Successful integration of newborn genetic testing into UK routine screening using prospective consent to determine eligibility for clinical trials.

OBJECTIVE: INGR1D (INvestigating Genetic Risk for type 1 Diabetes) was a type 1 diabetes (T1D) genetic screening study established to identify participants for a primary prevention trial (POInT, Primary Oral Insulin Trial). METHODS: The majority of participants were recruited by research midwives in antenatal clinics from 18 weeks' gestation. Using the NHS Newborn Bloodspot Screening Programme (NBSP) infrastructure, participants enrolled in INGR1D had an extra sample taken from their day 5 bloodspot card sent for T1D genetic screening. Those at an increased risk of T1D were informed of the result, given education about T1D and the opportunity to take part in POInT. RESULTS: Between April 2018 and November 2020, 66% of women approached about INGR1D chose to participate. 15 660 babies were enrolled into INGR1D and 14 731 blood samples were processed. Of the processed samples, 157 (1%) had confirmed positive results, indicating an increased risk of T1D, of whom a third (n=49) enrolled into POInT (20 families were unable to participate in POInT due to COVID-19 lockdown restrictions). CONCLUSION: The use of prospective consent to perform personalised genetic testing on samples obtained through the routine NBSP represents a novel mechanism for clinical genetic research in the UK and provides a model for further population-based genetic studies in the newborn.

'Just have some IVF!': A longitudinal ethnographic study of couples' experiences of seeking fertility treatment.

We present findings from a longitudinal ethnographic study of infertile couples seeking treatment following initial GP referral to specialist fertility services. Repeated observations and interviews were undertaken with the same 14 heterosexual participants over an 18-month period. Heterosexual, non-donor couples comprise the majority of fertility clinic patients; however, research interest in this group has dwindled over time as IVF cycles have increased. In the United Kingdom, IVF is presented as a logical response to involuntary childlessness, and as an entirely predictable, and linear, course of action. The market is well-developed and often patients' first experience of privatised health care in the NHS. Our couples were challenged by this, and while they felt expected to move on to IVF, some wished to explore other options. While IVF is ubiquitous, the discomfort and challenge around fertility treatments remain; experiences are prolonged and characterised by recursive narratives and expressions of disequilibrium, which are rarely acknowledged and reflected in ongoing clinic-patient interactions. Our findings develop understanding of the process of 'mazing' (Image - The Journal of Nursing Scholarship, 1989, 21, 220), the pursuit of parenthood, by showing that the routine and normative status of IVF, at least in the current health care context, is at odds with the lived experiences of individuals.

Antigen unmasking does not improve the visualization of phospholipase C zeta in human spermatozoa.

Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard "in-house" assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.

Transition to parenthood after successful non-donor in vitro fertilisation: The effects of infertility and in vitro fertilisation on previously infertile couples' experiences of early parenthood.

Recent social science research in the field of parenting following assisted conception has focused on the experiences of donor-assisted conception and surrogacy. This article draws from a study which explored the experiences of the transition to early parenthood in 16 heterosexual non-donor couples and includes a specific consideration of the experiences of men as they navigate this journey. We argue that these couples' transition to early parenthood can be as complex and provisional as in other newer forms of family making as they struggle with an emerging identity as a parent after successful non-donor in vitro fertilisation following their experiences of infertility. Their family making is contingent upon their ability to work at integrating their experiences of infertility and in vitro fertilisation into their emerging identity as a parent. This struggle is prominent when they contemplate a further pregnancy. Considering a sibling causes them further uncertainty and anxiety because it reminds them of their infertile identify and the possibility of further in vitro fertilisation. We report novel findings about the experiences of this transition to parenthood: how couples' identity as parents is shaped by the losses and grief of infertility and the anxiety of in vitro fertilisation. We argue that their struggle with an emerging parenthood identity challenges the normative, naturalised view of non-donor heterosexual in vitro fertilisation parenthood. Our work contributes to the work on identity in parenthood after in vitro fertilisation in an ongoing effort to understand how assisted technologies shape infertile parents' lives. This article reports a small study with a relatively homogeneous sample recruited from one fertility clinic. Nevertheless as an exploratory study of an under researched topic, we discuss useful insights and ideas for further research with larger and more diverse samples.

An integrative literature review of psychosocial factors in the transition to parenthood following non-donor-assisted reproduction compared with spontaneously conceiving couples.

The paper reports an integrative literature review of research into the psychosocial factors which shape the transition to parenthood in couples following non-donor in vitro fertilization in comparison with those conceiving spontaneously. Nineteen papers of non-donor IVF and SC mothers and fathers were included. Differences between groups were reported for a range of psychosocial measures during the transition from pregnancy to parenthood including: the control couples feel they have over their lives (locus of control), parental adjustment and child behaviour, parental stress, parental investment in the child, self-esteem and self-efficacy, greater levels of protectiveness (separation anxiety) towards child, marital and family functioning, family alliance, marital satisfaction and communication, as well as anxiety, indirect aggression and lowered respect for the child. We have conceptualised these differences as three substantive themes which reflect psychosocial factors shaping transition to parenthood in parents after non-donor AR: namely social support, relationships and emotional well-being, which are in turn influenced by gender differences. These findings have implications for health care professionals' assessment of individual couples' support needs.

