Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Association of serum vitamin D concentration and miscarriage rate in women with first-trimester threatened miscarriage.

RESEARCH QUESTION: Is low serum 25-hydroxyvitamin D (25(OH)D) associated with an increased risk of miscarriage in women who presented with threatened miscarriage to the Early Pregnancy Assessment Clinic (EPAC)? DESIGN: This was a secondary retrospective analysis using archived serum samples from a randomized, double-blind, placebo-controlled trial. Stored serum samples from 371 women presenting to the EPAC with threatened miscarriage during the first trimester were assayed for 25(OH)D by liquid chromatography-mass spectrometry. RESULTS: The overall miscarriage rate was 45/371 (12.1%) in the whole cohort. After grouping vitamin D insufficiency and vitamin D sufficiency together into a 'non-deficient' group and excluding participants who underwent termination of pregnancy, there was no difference in the miscarriage rate between those who were vitamin D deficient compared with those who were not (25/205, 12.2% versus 20/157, 12.7%, P= 0.877, odds ratio 0.951, 95% CI 0.507-1.784). When analysed according to the number of gestational weeks, the miscarriage rate was significantly higher in the vitamin D non-deficient group than the vitamin D-deficient group in women who presented at 6 gestational weeks or earlier (13/33 [39.4%] versus 10/58 [17.2%], P= 0.019), but there were no statistically significant differences between the two groups presenting at later gestations. There was no difference in the vitamin D level in women who had a miscarriage compared with those who had a live birth (48 [37-57] versus 47 [37-58] nmol/l, P= 0.725 median [25th-75th percentile]). CONCLUSIONS: A low serum vitamin D concentration was not associated with an increased risk of miscarriage in women with threatened miscarriage presenting to the EPAC.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice.

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility.

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.

Comparing the intercycle variation of serum anti-Mullerian hormone and antral follicle count measurements over four consecutive menstrual cycles.

OBJECTIVE: To compare the intercycle variation of serum anti-Mullerian hormone (AMH) and antral follicle count (AFC) measurements over four consecutive menstrual cycles. DESIGN: Observational study with secondary analysis using data from two previous randomized controlled trials. PATIENTS: Seventy-eight women from two previous randomized trials on the effect of dehydroepiandrosterone pretreatment on ovarian response in women undergoing in vitro fertilization (IVF) treatment. MEASUREMENTS: The intraclass correlation coefficients (ICC) for AFC and AMH across the four study cycles, as well as their predictive performance on poor ovarian response, were compared. RESULTS: No significant difference was observed in AMH (p = .608) across the four study cycles. AFC was significantly higher at 4 weeks before ovarian stimulation compared with 0, 8 and 12 weeks before ovarian stimulation (p < .05, Conover posthoc test). Both single-measures and average-measures ICC were significantly higher with AMH than with AFC. The areas under the receiver operating characteristic curve of the four AFC measurements in predicting poor ovarian response (defined as three or less oocytes retrieved) in the IVF cycle ranged from 0.657 to 0.743 with no significant difference (p > .05) among the four cycles, whereas those of the four AMH measurement ranged from 0.730 to 0.780 with no significant difference (p > .05) among the four cycles. CONCLUSIONS: Although both AFC and AMH are good predictors of ovarian response, intercycle repeatability was significantly better with serum AMH than AFC measurement. Both have no significant difference in their predictive performance on poor ovarian response when assessed within three months before IVF treatment, hence allowing pre-IVF assessment at more flexible timing.

Effect of basal serum testosterone level on the ovarian response and the cumulative live birth rate in infertile women undergoing in vitro fertilization.

PURPOSE: To evaluate the effect of basal serum testosterone levels on the ovarian response and the cumulative live birth rate of infertile women undergoing in vitro fertilization (IVF). METHODS: It is a retrospective study in a university-affiliated assisted reproduction center in Hong Kong. Infertile women undergoing the first IVF cycle in the center between December 2012 and November 2016 with archived serum samples and available information on cumulative live birth were included for the analysis. RESULTS: A total of 1122 women were included for analysis. The median basal serum testosterone level was 0.53 (25-75th percentile: 0.40-0.67) nmol/L. Women with higher basal serum testosterone levels required a lower total dosage of gonadotrophin and a shorter duration of stimulation and had more oocytes retrieved. The cumulative live birth rates did not differ among women with serum testosterone levels in the four quartiles. Basal serum testosterone level was not a significant independent predictor of the cumulative live birth after adjusted for the women's age and number of normally fertilized oocytes in a binary logistic regression. The areas under the receiver operative characteristics (ROC) curves in predicting low or high ovarian response and the cumulative live birth were all below 0.6. CONCLUSION: Higher basal serum testosterone levels were associated with a better ovarian response but had no effect on the cumulative live birth rate of infertile women undergoing IVF.

