Automation in ART: Paving the Way for the Future of Infertility Treatment.

In vitro fertilisation (IVF) is estimated to account for the birth of more than nine million babies worldwide, perhaps making it one of the most intriguing as well as commoditised and industrialised modern medical interventions. Nevertheless, most IVF procedures are currently limited by accessibility, affordability and most importantly multistep, labour-intensive, technically challenging processes undertaken by skilled professionals. Therefore, in order to sustain the exponential demand for IVF on one hand, and streamline existing processes on the other, innovation is essential. This may not only effectively manage clinical time but also reduce cost, thereby increasing accessibility, affordability and efficiency. Recent years have seen a diverse range of technologies, some integrated with artificial intelligence, throughout the IVF pathway, which promise personalisation and, at least, partial automation in the not-so-distant future. This review aims to summarise the rapidly evolving state of these innovations in automation, with or without the integration of artificial intelligence, encompassing the patient treatment pathway, gamete/embryo selection, endometrial evaluation and cryopreservation of gametes/embryos. Additionally, it shall highlight the resulting prospective change in the role of IVF professionals and challenges of implementation of some of these technologies, thereby aiming to motivate continued research in this field.

Developing a novel device, Eggcell, to improve temperature stability during oocyte collection for IVF.

RESEARCH QUESTION: What temperature fluctuations are oocytes exposed to during oocyte retrieval? Can an alternative method of oocyte retrieval be designed to minimize these fluctuations? DESIGN: Mock oocyte retrieval procedures were performed to investigate the change in temperature when the follicular fluid is drained into collection tubes and when the fluid is subsequently poured into dishes to allow identification of the cumulus-oocyte complex (COC). A new device, the Eggcell, has been designed that addresses the problem of these temperature fluctuations. To confirm its safety and demonstrate the clinical applicability of Eggcell, laboratory validation was performed prior to use with human participants (n = 15). RESULTS: Eggcell meets its design specification to provide temperature stability within the physiological range for aspirated follicular fluid. The COC can be successfully retained within the chamber (n = 180) without evidence of loss or damage to the oocytes or compromise of fertilization rate, blastocyst development or clinical outcome. CONCLUSIONS: This study has demonstrated the successful first stages of development of a new medical device. Further studies are needed for comparative evaluation of clinical outcome with standard technology.

Platelet-derived growth factor BB is reduced in endometrial endothelial cells of women with abnormal uterine bleeding-endometrial disorder.

RESEARCH QUESTION: What is the expression pattern of platelet-derived growth factor BB (PDGF-BB), and its receptors, across the menstrual cycle in healthy control women and those with abnormal uterine bleeding-endometrial disorder (AUB-E)? DESIGN: Immunohistochemical staining for PDGF-BB, platelet-derived growth factor receptor alpha (PDGFRα) and platelet-derived growth factor beta (PDGFRß) was performed in control and AUB-E endometrium from the proliferative, early, mid- and late secretory phases of the menstrual cycle (n = 5 each group). Control proliferative phase endometrium was cultured in PDGF-BB (0, 10 ng/ml) and vascular maturation assessed (n = 3). Endothelial cell to vascular smooth muscle cell (VSMC) association was assessed after treatment with PDGF-BB (0, 1, 10 ng/ml). Secretion of angiogenic growth factors by endothelial cells or VSMC was determined. RESULTS: Endothelial cell immunoreactivity for PDGF-BB was reduced in the mid and late secretory phases in AUB-E (P = 0.008). PDGFRα was also reduced in mid secretory phase endothelial cells, proliferative and early secretory phase glandular epithelium in AUB-E (P = 0.008). PDGFRß expression was not altered. Treatment of proliferative phase endometrium with PDGF-BB (10 ng/ml) reduced the percentage of vessels expressing contractile VSMC markers. PDGF-BB had no effect on angiogenic growth factor secretion by endothelial cells or VSMC in vitro and did not affect their association in an in-vitro endothelial cell-VSMC association assay. CONCLUSIONS: Reduced endothelial cell expression of PDGF-BB in the AUB-E endometrium may contribute to the reduced vascular maturation previously observed in these women.

Transforming Growth Factor (TGF) ß and Endometrial Vascular Maturation.

Appropriate growth and development of the endometrium across the menstrual cycle is key for a woman's quality of life and reproductive well-being. Recurrent pregnancy loss (RPL) and heavy menstrual bleeding (HMB) affect a significant proportion of the female population worldwide. These endometrial pathologies have a significant impact on a woman's quality of life as well as placing a high economic burden on a country's health service. An underlying cause for both conditions is unknown in approximately 50% of cases. Previous research has demonstrated that aberrant endometrial vascular maturation is associated with both RPL and HMB, where it is increased in RPL but reduced in HMB. TGFß1 is one of the key growth factors that regulate vascular maturation, by inducing phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic phenotype to a more contractile one. Our previous data demonstrated an increase in TGFß1 in the endometrium of RPL, while others have shown a decrease in women with HMB. However, TGFß1 bioavailability is tightly controlled, and we therefore sought to perform an extensive immunohistochemical analysis of different components in the pathway in the endometrium of normal controls, women with HMB or RPL. In addition, two in vitro models were used to examine the role of TGFß1 in endometrial vascular maturation and endothelial cell (EC):VSMC association. Taken all together, the immunohistochemical data suggest a decrease in bioavailability, receptor binding capacity, and signaling in the endometrium of women with HMB compared with controls. In contrast, there is an increase in the bioavailability of active TGFß1 in the endometrium of women with RPL compared with controls. Endometrial explants cultured in TGFß1 had an increase in the number of vessels associated with contractile VSMC markers, although the total number of vessels did not increase. In addition, TGFß1 increased EC:VSMC association in an in vitro model. In conclusion, TGFß1 is a key regulator of endometrial vascular maturation and could be considered as a therapeutic target for women suffering from HMB and/or RPL.

Endometrial vascular development in heavy menstrual bleeding: altered spatio-temporal expression of endothelial cell markers and extracellular matrix components.

STUDY QUESTION: Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)? SUMMARY ANSWER: Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB. WHAT IS KNOWN ALREADY: HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB. STUDY DESIGN, SIZE, DURATION: Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serial sections (3µm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels. MAIN RESULTS AND THE ROLE OF CHANCE: In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008). LIMITATIONS, REASONS FOR CAUTION: The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed. WIDER IMPLICATIONS OF THE FINDINGS: Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Menstrual cycle distribution of uterine natural killer cells is altered in heavy menstrual bleeding.

Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation.

Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.

STUDY QUESTION: How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? SUMMARY ANSWER: The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. WHAT IS KNOWN ALREADY: Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. MAIN RESULTS AND THE ROLE OF CHANCE: Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. LIMITATIONS, REASONS FOR CAUTION: This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. WIDER IMPLICATIONS OF THE FINDINGS: Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo.

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most - but not all - cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo.