Cell-free fetal DNA for genetic evaluation in Copenhagen Pregnancy Loss Study (COPL): a prospective cohort study.

BACKGROUND: One in four pregnancies end in a pregnancy loss. Although the effect on couples is well documented, evidence-based treatments and prediction models are absent. Fetal aneuploidy is associated with a higher chance of a next successful pregnancy compared with euploid pregnancy loss in which underlying maternal conditions might be causal. Ploidy diagnostics are therefore advantageous but challenging as they require collection of the pregnancy tissue. Cell-free fetal DNA (cffDNA) from maternal blood has the potential for evaluation of fetal ploidy status, but no large-scale validation of the method has been done. METHODS: In this prospective cohort study, women with a pregnancy loss were recruited as a part of the Copenhagen Pregnancy Loss (COPL) study from three gynaecological clinics at public hospitals in Denmark. Women were eligible for inclusion if older than 18 years with a pregnancy loss before gestational age 22 weeks (ie, 154 days) and with an intrauterine pregnancy confirmed by ultrasound (including anembryonic sac), and women with pregnancies of unknown location or molar pregnancies were excluded. Maternal blood was collected while pregnancy tissue was still in situ or within 24 h after pregnancy tissue had passed and was analysed by genome-wide sequencing of cffDNA. Direct sequencing of the pregnancy tissue was done as reference. FINDINGS: We included 1000 consecutive women, at the time of a pregnancy loss diagnosis, between Nov 12, 2020, and May 1, 2022. Results from the first 333 women with a pregnancy loss (recruited between Nov 12, 2020, and Aug 14, 2021) were used to evaluate the validity of cffDNA-based testing. Results from the other 667 women were included to evaluate cffDNA performance and result distribution in a larger cohort of 1000 women in total. Gestational age of fetus ranged from 35-149 days (mean of 70·5 days [SD 16·5], or 10 weeks plus 1 day). The cffDNA-based test had a sensitivity for aneuploidy detection of 85% (95% CI 79-90) and a specificity of 93% (95% CI 88-96) compared with direct sequencing of the pregnancy tissue. Among 1000 cffDNA-based test results, 446 (45%) were euploid, 405 (41%) aneuploid, 37 (4%) had multiple aneuploidies, and 112 (11%) were inconclusive. 105 (32%) of 333 women either did not manage to collect the pregnancy tissue or collected a sample classified as unknown tissue giving a high risk of being maternal. INTERPRETATION: This validation of cffDNA-based testing in pregnancy loss shows the potential and feasibility of the method to distinguish euploid and aneuploid pregnancy loss for improved clinical management and benefit of future reproductive medicine and women's health research. FUNDING: Ole Kirks Foundation, BioInnovation Institute Foundation, and the Novo Nordisk Foundation.

SARS-CoV-2 Antibodies in Breastmilk Three and Six Months Postpartum in Relation to the Trimester of Maternal SARS-CoV-2 Infection-An Exploratory Study.

The immune system of neonates is immature and therefore knowledge of possible early-life protection against SARS-CoV-2 infection, such as breastfeeding, is of great importance. Few studies have investigated the presence and duration of SARS-CoV-2 antibodies in breastmilk in relation to the trimester of maternal infection during pregnancy, and none with successful participation from all three trimesters. This study has dual objectives (1) in relation to the trimester of infection to examine the frequency, concentration and duration of IgA and IgG antibodies in breastmilk and blood serum in the third and sixth month post-partum in former SARS-CoV-2-infected mothers and (2) to examine the association in pediatric emergency admission of children within the first six months of life compared to children of non-SARS-CoV-2-infected women. The first objective is based on a prospective cohort and the second is based on a nested case-control design. The study participants are women with a former SARS-CoV-2 infection during pregnancy, whose serology IgG tests at delivery were still positive. Maternal blood and breastmilk samples were collected at three and six months postpartum. Serum IgA frequency three months pp was 72.7% (50%, 90% and 60% in the first, second and third trimester) and 82% six months pp (67%, 91% and 82% in the first, second and third trimester). Breastmilk IgA frequency three months pp was 27% (16.6%, 36% and 20% in first, second and third trimester) and 28% six months pp (0%, 38% and 28% in the first, second and third trimester). The highest IgA concentration in breastmilk was found six months post-partum with infection in the third trimester. Serum IgA was detectable more than 400 days post infection, and serum IgG above threshold was found 430 days after date of infection. We found no correlation between serum IgA and breastmilk IgA, nor between serum IgG and breastmilk IgA regardless of the trimester of infection.

Cross-species comparison of pregnancy-induced GDF15.

Growth differentiation factor 15 (GDF15) is a stress-induced cytokine. Although the exact physiological function of GDF15 is not yet fully comprehended, the significant elevation of circulating GDF15 levels during gestation suggests a potential role for this hormone in pregnancy. This is corroborated by genetic association studies in which GDF15 and the GDF15 receptor, GDNF family receptor alpha like (GFRAL) have been linked to morning sickness and hyperemesis gravidarum (HG) in humans. Here, we studied GDF15 biology during pregnancy in mice, rats, macaques, and humans. In contrast to macaques and humans, mice and rats exhibited an underwhelming induction in plasma GDF15 levels in response to pregnancy (∼75-fold increase in macaques vs. ∼2-fold increase in rodents). The changes in circulating GDF15 levels were corroborated by the magnitude of Gdf15 mRNA and GDF15 protein expression in placentae from mice, rats, and macaques. These species-specific findings may help guide future studies focusing on GDF15 in pregnancy and on the evaluation of pharmacological strategies to interfere with GDF15-GFRAL signaling to treat severe nausea and HG.NEW & NOTEWORTHY In the present study pregnancy-induced changes in circulating growth differentiation factor 15 (GDF15) in rodents, rhesus macaques, and humans are mapped. In sum, it is demonstrated that humans and macaques exhibit a tremendous increase in placental and circulating GDF15 during pregnancy. In contrast, GDF15 is negligibly increased in pregnant mice and rats, questioning a physiological role for GDF15 in pregnancy in rodents.