A retrospective analysis of artificial oocyte activation in patients with low or no fertilisation in intracytoplasmic sperm injection cycles.

Intracytoplasmic sperm injection (ICSI) is commonly used to treat severe male factor infertility in assisted reproduction. A small percentage of patients face suboptimal fertilisation rate or even fertilisation failure despite having ICSI. Artificial oocyte activation (AOA) has been proposed as a suitable method to overcome their problem. This is a retrospective cohort analysis of ICSI cycles undergoing AOA. Injected metaphase II oocytes were exposed to either calcium ionophore (A23187) after ICSI or injection of calcium chloride during ICSI followed by incubation with A23187 after ICSI. The previous ICSI cycles of the patients formed the historical control group. Thirty-four AOA cycles were analysed. The normal fertilisation rate (52.1%) was significantly improved in the AOA group. The percentage of failed fertilisation cycles (11.8%) were significantly reduced in the AOA group. The cumulative clinical pregnancy rate (47.1%) and live birth rate (29.4%) were significantly increased when compared to the previous cycles. Subgroup analysis revealed that the performance of the A23187 only protocol and the concomitant injection of calcium chloride protocol were comparable in terms of laboratory parameters and pregnancy outcomes. AOA is an effective method to improve the fertilisation rate and pregnancy outcome of infertile couples with previous fertilisation problem after ICSI.IMPACT STATEMENTWhat is already known on this subject? A failed and low fertilisation rate after ICSI is not uncommon in assisted reproduction. AOA is normally used to improve fertilisation but there are discrepancies in the efficacy of the treatment.What do the results of this study add? AOA improves the fertilisation rate and pregnancy outcomes of couples with suboptimal fertilisation rate and fertilisation failure in previous ICSI cycles. The efficacies of two AOA protocols were comparable. The A23187 only protocol was recommended because of its simplicity.What are the implications of these findings for clinical practice and/or further research? AOA should be considered as a routine procedure for infertile couples with compromised fertilisation rates in previous ICSI cycles.

Myometrial Cells Stimulate Self-Renewal of Endometrial Mesenchymal Stem-Like Cells Through WNT5A/ß-Catenin Signaling.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and self-renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active ß-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/ß-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/ß-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/ß-catenin signaling. Stem Cells 2019;37:1455-1466.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

STUDY QUESTION: Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? SUMMARY ANSWER: GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. WHAT IS KNOWN ALREADY: dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. STUDY DESIGN, SIZE, DURATION: CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 µg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. PARTICIPANTS/MATERIALS, SETTING, METHODS: Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. MAIN RESULTS AND THE ROLE OF CHANCE: GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

Knowledge, attitude and ethical consideration of Chinese couples requesting preimplantation genetic testing in Hong Kong.

AIM: Increasing preimplantation genetic testing (PGT) cycles are being performed in Hong Kong. This study aims to evaluate the knowledge, attitude and ethical consideration of Chinese couples toward PGT. METHODS: Couples requesting PGT between June 2013 and March 2014 were invited to complete a questionnaire. RESULTS: Total 49 couples (49 women, 47 men) completed the questionnaires. Eighteen couples (37%) were waiting for PGT (pre-PGT group), 15 couples (31%) were undergoing PGT (PGT group) and 16 couples (32%) had completed at least one PGT cycle (post-PGT group). Only 53% of the couples could tell the recurrent risk, and 31% (with monogenic disorders) could tell the mode of inheritance of their condition. The acceptability of PGT (>80%) and attitude toward the embryo fate (58-78%) were good. The post-PGT group had more concern than the PGT and pre-PGT groups on the prenatal diagnostic testing (**P = 0.007). 12.5% of the couples worried about the transfer of healthy embryos with carrier state and they all had monogenic disorders. If the prenatal testing confirmed an affected fetus, a higher percentage (32%) in the Post-PGT group disagreed to terminate the pregnancy in contrast to a much lower 6% in the pre-PGT group (**P = 0.02). Three-quarter of the couples opted to tell their child about their conception through PGT. CONCLUSION: Chinese couples in Hong Kong had an overall good acceptability and positive attitude toward PGT. We appreciate the difficulties the couples have gone through PGT. A checklist on what to cover pre-during-post-PGT in the counseling process is needed.

