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Normal reproductive functioning is critically dependent on pulsatile secretion of luteinising hormone (LH). Assessment of LH pulsatility is important for the clinical diagnosis of reproductive disorders, but current methods are hampered by frequent blood sampling coupled to expensive serial immunochemical analysis. Here, we report the development and application of a Robotic APTamer-enabled Electrochemical Reader (RAPTER) electrochemical analysis system to determine LH pulsatility. Through selective evolution of ligands by exponential enrichment (SELEX), we identify DNA aptamers that bind specifically to LH and not to related hormones. The aptamers are integrated into electrochemical aptamer-based (E-AB) sensors on a robotic platform. E-AB enables rapid, sensitive and repeatable determination of LH concentration profiles. Bayesian Spectrum Analysis is applied to determine LH pulsatility in three distinct patient cohorts. This technology has the potential to transform the clinical care of patients with reproductive disorders and could be developed to allow real-time in vivo hormone monitoring. Assessment of luteinising hormone pulsatility is important in the diagnosis of reproductive disorders. Here the authors develop a DNA aptamer-based electrochemical analysis integrated into a robotic platform for high-throughput and sensitive analysis.

Background This study aims to investigate the association of pre-conception vitamin D levels on adverse pregnancy outcomes in women undergoing in vitro fertilization with fresh embryo transfer. Methods This was a retrospective cohort study using archived serum 25-hydroxyvitamin D measured in the pre-conception period before ovarian stimulation in patients undergoing in vitro fertilization with fresh autologous embryo transfer. A total of 306 women were included and adverse pregnancy outcomes in their resulting pregnancy were recorded. Patients who were vitamin D deficient (< 20ng/ml) were compared with those who were non-deficient (≥20ng/ml) and analysed for any association with adverse pregnancy outcomes. Results A total of 16/306 (5.3%) patients had hypertensive disorders of pregnancy (gestational hypertension and/or pre-eclampsia). The adjusted odds ratio for hypertensive disorders of pregnancy using vitamin D deficiency as a reference was 0.190 (95% CI 0.042–0.852) ( p  = 0.030). Other pregnancy complications were not significantly different with regards to pre-conception vitamin D status. Conclusions Pre-conception vitamin D deficiency is associated with an increased risk of hypertensive disorders in pregnancy in women undergoing in vitro fertilization with fresh embryo transfer. Trial registration HKUCTR 2361 (9th March 2018).

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.

Background Manual counting for semen analysis is recommended by the World Health Organization. Technicians performing this usually record their results on a paper worksheet and then enter the data into an electronic laboratory information system. One disadvantage of this approach is the chance of post-analytical transcription errors, which can be reduced by checking the computer entries before reporting by another technician. Such practice inevitably increases the running cost and delays the reporting time. The present study was to establish a paperless electronic data entry system for semen analysis and compare its precision with the conventional paper method. During semen analysis, readings on the cell counter were video recorded. The precision of the paper record entries was determined by comparing them with the corresponding video records. Patient characteristics and semen analysis results were input directly into an in-house developed data entry system via a tablet computer immediately after analysis. The same set of data was also handwritten on a paper form and was subsequently input into a standard computerized database according to routine practice. The agreement of the data entries between the two systems was then compared. Results A total of 787 semen analyses were included in the study, involving 201 samples in Phase I and 586 samples in Phase II of the study. Phase I was the initial learning period. The overall rate of transcription error of the paper form was 0.07%, whereas that of the paperless system was 0.17%. In phase II, the paperless system was modified according to users’ comments. The transcription error rate of the paper form was 0.05%, while that of the paperless system was substantially reduced to 0.01% ( p  = 0.008). Conclusion The paperless system is a reliable tool for recording data from semen analysis compared with the conventional paper form. However, training is needed to reduce the error rate of the paperless system.

Background In vitro fertilization (IVF) is a well-established method to treat various causes of infertility. Some previous retrospective studies suggested a lower ovarian response in Asian women compared to Caucasian women. However, the ovarian stimulation regimens were not standardized, potentially confounding the findings. The objective of this study is to compare the number of oocytes obtained after ovarian stimulation between Chinese and Caucasian women undergoing IVF using a standardized stimulation regimen. Methods This is a prospective cohort study conducted in two tertiary IVF units in Hong Kong, China and Sydney, Australia from October 2016 to August 2019. A total of 192 women aged 18–42 years with a body weight > 60 kg underwent IVF with a standard ovarian stimulation regimen of 150 micrograms corifollitropin alfa (Elonva®) followed by 200 IU follitropin beta (Puregon®) per day. The number of oocytes retrieved in Chinese women treated in the Hong Kong center was compared to that of Caucasian women treated in the Australian center. Results Serum AMH levels were similar between the two groups. Although women in the Chinese cohort were older and had a higher body mass index (BMI), longer duration of infertility and lower antral follicle count (AFC) than those in the Caucasian cohort in this study, no differences in the number of oocytes retrieved [11 (8–17) vs. 11 (6–17), p=0.29], total dosage and duration of stimulation and number of follicles aspirated were noted between the two ethnic cohorts. The peak estradiol level was greater in Chinese women than in Caucasian women. After controlling for age, BMI and AFC, ethnicity was a significant independent determinant of the number of oocytes obtained. Conclusions Chinese women had a higher number of oocytes after ovarian stimulation using a standardized stimulation regimen compared with Caucasian women undergoing IVF after controlling for age, BMI, AFC and AMH despite presenting later after a longer duration of infertility. Trial registration number: NCT02748278