Psychosocial wellbeing shortly after allocation to a freeze-all strategy compared with a fresh transfer strategy in women and men: a sub-study of a randomized controlled trial.

STUDY QUESTION: Is the psychosocial wellbeing affected in women and men shortly after allocation to a freeze-all strategy with postponement of embryo transfer compared to a fresh transfer strategy? SUMMARY ANSWER: In general, psychosocial wellbeing (i.e. emotional reactions to the treatment, quality-of-life, infertility-related stress, and marital benefit) was similar in women and men allocated to a freeze-all versus those allocated to a fresh-transfer strategy 6 days after disclosure of treatment strategy (i.e. 4 days after oocyte retrieval), although women in the freeze-all group reported a slightly higher degree of depressive symptoms and mood swings compared to women in the fresh transfer group. WHAT IS KNOWN ALREADY: The use of a freeze-all strategy, i.e. freezing of the entire embryo cohort followed by elective frozen embryo transfer in subsequent cycles has increased steadily over the past decade in assisted reproductive technology (ART). This strategy essentially eliminates the risk of ovarian hyperstimulation syndrome and has proven beneficial regarding some reproductive outcomes in subgroups of women. However, patients experience a longer time interval between oocyte retrieval and embryo transfer, hence a longer time to pregnancy, possibly adding additional stress to the ART treatment. So far, little focus has been on the possible psychosocial strains caused by postponement of embryo transfer. STUDY DESIGN, SIZE, DURATION: This is a self-reported questionnaire based sub-study of a multicentre randomized controlled trial (RCT) including 460 women and 396 male partners initiating their first, second, or third treatment cycle of invitro fertilisation or intracytoplasmic sperm injection (ICSI) from May 2016 to September 2018. This sub-study was included in the primary project protocol and project plan for the RCT, as psychosocial wellbeing was considered a secondary outcome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women from eight public fertility clinics in Denmark and Sweden and one private clinic in Spain were randomized in a 1:1 ratio on the day of inclusion (menstrual cycle day 2 or 3) to either a freeze-all strategy with postponement of embryo transfer to a subsequent modified natural menstrual cycle or a fresh transfer strategy with embryo transfer in the hormone stimulated cycle. Treatment allocation was blinded until the day of the ovulation trigger. Women and their male partners were asked to complete a validated self-reported questionnaire 6 days after unblinding of treatment group allocation, corresponding to 4 days after oocyte retrieval, investigating their psychosocial wellbeing related to the treatment defined as emotional reactions to the treatment, quality-of-life, infertility-related stress, and marital benefit. The questionnaire included items from the Copenhagen Multi-Centre Psychosocial Infertility (COMPI) Fertility Problem Stress Scales and the COMPI Marital Benefit Measure. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were comparable between the two groups for both women and men. In total, response rates were 90.7% for women and 90.2% for men. In the freeze-all group, 207 women and 179 men completed the questionnaire compared with 204 women and 178 men in the fresh transfer group. Men in the two treatment groups did not differ in any of the explored aspects of psychosocial wellbeing (i.e. emotional reactions to the treatment, quality-of-life, infertility-related stress, and marital benefit) 6 days after disclosure of treatment strategy. Women in the freeze-all group reported a slightly higher degree of depressive symptoms (P = 0.045) and mood swings (P = 0.001) (i.e. variables included in 'emotional reactions to treatment') compared to women in the fresh transfer group. When adjusted for multiple testing, depressive symptoms were no longer significantly different between the two groups. No additional differences in psychosocial wellbeing were found. Self-reported quality-of-life during treatment was also rated as similar between the two groups in both women and men, but was slightly lower than they would rate their quality-of-life when not in fertility treatment. LIMITATIONS, REASONS FOR CAUTION: Although response rates were high, selection bias cannot be excluded. As this study was an RCT, we assume that psychosocial characteristics of the participants were equally distributed in the two groups, thus it is unlikely that the identified psychosocial differences between the freeze-all and fresh transfer group were present already at baseline. Furthermore, the questionnaire was completed as a one-time assessment 4 days after oocyte retrieval, thus not reflecting the whole treatment process, whereas an assessment after the full completed treatment cycle is needed to draw firm conclusions about the psychosocial consequences of the whole waiting period. However, a question posted that late would be highly biased on whether or not a pregnancy had been achieved. WIDER IMPLICATIONS OF THE FINDINGS: The results indicate that individuals in the freeze-all group exhibited slightly higher levels of depressive symptoms and mood swings compared to those in the fresh transfer group. Nevertheless, it is important to note that any worries related to potential emotional strains stemming from delaying embryo transfer should not overshadow the adoption of a freeze-all approach in cases where it is clinically recommended. As long as patients are provided with comprehensive information about the treatment strategy before initiating the process, it is worth emphasising that other aspects of psychosocial wellbeing were comparable between the two groups. STUDY FUNDING/COMPETING INTEREST(S): The study is part of the Reprounion collaborative study, co-financed by the European Union, Interreg V Öresund-Kattegat-Skagerrak. L.P. reports financial support from Merck A/S. H.S.N. reports grants from Freya Biosciences ApS, Ferring Pharmaceuticals, BioInnovation Institute, Ministry of Education, Novo Nordic Foundation, Augustinus Fonden, Oda og Hans Svenningsens Fond, Demant Fonden, Ole Kirks Fond and Independent Research Fund Denmark and personal fees from Ferring Pharmaceuticals, Merck A/S, Astra Zeneca, Cook Medical, IBSA Nordic and Gedeon Richter. H.S.N is founder and chairman of the Maternity Foundation and co-developed the Safe Delivery App (non-profit). N.C.F. reports grants from Gedeon Richter, Merck A/S, Cryos International and financial support from Ferring Pharmaceuticals, Merck A/S and Gedeon Richter. N.C.F. is chairman in the steering committee for the guideline groups for The Danish Fertility Society (non-profit). P.H. reports honoraria from Merch A/S, IBSA Nordic and Gedeon Richter. A.L.M.E. reports grants and financial support from Merck A/S and Gedeon Richter. A.P. reports grants from Gedeon Richter, Ferring Pharmaceuticals, Merck A/S and personal fees from Preglem S.A., Novo Nordic Foundation, Ferring Pharmaceuticals, Gedeon Richter, Cryos International, Merch A/S, Theramex and Organon and the lend of embryoscope to the institution from Gedeon Richter. All other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov NCT02746562.