Two-dimensional versus three-dimensional laparoscopy: evaluation of physicians' performance and preference using a pelvic trainer.

STUDY OBJECTIVE: To compare the proficiency and preference of physicians in performing standard tasks in a box trainer using 2-dimensional (2D) versus 3-dimensional (3D) laparoscopy. DESIGN: Prospective randomized controlled trial. DESIGN CLASSIFICATION: Prospective, randomized controlled trial (Canadian Task Force classification I). SETTING: Tertiary care teaching hospital. PARTICIPANTS: Thirty physicians from the Department of Obstetrics and Gynecology. INTERVENTION: Participants were randomly assigned to perform a set of 3 to 4 tasks in a pelvic trainer using 2D laparoscopy first and then 3D laparoscopy, and vice versa. MAIN RESULTS AND MEASUREMENTS: The time taken to complete the tasks and side effects experienced were noted. Participants were asked to complete a modified Global Operative Assessment of Laparoscopic Skills (GOALS) form at the end of their tasks to evaluate their experiences and to assess their own proficiency with both visual modalities. RESULTS: The time taken for peg transfer, duct cannulation, and suturing was significantly faster using 3D laparoscopy compared with 2D laparoscopy. There were no significant differences in the time taken for pattern cutting with both visual modalities. Participants experienced more dizziness using 3D laparoscopy (6.9% vs 37.9%; p = .004). The GOALS self-evaluation score was significantly higher for 3D compared with 2D laparoscopy. After the study, 11 of 29 (37.9%) participants preferred 2D, 16 of 29 (55.2%) preferred 3D, and 1 of 29 (8.3%) had no preference. CONCLUSION: Although 3D laparoscopy scored higher on self-evaluation and was preferred by more participants, it only gave better objective performance in the completion of some selected tasks by participants with intermediate skill levels and was associated with more dizziness. Further studies are needed to determine the value of 3D laparoscopy, especially when used in the clinical setting.

Caesarean scar pregnancy: a 10-year experience.

AIMS: To review the management and outcome of all women with caesarean scar pregnancy (CSP) at a single centre. METHODS: A retrospective review of women diagnosed with CSP between January 2004 and December 2013 was performed. Women were identified from the admission and Early Pregnancy Assessment Clinic records. RESULTS: Twenty-two cases of CSP were identified during the study period. The median gestational age at diagnosis was 6.7 weeks (range 4.7-11.8 weeks). All diagnoses were made by ultrasound, the most common finding of which was a heterogeneous mass at the site of the caesarean scar (100%). The median duration for human chorionic gonadotropin to return to undetectable level was 10 weeks (range 2-20 weeks), although the duration for sonographic resolution of the CSP can take much longer. The success rate of intralesional methotrexate was 80% in our series. CONCLUSION: There is no consensus regarding the method of choice for managing CSP. Medical management using intralesional methotrexate appears to be an acceptable treatment in clinically stable women.

Live birth rate, multiple pregnancy rate, and obstetric outcomes of elective single and double embryo transfers: Hong Kong experience.

OBJECTIVE: To compare the live birth rate, multiple pregnancy rate, and obstetric outcomes of elective single and double embryo transfers. DESIGN: Case series with internal comparisons. SETTING: University affiliated hospital, Hong Kong. PARTICIPANTS: Between October 2009 and December 2011, 206 women underwent their first in-vitro fertilisation cycle. Elective single embryo transfer was offered to women who were aged 35 years or below, and had endometrial thickness of 8 mm or more and at least two embryos of good quality. MAIN OUTCOME MEASURES: Live birth rate, multiple birth rate, and obstetric outcomes. RESULTS: Among the 206 eligible women, 74 underwent an elective single embryo transfer and 132 a double embryo transfer. The live birth rate was comparable in the two groups, being 39.2% in the elective single embryo transfer group and 43.2% in the double embryo transfer group, while the multiple pregnancy rate was significantly lower in the elective single embryo transfer group than the double embryo transfer group (6.9% vs 40.4%; P<0.001). Gestational ages and birth weights were comparable in the two groups. There was no significant difference between the two groups with respect to the rate of preterm delivery and antenatal complications (27.6% vs 43.9%, respectively; P>0.05). CONCLUSION: In this selected population, an elective single embryo transfer policy decreases the multiple pregnancy rate without compromising the live birth rate. The non-significant difference in antenatal complications may be related to the small sample size.

Interleukin 6 at menstruation promotes the proliferation and self-renewal of endometrial mesenchymal stromal/stem cells through the WNT/ß-catenin signaling pathway.