Sperm selection in the female reproductive tract (FRT) is sophisticated. Only about 1,000 sperm out of millions in an ejaculate reach the fallopian tube and thus have a chance of fertilizing an oocyte. In assisted reproduction techniques, sperm are usually selected using their density or motility, characteristics that do not reflect their fertilization competence and, therefore, might result in failure to fertilize the oocyte. Although sperm processing in in vitro fertilization (IVF) and intrauterine insemination (IUI) bypasses many of the selection processes in the FRT, selection by the cumulus mass and the zona pellucida remain intact. By contrast, the direct injection of a sperm into an oocyte in intracytoplasmic sperm injection (ICSI) bypasses all natural selection barriers and, therefore, increases the risk of transferring paternal defects such as fragmented DNA and genomic abnormalities in sperm to the resulting child. Research into surrogate markers of fertilization potential and into simulating the natural sperm selection processes has progressed. However, methods of sperm isolation — such as hyaluronic acid-based selection and microfluidic isolation based on sperm tactic responses — use only one or two parameters and are not comparable with the multistep sperm selection processes naturally occurring within the FRT. Fertilization-competent sperm require a panel of molecules, including zona pellucida-binding proteins and ion channel proteins, that enable them to progress through the FRT to achieve fertilization. The optimal artificial sperm selection method will, therefore, probably need to use a multiparameter tool that incorporates the molecular signature of sperm with high fertilization potential, and their responses to external cues, within a microfluidic system that can replicate the physiological processes of the FRT in vitro.In this Review, the authors describe current sperm selection methods and the advances in selection technologies for assisted reproductive techniques, highlighting their mechanisms of selection, advantages, limitations and clinical outcomes. They also propose a conceptual sperm selection model that uses multiple selection mechanisms.

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.

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/β-catenin signaling pathway. 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. 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. 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/β-catenin signaling pathway was observed upon IL-6 treatment, while suppression of the WNT/β-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. 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/β-catenin pathway. Treatment of eMSC with IL-6 and WNT5A might enhance their therapeutic potential in the regeneration of injured endometrium.

The objective of this study was to assess the effects of freezing and storage on serum vitamin D levels in women undergoing ovarian stimulation for in vitro fertilization and in the natural cycle. This is a prospective observational in vitro study in a tertiary reproductive medicine center and an accredited hospital-based Chemical Pathology Laboratory. Each serum sample was divided into equal volume aliquots and stored at -20 o C and −80 o C. The samples were analyzed for 25-hydroxyvitamin D [25(OH)D] levels using liquid chromatography‒mass spectrometry and immunoassay at baseline and after 2 weeks and 7 months of storage. The overall serum 25(OH)D levels measured by liquid chromatography‒mass spectrometry showed a statistically significant reduction over 7 months of storage at -80 °C [median % change: -4.9 (25th, 75th percentile: -10.1, 2.5), p  = 0.020] but not at -20 °C compared with the baseline. With the immunoassay, the overall serum 25(OH)D levels also significantly decreased over 7 months of storage at -80 °C [median % change: -4.0 (25th, 75th percentile: -15.6, 1.4), p  < 0.001] but not at -20 °C compared with the baseline. In conclusion, the 25(OH)D levels of the serum samples stored at -20 o C and − 80 o C were lower at 7 months of storage when compared with baseline levels, but the difference was small. Nevertheless, vitamin D deficiency is usually assessed based on clinical thresholds. Awareness of potential degradation of 25(OH)D is needed, and the method used for measurement should be taken into account when interpreting retrospective studies of 25(OH)D using archived serum samples, as even small variations can lead to women being ‘reclassified’ into different vitamin D status categories.

B cells constitute a diverse and adaptable immune cell population with functions that can vary according to the environment and circumstances. The involvement of B cells in pregnancy, as well as the associated molecular pathways, has yet to be investigated. This review consolidates current knowledge on B cell activities and regulation during pregnancy, with a particular focus on the roles of various B cell subsets and the effects of B cell-derived factors on pregnancy outcomes. Moreover, the review examines the significance of B cell-associated autoantibodies, cytokines, and signaling pathways in relation to pregnancy complications such as pregnancy loss, preeclampsia, and preterm birth.