Prospective reproductive outcomes according to sperm parameters, including DNA fragmentation, in recurrent pregnancy loss.

RESEARCH QUESTION: Are the prospective reproductive outcomes in couples experiencing recurrent pregnancy loss (RPL) related to the sperm DNA fragmentation index (DFI), as measured by sperm chromatin structure assay, sperm morphology and sperm concentration at referral? DESIGN: This prospective cohort study included 95 couples seen between 1 April 2018 and 1 December 2019 at the tertiary Copenhagen RPL Unit, Copenhagen University Hospital, Rigshospitalet and Hvidovre Hospital, Denmark. The couples had experienced three or more unexplained consecutive pregnancy losses or two late pregnancy losses (>12 weeks gestation). Follow-up was 12-31 months. RESULTS: Eighty-one of 95 (85.3%) couples achieved pregnancy after referral. In the first pregnancy after referral, 46 (56.8%) couples achieved a live birth, and 35 (43.2%) couples experienced another pregnancy loss. There was no significant difference in baseline DFI between couples that experienced pregnancy loss [median 11.7, interquartile range (IQR) 9.1-17.3] and couples that achieved a live birth (median 12.5, IQR 9.3-16.5; P = 0.971). Improving sperm morphology increased the odds of a live birth after referral (adjusted OR 1.26, 95% CI 1.05-1.52; P = 0.014). DFI and sperm concentration were not associated with the outcome of the first pregnancy after referral. Overall, 35.9% of the men had DFI ≥15 at inclusion. Couples that failed to achieve pregnancy had a higher median DFI of 17.7 (IQR 7.7-27.2) compared with the rest of the cohort (median 12.0, IQR 9.3-16.5; P = 0.041). CONCLUSIONS: At referral, sperm DFI, morphology and concentration cannot be used to identify RPL couples at risk of another pregnancy loss. Increased baseline DFI was associated with difficulty achieving another pregnancy, and improving sperm morphology was associated with increased odds of a live birth.

Biopsy-free profiling of the uterine immune system in patients with recurrent pregnancy loss and unexplained infertility.

RESEARCH QUESTION: What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) and patients with unexplained infertility (uINF)? DESIGN: Prospective study including 46 healthy controls, 28 RPL and 11 uINF patients. A feasibility study compared lymphocyte compositions of endometrial biopsies and menstrual blood collected during the first 48 h of menstruation in seven controls. In all patients, peripheral and menstrual blood from the first and subsequent 24 h were analysed separately by flow cytometry, focusing on the main lymphocyte populations and natural killer (NK) cell subsets. RESULTS: The first 24 h of menstrual blood resembles the uterine immune milieu as tested by endometrial biopsy. RPL patients showed significantly higher menstrual blood CD56+ NK cell numbers than controls (mean ± SD: 31.13 ± 7.52% versus 36.73 ± 5.4%, P = 0.002). Menstrual blood CD56dimCD16bright NK cells within the CD56+ NK cell population were decreased in RPL (16.34 ± 14.65%, P = 0.011) and uINF (15.7 ± 5.91%, P = 0.02) patients versus control (20.42 ± 11.53%). uINF patients had the lowest menstrual blood CD3+ T cell counts (38.81 ± 5.04%, control versus uINF: P = 0.01) and cytotoxicity receptors NKp46 and NKG2D on CD56brightCD16dim cells were higher in uINF (68.12 ± 11.84%, P = 0.006; 45.99 ± 13.83%, P = 0.01, respectively) and RPL (NKp46: 66.21 ± 15.36%, P = 0.009) patients versus controls. RPL and uINF patients had higher peripheral CD56+ NK cell counts versus controls (11.42 ± 4.05%, P = 0.021; 12.86 ± 4.29%, P = 0.009 versus 8.4 ± 3.5%). CONCLUSIONS: Compared with controls, RPL and uINF patients had a different menstrual blood-NK-subtype profile, indicating an altered cytotoxicity. In future studies, this non-invasive analysis might enable identification and monitoring of patients receiving immunomodulatory medications.