Background: At menstruation, the functional layer of the human endometrium sheds off due to the trigger of the release of inflammatory factors, including interleukin 6 (IL-6), as a result of a sharp decline in progesterone levels, leading to tissue breakdown and bleeding. The endometrial mesenchymal stem-like cells (CD140b+CD146+ eMSC) located in the basalis are responsible for the cyclical regeneration of the endometrium after menstruation. Endometrial cells from the menstruation phase have been proven to secrete a higher amount of IL-6 and further enhance the self-renewal and clonogenic activity of eMSC. However, the IL-6-responsive mechanism remains unknown. Thus, we hypothesized that IL-6 secreted from niche cells during menstruation regulates the proliferation and self-renewal of eMSC through the WNT/ß-catenin signaling pathway. Methods: In this study, the content of IL-6 across the menstrual phases was first evaluated. Coexpression of stem cell markers (CD140b and CD146) with interleukin 6 receptor (IL-6R) was confirmed by immunofluorescent staining. In vitro functional assays were conducted to investigate the effect of IL-6 on the cell activities of eMSC, and the therapeutic role of these IL-6- and WNT5A-pretreated eMSC on the repair of injured endometrium was observed using an established mouse model. Results: The endometrial cells secrete a high amount of IL-6 under hypoxic conditions, which mimic the physiological microenvironment in the menstruation phase. Also, the expression of IL-6 receptors was confirmed in our eMSC, indicating their capacity to respond to IL-6 in the microenvironment. Exogenous IL-6 can significantly enhance the self-renewal, proliferation, and migrating capacity of eMSC. Activation of the WNT/ß-catenin signaling pathway was observed upon IL-6 treatment, while suppression of the WNT/ß-catenin signaling impaired the stimulatory role of IL-6 on eMSC activities. IL-6- and WNT5A-pretreated eMSC showed better performance during the regeneration of the injured mouse endometrium. Conclusion: We demonstrate that the high level of IL-6 produced by endometrial cells at menstruation can induce the stem cells in the human endometrium to proliferate and migrate through the activation of the WNT/ß-catenin pathway. Treatment of eMSC with IL-6 and WNT5A might enhance their therapeutic potential in the regeneration of injured endometrium.

Effect of vitamin D in addition to letrozole on the ovulation rate of women with polycystic ovary syndrome: protocol of a multicentre randomised double-blind controlled trial.

INTRODUCTION: Low vitamin D status is prevalent among women with polycystic ovary syndrome (PCOS). The objective of the study is to assess the effect of vitamin D supplementation on (1) the ovulation rate to letrozole and (2) other reproductive, endocrine and metabolic outcomes after 1 year of supplementation in women with PCOS. METHODS AND ANALYSIS: This is a multicentre, randomised, double-blind, controlled clinical trial. A total of 220 anovulatory women with PCOS diagnosed by the Rotterdam criteria will be recruited. They will be randomly assigned to either the (1) vitamin D supplementation group or (2) placebo group. Those in the vitamin D group will take oral Vitamin D3 50 000 IU/week for 4 weeks, followed by 50 000 IU once every 2 weeks for 52 weeks. Those who remain anovulatory after 6 months will be treated with a 6-month course of letrozole (2.5 mg to 7.5 mg for 5 days per cycle titrated according to response) for ovulation induction. The primary outcome is the ovulation rate. All statistical analyses will be performed using intention-to-treat and per protocol analyses. ETHICS AND DISSEMINATION: Ethics approval was sought from the Institutional Review Board of the participating units. All participants will provide written informed consent before joining the study. The results of the study will be submitted to scientific conferences and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04650880.

Placenta exosomal miRNA-30d-5p facilitates decidual macrophage polarization by targeting HDAC9.

Pregnancy involves a wide range of adaptations in the maternal body. Maternal immune tolerance toward the foreign fetus is critical for a successful pregnancy. Decidual macrophages are the primary antigen-presenting and phagocytic cells responsible for antigen presentation and apoptotic cell removal. Their phenotype changes dynamically during pregnancy. Placenta-derived exosomes are small vesicles carrying active biological molecules such as microRNAs, proteins, and lipids. The placenta-derived exosomes have been implicated in endothelial cell activation, smooth muscle cell migration, and T-cell apoptosis, but it is unknown whether placenta-derived exosomes would affect the development and functions of decidual macrophages. In this study, we reported that placenta-derived exosomes stimulated macrophage polarization into alternatively activated (M2) macrophages. Mechanistically, miRNA-30d-5p from the placenta-derived exosomes induced macrophage polarization to the M2 phenotype by targeting histone deacetylase 9. Furthermore, the conditioned medium of placenta-derived exosome-treated macrophages promoted trophoblast migration and invasion. By contrast, the conditioned medium impaired the ability of endothelial cell tube formation and migration. Placenta-derived exosome-treated macrophages had no impact on T-cell proliferation. Together, we demonstrated that placenta-derived exosomes polarize macrophages to acquire a decidua-like macrophage phenotype to modulate trophoblast and endothelial cell functions.

