Smoking and recurrent pregnancy loss: A cohort study of 2829 women.

Smoking during pregnancy is associated with negative reproductive outcome. Less is known about the impact of smoking or previous smoking in women with recurrent pregnancy loss (RPL) which this study aimed to investigate. We included all women <42 years (n=2829) referred to a RPL unit at Copenhagen University Hospital between January 2000 and December 2021 in the cohort with follow-up until June 2022. Patients were categorized as 'smokers at time of referral', 'never-smokers' or 'former smokers'. The main outcomes were pregnancy history prior to referral, prospective pregnancy rate, live birth rate, rates of ectopic pregnancy, and stillbirth. At referral, smokers (n=373) were on average 2.0 years younger (P<0.001) and had experienced significantly more pregnancy losses (P<0.001), and stillbirths (P=0.01) compared to never-smokers (n=2100). Former smokers had a higher risk of stillbirth prior to referral compared to never-smokers but no differences in pregnancy rate or other outcomes. Prospective pregnancy rates were lower for smokers compared with never-smokers (71.8% vs. 77.5%, P=0.02). Live birth rate was 58.0% for the 243 women who smoked at referral compared to 61.4% for the 1488 never-smokers (P=0.32). Stillbirth and ectopic pregnancies were significantly more common for smokers (2.8% vs. 0.4%, P=0.01; 6.0% vs. 2.0%, P<0.008). Women with RPL who smoked at referral were referred younger with a higher number of previous pregnancy losses and stillbirths compared with never-smokers. Fewer smokers achieved a pregnancy after referral but those who did had a similar live birth rate compared to never-smokers, although stillbirths and ectopic pregnancies were more common.

The development of an indel panel for microchimerism detection.

OBJECTIVES: The aim of the study was to create a simple assay for microchimerism detection independent of sex and without HLA genotyping. METHODS: The method is based on detection of insertion or deletions utilizing a multiplex PCR followed by fragment analysis by capillary electrophoresis, and probe-based qPCR assays. A total of 192 samples, taken either before pregnancy, during 1st trimester, or either during 2nd trimester or at miscarriage, obtained from a cohort of 97 female patients with either primary or secondary recurrent pregnancy loss, were screened for fetal microchimerism by the indel panel as well as an existing assay based on detection of the Y-chromosome marker; DYS14. RESULTS: The overall prevalence of DYS14 positive samples was 29% (55/192) whereas 32% (61/192) tested positive by the indel method. There was an overall agreement of 64% (122/192) between the results obtained by the two methods. A Fisher's Exact test showed no statistic significant difference in the prevalence of microchimerism detected by the two methods at any of the three times of sampling. The distribution of the number of positive wells detected by both methods were compared by a Mann-Whitney U test, which showed no statistically significant difference at any of the three times of sampling. CONCLUSION: The data indicates that microchimerism can be detected efficiently by the indel method. This makes it possible to detect both female and male cells without the need of HLA-genotyping. Furthermore, the indel method has potential to be implemented as a routine analysis. This will remove the sex bias in future explorations of the role microchimerism plays in health and disease.

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.

The microbiome in reproductive health: protocol for a systems biology approach using a prospective, observational study design.