Anti-Müllerian hormone and live birth in unexplained recurrent pregnancy loss.

RESEARCH QUESTION: Is anti-Müllerian hormone (AMH) associated with live birth rate (LBR) in women with unexplained recurrent pregnancy loss (RPL)? DESIGN: Cohort study of women with unexplained RPL attending the RPL Unit, Copenhagen University Hospital, Denmark, between 2015 and 2021. AMH concentration was assessed upon referral, and LBR in the next pregnancy. RPL was defined as three or more consecutive pregnancy losses. Regression analyses were adjusted for age, number of previous losses, body mass index, smoking, treatment with assisted reproductive technology (ART) and RPL treatments. RESULTS: A total of 629 women were included; 507 (80.6%) became pregnant after referral. Pregnancy rates were similar for women with low and high AMH compared to women with medium AMH (81.9, 80.3 and 79.7%, respectively) (low AMH: adjusted odds ratio [aOR] 1.44, 95% confidence interval [CI] 0.84-2.47, P = 0.18; high AMH: aOR 0.98, 95% CI 0.59-1.64, P = 0.95). AMH concentrations were not associated with live birth. LBR was 59.5% in women with low AMH, 66.1% with medium AMH and 65.1% with high AMH (low AMH: aOR 0.68, 95% CI 0.41-1.11, P = 0.12, high AMH: aOR 0.96, 95% CI 0.59-1.56, P = 0.87). Live birth was lower in ART pregnancies (aOR 0.57, 95% CI 0.33-0.97, P = 0.04) and with higher numbers of previous losses (aOR 0.81, 95% CI 0.68-0.95, P = 0.01). CONCLUSION: In women with unexplained RPL, AMH was not associated with the chances of live birth in the next pregnancy. Screening for AMH in all women with RPL is not supported by current evidence. The chance of live birth among women with unexplained RPL achieving pregnancy by ART was low and needs to be confirmed and explored in future studies.

Low-grade inflammation is negatively associated with live birth in women undergoing IVF.

RESEARCH QUESTION: Is low-grade inflammation, detected by C-reactive protein (CRP), a marker of IVF outcome addressing both blastocyst quality and pregnancy outcome? DESIGN: This sub-study of a multicentre randomized controlled trial included 440 women undergoing IVF treatment with a gonadotrophin-releasing hormone (GnRH) antagonist protocol. Serum CRP was measured on cycle day 2-3 (baseline) and on the day of ovulation triggering. The association between CRP concentrations and reproductive outcomes (number of retrieved oocytes, number of good-quality blastocysts, pregnancy, pregnancy loss and live birth), were analysed, adjusting for relevant confounders. RESULTS: A negative association was found between higher baseline CRP concentrations and live birth rate (odds ratio [OR] 0.77, 95% confidence interval [CI] 0.62-0.96, P = 0.02) and higher CRP concentrations at baseline were associated with pregnancy loss among women who conceived (OR 1.37, 95% CI 1.07-1.76, P = 0.01). When testing for a specific cut-off, CRP concentrations above 2.34 (the highest quartile) were more likely to be associated with pregnancy loss (P = 0.02) and a lower chance of live birth (P = 0.04) compared with the lowest quartile. No associations were found between CRP concentrations and pregnancy outcomes on the day of ovulation triggering, and there were no associations between CRP concentrations and the number of good-quality blastocysts. CONCLUSIONS: Higher CRP concentrations at cycle day 2-3, before starting ovarian stimulation, are negatively associated with chance of live birth, possibly because of an increased risk of pregnancy loss. No association was found between the number of good-quality blastocysts and CRP concentration. More studies are needed to investigate the impact of low-grade inflammation.

The impact of the Safe Delivery Application on knowledge and skills managing postpartum haemorrhage in a low resource setting: a cluster randomized controlled trial in West Wollega region, Ethiopia.

BACKGROUND: Postpartum haemorrhage is one of the leading causes of maternal mortality in low-income countries. Improving health workers' competencies in obstetric emergencies in low-income settings, has been recognized as an important factor in preventing maternal mortality and morbidity. mHealth interventions in maternal and newborn health care has shown the potential to improve health service delivery. Strong study designs such as randomized controlled trials are missing to estimate the effectiveness of the mHealth interventions. METHODS: Between August 2013 and August 2014, 70 health facilities in West Wollega Region, Ethiopia were included and randomized to intervention or control in a cluster randomized controlled trial. At intervention facilities birth attendants were provided with a smartphone with the SDA installed. Of 176 midwives and "health extension workers," 130 completed at 12 months follow-up. At baseline and after 6- and 12-months participants were assessed. Knowledge was tested by a Key Feature Questionnaire, skills by an Objective Structured Assessment of Technical Skills in a structured role-play scenario. RESULTS: Baseline skills scores were low and comparable with a median of 12/100 in the intervention and the control group. After 6 months skills had doubled in the intervention group (adjusted mean difference 29.6; 95% CI 24.2-35.1 compared to 1·8; 95% CI - 2.7 to 6.3 in the control group). At 12 months skills had further improved in the intervention group (adjusted mean difference 13.3; 95% CI 8.3-18.3 compared to 3.1; 95% CI - 1.0 to 7.3 in the control group). Knowledge scores also significantly improved in the intervention group compared to the control (adjusted mean difference after 12 months 8.5; 95% CI 2.0-15.0). CONCLUSION: The Safe Delivery App more than doubled clinical skills for managing postpartum haemorrhage among birth attendants making it an attractive tool to reduce maternal mortality. TRIAL REGISTRATION: Clinicaltrial.gov Identifier NCT01945931. September 5, 2013.

