medical termination of pregnancy on the expression of steroid receptors in the placenta.

STUDY QUESTION: What is the effect of letrozole on the expression of steroid receptors in the placentae in cases of termination of pregnancies? SUMMARY ANSWER: The expression of estrogen receptor-α (ERα) and progesterone receptor (PR) transcripts, as well as ERα protein, in placentae was suppressed by letrozole pretreatment in second trimester termination of pregnancy. WHAT IS KNOWN ALREADY: There have been no data in the literature on the effect of letrozole in termination of human pregnancies. STUDY DESIGN, SIZE, DURATION: This study is part of a clinical randomized trial in which 50 subjects were recruited and 44 placentae were collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women (n = 50) requesting second trimester abortion between 12 and 20 gestational weeks were randomized to receive either letrozole or placebo pretreatment for 3 days before administration of vaginal misoprostol. Placentae were collected from both groups of women after the abortion. Total RNA from the frozen placenta samples was extracted and subjected to real-time RT-PCR analysis of ERα and estrogen receptor-ß (ERß), PR and glucocorticoid receptor (GR) transcripts. Immunohistochemical studies of ERα, ERß, PR and GR expression, as well as Ki67 and PCNA staining for proliferation, were performed. TUNEL assays were performed to determine the extent of apoptosis. MAIN RESULTS AND THE ROLE OF CHANCE: Real-time RT-PCR demonstrated that the median ERα {3.900 [95% confidence interval (CI): -0.643-8.443] in the letrozole group versus 4.714 (95% CI: 1.776-7.652) in the control group; P = 0.005} and the median PR [0.701 (95% CI: 0.333-1.069) in the letrozole group versus 1.774 (95% CI: 1.07-2.478) in the control group; P = 0.003] were significantly lower in the letrozole group compared with the control group. Furthermore, ERα protein levels, in both syncytiotrophoblasts and cytotrophoblasts but not in villous stromal cells, were significantly reduced [H-score of 113 (95% CI: 103-119) in the letrozole group versus 217 (95% CI: 214-290) in the control group, in syncytiotrophoblasts; 100 (95% CI: 98-105) in the letrozole group versus 210 (95% CI: 200-286) in the control group, in cytotrophoblasts; P = 0.004], while the expression levels of ERß, PR, GR, PCNA, Ki67 and TUNEL were not significantly different between the two groups. LIMITATIONS, REASONS FOR CAUTION: Only the placentae from the second trimester termination of pregnancy were collected in this study. Information from first trimester terminations is still lacking. WIDER IMPLICATIONS OF THE FINDINGS: The results shed some light on the mechanism of action of letrozole pretreatment in termination of pregnancies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the GRF/RGC and CRCG grants of the University of Hong Kong. TRIAL REGISTRATION NUMBER: HKClinicalTrials.com with trial number HKCTR-695.

The involvement of let-7 in hCG-induced progesterone synthesis via regulating p27Kip1 and p21Cip1 expression.

Progesterone is essential in females to maintain a regular menstrual cycle and pregnancy. The luteinizing hormone (LH) surge induces the luteinization of granulosa cells and thecal cells to form the corpus luteum, which is responsible for progesterone synthesis. However, the specific mechanism of how hCG, the analog of LH, regulates progesterone synthesis has yet to be fully discovered. In this study, we found that progesterone level was increased in adult wild-type pregnant mice 2 and 7 days post-coitum, along with a decrease in let-7 expression compared with the estrus stage. Besides, the let-7 expression was negatively correlated with progesterone level in post-delivery day 23 wild-type female mice after being injected with PMSG and hCG. Then, using let-7 transgenic mice and a human granulosa cell line, we found that overexpression of let-7 antagonized progesterone level via targeting p27Kip1 and p21Cip1 and steroidogenic acute regulatory protein (StAR) expression, which is a rate-limiting enzyme in progesterone synthesis. Furthermore, hCG suppressed let-7 expression by stimulating the MAPK pathway. This study elucidated the role of microRNA let-7 in regulating hCG-induced progesterone production and provided new insights into its role in clinical application.

Current progress on in vitro differentiation of ovarian follicles from pluripotent stem cells.

Mammalian female reproduction requires a functional ovary. Competence of the ovary is determined by the quality of its basic unit-ovarian follicles. A normal follicle consists of an oocyte enclosed within ovarian follicular cells. In humans and mice, the ovarian follicles are formed at the foetal and the early neonatal stage respectively, and their renewal at the adult stage is controversial. Extensive research emerges recently to produce ovarian follicles in-vitro from different species. Previous reports demonstrated the differentiation of mouse and human pluripotent stem cells into germline cells, termed primordial germ cell-like cells (PGCLCs). The germ cell-specific gene expressions and epigenetic features including global DNA demethylation and histone modifications of the pluripotent stem cells-derived PGCLCs were extensively characterized. The PGCLCs hold potential for forming ovarian follicles or organoids upon cocultured with ovarian somatic cells. Intriguingly, the oocytes isolated from the organoids could be fertilized in-vitro. Based on the knowledge of in-vivo derived pre-granulosa cells, the generation of these cells from pluripotent stem cells termed foetal ovarian somatic cell-like cells was also reported recently. Despite successful in-vitro folliculogenesis from pluripotent stem cells, the efficiency remains low, mainly due to the lack of information on the interaction between PGCLCs and pre-granulosa cells. The establishment of in-vitro pluripotent stem cell-based models paves the way for understanding the critical signalling pathways and molecules during folliculogenesis. This article aims to review the developmental events during in-vivo follicular development and discuss the current progress of generation of PGCLCs, pre-granulosa and theca cells in-vitro.

Role of small RNAs harbored by sperm in embryonic development and offspring phenotype.

BACKGROUND: RNA harbored by mammalian sperm is increasingly considered to be an additional source of paternal hereditary information, beyond DNA. Recent studies have demonstrated the role of sperm small noncoding RNAs (sncRNAs) in modulating early embryonic development and offspring phenotype. The biogenesis of the sperm sRNA payload of mammalian sperm has been explored in many studies. AIMS: To summarize the possible mechanisms underpinning sperm sncRNAs regulating embryonic development and offspring phenotypes. MATERIALS AND METHODS: PubMed database (papers published from 2002 to 2022) was searched for studies reporting the impact of sperm sncRNAs on early embryonic development and offspring phenotype. RESULTS: The sncRNAs categories and source (such as tRNA-derived small RNAs, ribosomal RNA-derived small RNAs, microRNAs, and PIWI-interacting RNAs), and RNA modification upon different types of environmental exposure or by paternally-acquired factors were summarized. The potential mechanisms whereby the modifications of sperm sncRNAs modulate embryonic development and offspring phenotype under normal and pathological conditions (such as obesity, altered glucose metabolism, and psychological stress) were discussed. DISCUSSION AND CONCLUSION: Sperm sncRNAs modulate embryo development and offspring phenotype, and the resulting modifications may be transgenerationally inherited.