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

Adrenomedullin has a pivotal role in trophoblast differentiation: A promising nanotechnology-based therapeutic target for early-onset preeclampsia.

Early-onset preeclampsia (EOPE) is a severe pregnancy complication associated with defective trophoblast differentiation and functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of EOPE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast and syncytiotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed EOPE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to EOPE-like phenotypes. The EOPE-like phenotypes in a mouse PE model were reduced by a placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, EOPE pathogenesis, and its potential clinical uses.

100 YEARS OF VITAMIN D: Effect of serum vitamin D level before ovarian stimulation on the cumulative live birth rate of women undergoing in vitro fertilization: a retrospective analysis.

Objective: Vitamin D receptors are present in the female reproductive tract. Studies on the association between serum vitamin D level and pregnancy rate of in vitro fertilization (IVF) showed inconsistent results and focused on a single fresh or frozen embryo transfer cycle. The objective of our study was to evaluate if serum vitamin D level before ovarian stimulation was associated with the cumulative live birth rate (CLBR) of the first IVF cycle. Design: Retrospective cohort study. Methods: Women who underwent the first IVF cycle from 2012 to 2016 at a university-affiliated reproductive medicine center were included. Archived serum samples taken before ovarian stimulation were analyzed for 25(OH)D levels using liquid chromatography-mass spectrometry. Results: In total, 1113 had pregnancy outcome from the completed IVF cycle. The median age (25th-75th percentile) of the women was 36 (34-38) years and serum 25(OH)D level was 53.4 (41.9-66.6) nmol/L. The prevalence of vitamin D deficiency (less than 50 nmol/L) was 42.2%. The CLBR in the vitamin D-deficient group was significantly lower compared to the non-deficient group (43.9%, 208/474 vs 50.9%, 325/639, P = 0.021, unadjusted), and after controlling for women's age, BMI, antral follicle count, type and duration of infertility. There were no differences in the clinical/ongoing pregnancy rate, live birth rate and miscarriage rate in the fresh cycle between the vitamin D deficient and non-deficient groups. Conclusions: Vitamin D deficiency was prevalent in infertile women in subtropical Hong Kong. The CLBR of the first IVF cycle in the vitamin D-deficient group was significantly lower compared to the non-deficient group.

WNT5A Interacts With FZD5 and LRP5 to Regulate Proliferation and Self-Renewal of Endometrial Mesenchymal Stem-Like Cells.

Endometrial mesenchymal stem-like cells (eMSC) reside in the basal layer of the endometrium and are responsible for cyclic regeneration during the reproductive lives of women. Myometrial cells act as a component of the niche and regulate the stem cell fate through the activation of WNT/ß-catenin signaling via WNT5A. Since WNT5A-responsive mechanisms on eMSC are still uncertain, we hypothesize that the WNT ligand-WNT5A works to activate WNT/ß-catenin signaling through binding to Frizzled receptors (FZDs) and co-receptor low-density lipoprotein receptor-related protein 5 (LRP5). Among the various receptors that have been reported to interact with WNT5A, we found FZD5 abundantly expressed by eMSC when compared to unfractionated stromal cells. Neutralizing the protein expression by using anti-FZD5 antibody suppressed the stimulatory effects on phenotypic expression and the clonogenicity of eMSC in a myometrial cell-eMSC co-culture system as well as in an L-Wnt5a conditioned medium. Gene silencing of FZD5 not only reduced the binding of WNT5A to eMSC but also decreased the TCF/LEF transcriptional activities and expression of active ß-catenin. Inhibition of LRP coreceptors with recombinant Dickkopf-1 protein significantly reduced the binding affinity of eMSC to WNT5A as well as the proliferation and self-renewal activity. During postpartum remodeling in mouse endometrium, active ß-catenin (ABC) was detected in label-retaining stromal cells (LRSCs), and these ABC+ LRSCs express FZD5 and LRP5, suggesting the activation of WNT/ß-catenin signaling. In conclusion, our findings demonstrate the interaction of WNT5A, FZD5, and LRP5 in regulating the proliferation and self-renewal of eMSC through WNT/ß-catenin signaling.