Maternal mortality caused by postpartum haemorrhage is a major public health concern in many low-income countries. Having access to skilled health care professionals during pregnancy and childbirth can prevent maternal deaths related to postpartum haemorrhage. mHealth interventions like the Safe Delivery App (SDA), a smartphone application, has shown the potential to improve the quality of care in emergency situations related to childbirth in low-income health system settings.This study examines the SDA as a training/education tool for improving health workers' competencies and performances in managing postpartum haemorrhage. The SDA contained animated instruction videos on how to prevent and treat postpartum haemorrhage and a list of essential drugs and basic equipment.In Ethiopia, 70 health facilities and 176 birth attendants were included in a randomized controlled trial. The intervention group received a smartphone with the SDA installed and half a day of introduction to the use of the app. Birth attendants' skills and knowledge in managing postpartum haemorrhage in the control and the intervention group were tested at baseline, 6 and 12 months after intervention.Our study found that the SDA is an effective tool to improve and sustain birth attendants' knowledge and skills in the management of postpartum haemorrhage in a rural, low-resource health system setting in Ethiopia, which confirms findings in other non-randomized studies examining the SDA on the management of postpartum haemorrhage.

The healthy female microbiome across body sites: effect of hormonal contraceptives and the menstrual cycle.

STUDY QUESTION: How does hormonal contraceptive use and menstrual cycle phase affect the female microbiome across different body sites? SUMMARY ANSWER: The menstrual cycle phase, but not hormonal contraceptive use, is associated with the vaginal and oral but not the gut microbiome composition in healthy young women. WHAT IS KNOWN ALREADY: Women with low vaginal levels of Lactobacillus crispatus are at increased risk of pre-term birth, fertility treatment failure, sexually transmitted infections and gynaecological cancers. Little is known about the effect of hormonal fluctuations on other body site's microbiomes as well as the interplay between them. STUDY DESIGN, SIZE, DURATION: This study includes a cohort of 160 healthy young Danish women using three different contraceptive regimens: non-hormonal methods (n = 54), combined oral contraceptive (COC, n = 52) or levonorgestrel intrauterine system (LNG-IUS, n = 54). Samples were collected from four body sites during the menstrual cycle (menses, follicular and luteal phases) at Copenhagen University Hospital, Rigshospitalet, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: The oral, vaginal, rectal and faecal microbiomes were characterized by shotgun sequencing. Microbial diversity and community distance measures were compared between study groups, menstrual phase timepoints and body sites. All participants answered an extensive questionnaire on current health, lifestyle and sex life. Confounding factors such as smoking, BMI and diet were analysed by PERMANOVA. Plasma oestradiol and progesterone levels are correlated with microbiome composition. MAIN RESULTS AND THE ROLE OF CHANCE: The use of COC and LNG-IUS was not associated with the microbiome composition or diversity. However, increased diversity in the vaginal microbiome was observed during menses, followed by a subsequent expansion of Lactobacillus spp. during the follicular and luteal phases which correlated with measured serum oestradiol levels (r = 0.11, P < 0.001). During menses, 89 women (58%) had a dysbiotic vaginal microbiome with <60% Lactobacillus spp. This declined to 49 (32%) in the follicular phase (P < 0.001) and 44 (29%) in the luteal phase (P < 0.001). During menses, bacterial richness and diversity in saliva reached its lowest point while no differences were observed in the faecal microbiome. The microbiome in different body sites was on average more similar within the same individual than between individuals, despite phase or hormonal treatment. Only the vagina presented a clear cluster structure with dominance of either L. crispatus, Lactobacillus iners, Gardnerella vaginalis or Prevotella spp. LARGE SCALE DATA: The microbiome samples analysed in this study were submitted to the European Nucleotide Archive under project number PRJEB37731, samples ERS4421369-ERS4422941. LIMITATIONS, REASONS FOR CAUTION: The cohort is homogenous which limits extrapolation of the effects of ethnicity and socio-economic status on the microbiome. We only present three defined timepoints across the menstrual phase and miss potential important day to day fluctuations. WIDER IMPLICATIONS OF THE FINDINGS: The use of hormonal contraception did not significantly associate with the microbiome composition in the vagina, faeces, rectum or saliva in healthy young women. This is a welcome finding considering the widespread and prolonged use of these highly efficient contraceptive methods. The menstrual cycle is, however, a major confounding factor for the vaginal microbiome. As such, the time point in the menstrual cycle should be considered when analysing the microbiome of women of reproductive age, since stratifying by vaginal dysbiosis status during menstruation could be misleading. This is the first study to confirm by direct measurements of oestradiol, a correlation with the presence of L. crispatus, adding evidence of a possible hormonal mechanism for the maintenance of this desirable microbe. STUDY FUNDING/COMPETING INTEREST(S): This work was partly funded by the Ferring Pharmaceuticals through a research collaboration with The Centre for Translational Microbiome Research (CTMR) at the Karolinska Institutet (L.W.H., E.F., G.E. and I.S.-K.). Ferring Pharmaceuticals also funded the infrastructure to obtain the clinical samples at Copenhagen University Hospital ([#MiHSN01], M.C.K., Z.B., and H.S.N.). This work was also supported by funding from Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K.) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). M.C.K., L.W.H., E.F., Z.B., G.E., L.E., I.S.-K. and H.S.N., are partially funded by Ferring Pharmaceuticals, which also provided funds for the collection and processing of the samples analysed in this study. H.S.N.'s research is further supported by Freya Biosciences and the BioInnovation Institute. H.S.N. has received honoraria from Ferring Pharmaceuticals, Merck A/S, Astra-Zeneca, Cook Medical and Ibsa Nordic. A.N.A. reports no competing interests.