Use of phospholipase C zeta analysis to identify candidates for artificial oocyte activation: a case series of clinical pregnancies and a proposed algorithm for patient management.

OBJECTIVE: To investigate the applicability of phospholipase C zeta (PLCζ) analysis in assisting the clinical decision-making process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology. DESIGN: Fifty-six males (43 infertile/13 fertile) were screened using our PLCζ assay. SETTING: Fertility unit/university laboratory. PATIENT(S): Infertile males with abnormal sperm morphology or total fertilization failure, low fertilization rate (<50%), or repeated fertilization failure in assisted reproductive technology. INTERVENTION(S): We analyzed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent intracytoplasmic sperm injection (ICSI)/artificial oocyte activation (AOA) with calcimycin (GM508). MAIN OUTCOME MEASURE(S): PLCζ localization and level and the proportion of sperm expressing PLCζ. Thresholds of PLCζ deficiency, fertilization rates, pregnancy rates, and live birth rates of AOA and non-AOA cycles. RESULT(S): Compared with 13 fertile controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed "PLCζ deficiency." Five PLCζ-deficient patients opted for AOA; all five achieved fertilization, and four achieved clinical pregnancies and live births. The fertilization rate improved significantly from 18.6% (ICSI) to 56.8% (ICSI/AOA). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cutoffs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level. CONCLUSION(S): PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment.

In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles.

Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR-/-: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFß: transforming growth factor ß; uNK: uterine natural killer.

Preconception care for infertile couples: Nurses' and midwives' roles in promoting better maternal and birth outcomes.

INTRODUCTION: Preconception care promotes better maternal outcomes, may assist in preventing birth defects and improves fertility awareness among healthy childbearing couples. Yet, the significance of preconception care for infertile couples is undeveloped area of practice in Europe. AIMS AND OBJECTIVES: To discuss the importance of nurses and midwives in providing preconception care to infertile couples in the United Kingdom and Spain. DESIGN: Discursive paper. METHOD: A comparison of different midwifery and nursing approaches to preconception care for infertile couples in two European countries. FINDINGS: At present, infertile couples' needs for preconception care are not routinely identified or understood. There is an opportunity for these needs to be considered and identified by nurses at the time of investigation for infertility or when planning pregnancy with assisted conception. CONCLUSIONS: We argue that, by providing preconception care, nurses and midwives have an opportunity to deliver important advice to infertile couples in both primary care and specialist infertility services.

Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22-54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ-related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm's fundamental ability to activate an oocyte.

Randomized, controlled pilot trial of natural versus hormone replacement therapy cycles in frozen embryo replacement in vitro fertilization.

OBJECTIVE: To determine whether there is any difference between the outcomes of two standard treatment protocols for frozen embryo replacement (FER): natural and down-regulated hormone replacement treatment (HRT). DESIGN: Open, single-center, randomized, controlled pilot trial. SETTING: Private fertility clinic. PATIENT(S): Women (n = 159) planning an FER cycle at the Oxford Fertility Unit, aged <40 years at the time their embryos were frozen; with at least one blastocyst or two cleavage-stage embryos in storage; regular ovulatory cycles; and at most two previous FER cycles. INTERVENTION(S): Eligible participants were recruited and randomized between March 2010 and July 2012 into one of two standard FER treatment groups: natural (n = 80) menstrual (Natural) or GnRH agonist/HRT (n = 79) cycles. MAIN OUTCOME MEASURE(S): Live birth rate after replacement of frozen-thawed embryos, clinical pregnancy rate, implantation rate, and cycle cancellations. RESULT(S): A total of 159 women were randomized (80 Natural; 79 HRT), and 145 had ET and completed the study (72 Natural; 73 HRT). Pregnancy outcomes were not significantly different between the two groups. The live birth rates were 26.3% (Natural) and 31.7% (HRT) per randomized patient. Per ET/protocol the live birth rates were 29.2% and 34.2%. The implantation rates were 24.3% and 26.0%, and there were three twin births in the Natural and five in the HRT arms. CONCLUSION(S): The findings of this pilot study support the suggestion that for women with ovulatory cycles undergoing FER, the outcomes are similar between natural and HRT protocols. CLINICAL TRIAL REGISTRATION NUMBER: NCT00843570. Registered at www.clinicaltrials.gov.

Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection.

OBJECTIVE: To study the relationship of total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta, with fertilization rates after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). DESIGN: Laboratory study; controls vs. patients after IVF (n = 27) or ICSI (n = 17) treatment. SETTING: Fertility center. PATIENT(S): A total of 44 semen samples, subjected to either IVF or ICSI treatment. Oocyte collection, ICSI or IVF, determination of sperm concentration and motility, and immunocytochemical analyses of phospholipase C zeta (PLCζ). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Percentages of sperm exhibiting PLCζ. RESULT(S): Significant positive correlation between ICSI fertilization rates and total levels, localization patterns, and the proportion (percentage) of sperm exhibiting PLCζ. Total levels, localization patterns, and the proportion of sperm exhibiting PLCζ are correlated with fertilization rates for ICSI, but not for IVF. CONCLUSION(S): Evaluating total levels, localization patterns, and proportions of PLCζ may represent a useful diagnostic tool for clinical purposes in men for whom IVF is not advised or has previously failed. This clinical study further supports the fundamental role of PLCζ in the oocyte activation process.