Pregnancy Loss and Cardiovascular Diseases in Women: Recent Findings and Potential Mechanisms.

PURPOSE OF REVIEW: Pregnancy loss (PL) has been acknowledged by the American Heart Association as a risk factor for cardiovascular diseases (CVD) later in life. This review aims to sum up recent findings (< ~ 5 years), concerning the link between PL and CVD. RECENT FINDINGS: The association between PL and risk of CVD increased with increasing number of PLs and is inversely correlated to maternal age, indicating that the association concerns euploid PLs. Likely mechanisms leading to PL and an increased risk of CVD include endothelial dysfunction, a pro-inflammatory state, antiphospholipid syndrome, autoimmunity, and genetic predisposition. PL as an independent risk factor for CVD constitutes an obvious gateway for a more targeted approach to future research, prevention, and treatment. Future research should clarify the following questions to which the answers are still unknown: whether PL is (a) directly causing the increased risk of CVD or (b) sharing pathophysiological mechanisms also leading to CVD.

Feelings of guilt and loss of control dominate in stress and depression inventories from women with recurrent pregnancy loss.

PURPOSE: Which feelings on the major depression inventory (MDI) and the perceived stress scale (PSS) are predominant among women with recurrent pregnancy loss (RPL)? MATERIALS AND METHODS: Prospective cohort study of women with RPL referred to the tertiary RPL Unit at Copenhagen University Hospital, Rigshospitalet, Denmark, from 2010-2013. All women answered the MDI and PSS at time of referral. RESULTS: In total, 298 women completed the MDI and the PSS, of which 162 had primary RPL and 136 secondary RPL. The most common feelings were low in energy (42%), loss of interest (35%), sadness (35%), and guilt (29%). Twenty-six (8.6%) women fulfilled the criteria for moderate to severe depression. Of the remaining 272 women, nine felt that life was not worth living. Among all women feeling angered of things outside their control (35%) and unable to control important things (27%) were predominant. Women with primary RPL compared to secondary RPL more often felt less self-confident and that life wasn't worth living (p = 0.007 and p = 0.002). CONCLUSIONS: Feelings of guilt and loss of control were predominant in women with RPL. Women with primary RPL could represent a particularly sensitive group. Addressing these specific feelings could help treating the psychological aspects of RPL.

Chance of live birth: a nationwide, registry-based cohort study.

STUDY QUESTION: Does the sequence of prior pregnancy events (pregnancy losses, live births, ectopic pregnancies, molar pregnancy and still birth), obstetric complications and maternal age affect chance of live birth in the next pregnancy and are prior events predictive for the outcome? SUMMARY ANSWER: The sequence of pregnancy outcomes is significantly associated with chance of live birth; however, pregnancy history and age are insufficient to predict the outcome of an individual woman's next pregnancy. WHAT IS KNOWN ALREADY: Adverse pregnancy outcomes decrease the chance of live birth in the next pregnancy, whereas the impact of prior live births is less clear. STUDY DESIGN, SIZE, DURATION: Nationwide, registry-based cohort study of 1 285 230 women with a total of 2 722 441 pregnancies from 1977 to 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women living in Denmark in the study period with at least one pregnancy in either the Danish Medical Birth Registry or the Danish National Patient Registry. Data were analysed using logistic regression with a robust covariance model to account for women with more than one pregnancy. Model discrimination and calibration were ascertained using 20% of the women in the cohort randomly selected as an internal validation set. MAIN RESULTS AND THE ROLE OF CHANCE: Obstetric complications, still birth, ectopic pregnancies and pregnancy losses had a negative effect on the chance of live birth in the next pregnancy. Consecutive, identical pregnancy outcomes (pregnancy losses, live births or ectopic pregnancies) immediately preceding the next pregnancy had a larger impact than the total number of any outcome. Model discrimination was modest (C-index = 0.60, positive predictive value = 0.45), but the models were well calibrated. LIMITATIONS, REASONS FOR CAUTION: While prior pregnancy outcomes and their sequence significantly influenced the chance of live birth, the discriminative abilities of the predictive models demonstrate clearly that pregnancy history and maternal age are insufficient to reliably predict the outcome of a given pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: Prior pregnancy history has a significant impact on the chance of live birth in the next pregnancy. However, the results emphasize that only taking age and number of losses into account does not predict if a pregnancy will end as a live birth or not. A better understanding of biological determinants for pregnancy outcomes is urgently needed. STUDY FUNDING/COMPETING INTEREST(S): The work was supported by the Novo Nordisk Foundation, Ole Kirk Foundation and Rigshospitalet's Research Foundation. The authors have no financial relationships that could appear to have influenced the work. TRIAL REGISTRATION NUMBER: N/A.