An overview of information giving in fertility clinics.

Information giving is a key aspect of the provision of high-quality patient-centred health care, resulting in patients who are well-informed, better adjusted to their circumstances and are compliant with their treatment. Fertility patients generally appear to be satisfied with the information they are given but a significant minority are not. Giving information to infertile patients is complicated by the nature of their condition, desire for a child and complexity of treatment options. Patients need detailed, well-timed information to support difficult decision-making, such as when to end treatment. The experiences of some individual patients and particular sub-groups in receiving and understanding information suggest that the quality of information or the way it is communicated could be improved. It is suggested that this may be achieved by reviewing the format and timing of the information provided, ensuring adequate staff training and facilitating flexibility in clinic organisation.

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

OBJECTIVE: To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. DESIGN: Laboratory study. SETTING: University laboratory. PATIENT(S): Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). INTERVENTION(S): Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. MAIN OUTCOME MEASURE(S): Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. RESULT(S): Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. CONCLUSION(S): The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis.

Motile sperm organelle morphology evaluation-selected globozoospermic human sperm with an acrosomal bud exhibits novel patterns and higher levels of phospholipase C zeta.

STUDY QUESTION: Does motile sperm organelle morphology examination (MSOME) affect levels and localization patterns of the oocyte activation factor phospholipase C zeta (PLCζ) in globozoospermic sperm with and without an acrosomal bud? SUMMARY ANSWER: MSOME identified round-headed globozoospermic sperm with increased levels of PLCζ relative to sperm from the same sample that did not undergo MSOME, and identified novel patterns of PLCζ localization in sperm exhibiting an acrosomal bud. WHAT IS KNOWN ALREADY: Absence or reduction in the level of PLCζ in the sperm head, abnormal localization patterning, or defective functional ability as a result of PLCζ gene mutation, have been linked to certain types of human male factor infertility in which oocyte activation is deficient. It has been determined that a subpopulation of sperm (1%) from a patient exhibiting 100% globozoospermia presented with an acrosome bud upon MSOME. A cycle of intracytoplasmic morphologically selected sperm injection, carried out with sperm exhibiting an acrosomal bud led to pregnancy and birth of a healthy baby boy, without the use of assisted oocyte activation (AOA). STUDY DESIGN, SIZE, DURATION: Immunofluorescent analysis of PLCζ in globozoospermic sperm from three patients, before and after MSOME. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative immunofluorescence was used to investigate PLCζ levels and localization patterns in individual sperm (n = 1 patient) identified by MSOME and isolated by micromanipulation, and presenting with and without the acrosomal bud. A secondary aim was to investigate levels and localization patterns of PLCζ in sperm before and after MSOME from two other globozoospermic men. MAIN RESULTS AND THE ROLE OF CHANCE: Non-globozoospermic control sperm exhibited characteristic localization patterns of PLCζ immunofluorescence. Completely round-headed globozoospermic sperm from patients 1-3 were either devoid of PLCζ immunofluorescence, or exhibited an abnormal, punctate, pattern of PLCζ localization. PLCζ immunofluorescence in sperm exhibiting an acrosomal bud was observed in the midpiece with varying fluorescent intensity and was detected in 28.5% of such sperm. The majority of sperm with an acrosomal bud (43.0%) exhibited punctate patterns of PLCζ localization within the sperm head. A further 28.5% of sperm exhibited PLCζ in both the head and the midpiece. Total levels of PLCζ, and the proportions of sperm exhibiting PLCζ immunoreactivity, showed significant variance (P ≤ 0.05) amongst control [45.8 arbitrary units (a.u.) and 95.7%, respectively], non-MSOME-selected (25.9 a.u. and 46.1%, respectively) and MSOME-selected globozoospermic sperm (33.4 a.u. and 65.0%, respectively). Total levels of PLCζ immunofluorescence, and proportions of sperm exhibiting PLCζ immunoreactivity, in control sperm was significantly higher (P≤ 0.05) compared with non-MSOME-selected sperm, but not significantly different from MSOME-selected sperm. LIMITATIONS, REASONS FOR CAUTION: The low numbers of sperm analysed may not be ideal for conclusive statistical analysis. Evaluation of the effects of MSOME on morphologically normal sperm would confirm conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The present findings provide hope for the future treatment of globozoospermia without the need for AOA, and provide further evidence for the clinical application of PLCζ as a therapeutic and prognostic tool. STUDY FUNDING/COMPETING INTEREST(S): The research described herein was funded by the Nuffield Department of Obstetrics and Gynaecology, University of Oxford. The authors report no conflict of interest.