Meditation and mindfulness reduce perceived stress in women with recurrent pregnancy loss: a randomized controlled trial.

RESEARCH QUESTION: Can participating in a tailored 7-week meditation and mindfulness programme with additional standard supportive care versus standard supportive care only reduce perceived stress for women with recurrent pregnancy loss (RPL)? DESIGN: A two-armed randomized controlled trial (RCT) with 12-month follow-up. In total 76 patients were enrolled and randomly assigned to either standard supportive care or to a 7-week meditation and mindfulness programme led by an instructor in addition to standard supportive care. RESULTS: At intervention completion (after 7 weeks), perceived stress decreased significantly both in the intervention group (P = 0.001) and in the control group (P = 0.006). The decrease in perceived stress in the intervention group was significantly larger (P = 0.027) compared with the control group. At the 12-month follow-up perceived stress was still significantly decreased in both groups compared with baseline (P < 0.0001 in the intervention group and P = 0.002 in the control group). CONCLUSION: This first RCT of a tailored meditation and mindfulness intervention for women with RPL documents that a 7-week daily at-home meditation and mindfulness programme combined with group sessions reduced perceived stress significantly more than a standard supportive care programme. Future studies should address the most effective format and the 'dose' needed for an impact on perceived stress levels.

Stress and depression among women and men who have experienced recurrent pregnancy loss: focusing on both sexes.

RESEARCH QUESTION: Are women and men suffering from recurrent pregnancy loss (RPL) more affected by psychological stress and depression than the general population? DESIGN: Cross-sectional study investigating the prevalence of stress and depression in women and men with RPL seen in the Danish national RPL Unit. Data were collected between 2015-2018. All newly referred couples were asked to complete the Major Depression Index (MDI) and Cohen's Perceived Stress Scale (PSS). The scores of both sexes were compared with scores from relevant cohorts of men and women from the general population. RESULTS: In total, 412 women with RPL (82% response rate) and 281 male partners (60% response rate) were included. Depression: 5/281 (1.8%) of men with RPL had moderate/severe depression vs. 5/253 (2.0%) of men in the comparison group (relative risk (RR) 0.90; 95% CI 0.26-3.07, p=0.99). Among women with RPL, 34/412 (8.3%) had a moderate/severe depression vs. 2.2% in the comparison group (RR 3.74; 95% CI 2.40-5.83, p<0.001). High stress levels were found in 30/281 men with RPL (10.7%) vs. 15.8% in the comparison group (co-habiting men) (RR 0.67; 95% CI 0.48-0.94, p=0.017). High stress level was found among 110/384 (28.6%) of RPL-women vs. 420/1813 (23.2%) of comparison women (RR 1.24; 95% CI 1.03-1.48, p=0.026). Both MDI and PSS scores, respectively, for a woman and a man in an RPL couple were significantly correlated. CONCLUSION: Male partners in RPL couples did not have increased prevalence of stress and depression compared with other men but we confirmed our previous finding of significantly increased frequencies among women with RPL.

The reproductive microbiome - clinical practice recommendations for fertility specialists.

The interest in and understanding of the human microbiome has grown remarkably over recent years. Advances in molecular techniques have allowed researchers to identify and study the microbiota and also use this information to develop therapeutic solutions for a spectrum of conditions. Alongside the growing interest in the microbiome, societal changes have resulted in many couples looking to start families later in life, therefore increasing the demand for assisted reproductive technologies. Combining these trends, it makes sense that clinicians are eager to understand and exploit the microbiome of their patients, i.e. the reproductive microbiome, in order to help them achieve their goal of becoming parents. This paper aims to provide an overview of the current and future research into the reproductive microbiome in relation to fertility and also share clinical practice recommendations for physicians who are new to this field or unsure about how they can utilise what is known to help their patients.

Ovarian reserve markers after discontinuing long-term use of combined oral contraceptives.

RESEARCH QUESTION: How early do the ovarian reserve markers anti-Müllerian hormone (AMH) and antral follicle count (AFC) normalize after discontinuation of the long-term use of combined oral contraceptives (COC). DESIGN: This was a prospective cohort study of 68 women with a history of long-term COC use. Serum AMH concentrations, ovarian volume and AFC were measured during COC use and serially in a 4-month period after discontinuing COC: 1 and 2 weeks after discontinuation, and on cycle day 2-5 during three consecutive menstrual cycles. Changes in AMH and AFC were investigated using linear mixed models of repeated measurements adjusted for relevant covariates. RESULTS: Mean age was 29.4 years and mean duration of COC use 8.0 years. Baseline median AMH concentrations during COC use of 13 pmol/l (interquartile range [IQR] 8.4-22 pmol/l) increased to a median of 22.5 pmol/l (IQR 11-37 mol/l) 3 months after discontinuation. The estimated average increase was 53% (95% confidence interval [CI] 1.40-1.68, P < 0.001). AFC increased from a median value of 17 (IQR 11-25) to 24 (IQR 17-34). The estimated average increase was 41% (95% CI 1.30-1.52, P < 0.001). Ovarian volume increased from 2.4 to 5.8 ml (P < 0.001). The ovarian reserve markers increased continuously from baseline measurements until 2 months after discontinuation. Thereafter a plateau was reached. CONCLUSION: After discontinuation of COC, AMH increased by 53% and AFC by 41%, with values returning to normal within 2 months. This study provides clinicians with the highly relevant knowledge that AMH and AFC can be measured 2 months after discontinuation of COC without having to account for their influence.

Smoking during pregnancy reduces vitamin D levels in a Finnish birth register cohort.

OBJECTIVE: Maternal vitamin D level in pregnancy may have implications for both the mother and fetus. Deficiency of vitamin D has been linked to several pregnancy complications and fetal skeletal health. Smoking has been associated with reduced serum level of the vitamin D metabolite, 25-hydroxyvitamin D (25(OH)D). DESIGN: A nested case-control study within the Finnish Maternity Cohort, a population-based cohort which includes first-trimester sera from 98 % of pregnancies in Finland since 1987. The selection consisted of women with uncomplicated pregnancies. We studied serum concentration of 25(OH)D in 313 non-smoking and forty-six self-reported smoking pregnant women. SETTING: We hypothesize that pregnant smokers may have an increased risk of low 25(OH)D levels especially during winter months. PARTICIPANTS: A control group from an unpublished pregnancy complication study consisting of 359 uncomplicated pregnancies. Individuals who reported that they do not smoke were considered 'non-smokers' (n 313) and those who reported continued smoking after the first trimester of pregnancy were considered 'smokers' (n 46). RESULTS: Smokers had significantly lower levels of 25(OH)D irrespective of sampling time (P<0·0001). Furthermore, during the low sun-exposure season, only 14 % of smokers met the guideline level of 40 nmol/l for serum 25(OH)D in comparison with 31 % of non-smokers. CONCLUSIONS: Expectant mothers who smoke have an increased risk of vitamin D deficiency during low sun-exposure months in northern regions. Further studies are needed to assess the associated risks for maternal and fetal health as well as possible long-term implications for the infant.

Ovarian reserve markers and endocrine profile during oral contraception: Is there a link between the degree of ovarian suppression and AMH?

The ovarian reserve markers anti-Müllerian hormone (AMH) and antral follicle count (AFC) are suppressed in varying degree during the use of combined oral contraceptives (COC). Further, long-term use of COC can mask a condition of premature ovarian insufficiency. A desirable clinical tool that could distinguish true low ovarian reserve markers from COC-induced low levels during use of COC is warranted. The aim of this multicenter study including 235 COC users was to assess whether low age-adjusted AMH levels during COC use were linked to concomitant low levels of LH, FSH, estradiol and androgens - as a potential future tool to differentiate between 'false', COC-induced low AMH levels vs. true low AMH. Study population and methods: In total, 235 COC users from the general population aged 19-40 years were included. AMH, AFC and a reproductive hormonal profile were measured during COC intake. Age-adjusted AMH levels (Z-scores) were calculated from a comparison group of 983 non-users of COC. Differences in hormonal profile were tested between women with low versus high age-adjusted AMH-quartiles based on non-parametric Wilcoxon rank sum tests. The outcomes of interest were levels of gonadotropins, estradiol and androgens according to the four the age-adjusted AMH quartiles to find out if women with low age-adjusted AMH levels had a stronger gonadotropin suppression compared with women with higher age-adjusted AMH levels. Mean age of COC users was 30.2 years (SD 3.8), median AMH 14 pmol/l (inter-quartile range (IQR) 8.7-23)), median AFC 16 (IQR 11-25). We found no significant differences across the age-adjusted AMH quartiles in either the levels of gonadotropins, estrogens or androgens, respectively. Thus, the degree of suppression of FSH, LH, androgens and estradiol are unlikely to be a useful tool to differentiate between false low and true low ovarian reserve markers during COC use. Presently, there seems to be no alternative to withdrawal of the COC and to re-test the ovarian reserve after 2-3 months. Trial registration Trial no. NCT02785809 (www.clinicaltrials.gov).

Ovarian reserve markers in women using various hormonal contraceptives.

Objectives: The aim of the study was to assess whether the ovarian reserve markers anti-Müllerian hormone (AMH) and antral follicle count (AFC) were lower among women using the progestin-only pill (POP) or levonorgestrel-releasing intrauterine system (LNG-IUS) and similar to the decrease observed in combined oral contraceptive (COC) pill users.Methods: This retrospective study comprised 565 hormonal contraceptive users (COC, POP, LNG-IUS or contraceptive vaginal ring) and 983 non-hormonal contraceptive users, who were seen in two Danish fertility assessment and counselling clinics between 2015 and 2019. Adjusted multiple regression analysis was used to examine the differences in AMH and AFC between hormonal and non-hormonal contraceptive users.Results: Compared with non-hormonal contraceptive users, AMH was 31.1% lower among COC users [95% confidence interval (CI) -39.6%, -25.9%; p < 0.001], 35.6% lower among POP users (95% CI -49.0%, -18.6%; p < 0.001) and 17.1% lower among LNG-IUS users (95% CI -31.4%, 0.002%; p = 0.052); no significant differences were seen among vaginal ring users. Compared with non-hormonal contraceptive users, AFC was 31.3% lower among COC users (95% CI -35.0%, -25.3%; p < 0.001) and 29.7% lower among POP users (-39.1%, -17.9%; p < 0.001); no significant differences were seen among LNG-IUS or vaginal ring users. Ovarian volume was more than 50% reduced among COC and vaginal ring users (p < 0.001) but was unchanged among POP and LNG-IUS users.Conclusion: Assessment of ovarian reserve markers among users of all types of hormonal contraception should be interpreted cautiously and the type of contraceptive method considered.