STUDY QUESTION: What is the microbiome profile across different body sites in relation to the normal menstrual cycle (with and without hormonal contraception), recurrent pregnancy loss (RPL) (before and during pregnancy, pregnancy loss or birth) and endometriosis (before, during and after surgery)? How do these profiles interact with genetics, environmental exposures, immunological and endocrine biomarkers? WHAT IS KNOWN ALREADY: The microbiome is a key factor influencing human health and disease in areas as diverse as immune functioning, gastrointestinal disease and mental and metabolic disorders. There is mounting evidence to suggest that the reproductive microbiome may be influential in general and reproductive health, fertility and pregnancy outcomes. STUDY DESIGN SIZE DURATION: This is a prospective, longitudinal, observational study using a systems biology approach in three cohorts totalling 920 participants. Since microbiome profiles by shot-gun sequencing have never been investigated in healthy controls during varying phases of the menstrual cycle, patients with RPL and patients with endometriosis, no formal sample size calculation can be performed. The study period is from 2017 to 2024 and allows for longitudinal profiling of study participants to enable deeper understanding of the role of the microbiome and of host-microbe interactions in reproductive health. PARTICIPANTS/MATERIALS SETTING METHODS: Participants in each cohort are as follows: Part 1 MiMens-150 healthy women with or without hormonal contraception; Part 2 MiRPL-200 couples with RPL, 50 healthy couples with prior uncomplicated pregnancy and 150 newborns; Part 3 MiEndo-120 patients with endometriosis requiring surgery with or without hormonal treatment. Microbiome profiles from saliva, faeces, rectal mucosa, vaginal fluid and endometrium will be studied, as well as the Omics profile, endocrine disrupting chemicals and endocrine and immune factors in blood, hair, saliva and urine. Pregnancy loss products, seminal microbiome, HLA types, endometriotic tissue and genetic risk and comprehensive questionnaire data will also be studied, where appropriate. Correlations with mental and physical health will be evaluated. STUDY FUNDING/COMPETING INTERESTS: This work is supported by funding from Ferring Pharmaceuticals ([#MiHSN01] to H.S.N., M.C.K., M.E.M., L.E.V., L.E., I.S.-K., F.B., L.W.H., E.F. and M.H.), Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K. and [#E-22222-06] to S.B.), Niels and Desiree Yde's Foundation (S.B., endocrine analyses [#2015-2784]), the Musikforlæggerne Agnes and Knut Mørk's Foundation (S.B., endocrine and immune analyses [#35108-001]) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). Medical writing assistance with this manuscript was provided by Caroline Loat, PhD, and funded by Ferring Pharmaceuticals. H.S.N. reports personal fees from Ferring Pharmaceuticals, Merck Denmark A/S, Ibsa Nordic, Astra Zeneca and Cook Medical outside the submitted work. K.W. is a full-time employee of Ferring Pharmaceuticals. No other conflicts are reported. TRIAL REGISTRATION NUMBER: N/A. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

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.

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.

Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar.

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.

Serum ferritin level is inversely related to number of previous pregnancy losses in women with recurrent pregnancy loss.

OBJECTIVE: To study whether low serum ferritin (s-ferritin) levels are associated with recurrent pregnancy loss (RPL), and whether low s-ferritin predicts the risk of another pregnancy loss or the ability to conceive. DESIGN: Cohort study. SETTING: Fertility clinic at a university hospital. PATIENT(S): Eighty-four women referred to the RPL Unit and 153 women of reproductive age with no known fertility problem. s-Ferritin levels were measured in serum samples taken before pregnancy attempt. INTERVENTION: None. MAIN OUTCOME MEASURE(S): s-Ferritin levels were correlated to pregnancy history, ability to conceive, and time to conception during the first 2 years after sampling. Furthermore, s-ferritin levels were correlated to outcome of the first pregnancy after referral for RPL. RESULT(S): Women with RPL had lower s-ferritin than the comparison group, 39.9 µg/L versus 62.2 µg/L, and had a higher prevalence of low iron stores (s-ferritin <30 µg/L), 35.7% versus 13.7%. We found an inverse relationship between s-ferritin level and number of pregnancy losses before referral. We did not find s-ferritin level to be associated with ability to conceive or time to pregnancy in either group. Nor did s-ferritin level predict the risk of losing the first pregnancy after referral for RPL. CONCLUSION(S): The inverse relationship between s-ferritin levels and previous pregnancy losses suggests that low s-ferritin is associated with a more severe reproductive disturbance in women with RPL. Whether low s-ferritin is causally related to RPL and if such women could benefit from iron supplementation to achieve a live birth warrants further investigation.

[Recurrent pregnancy loss].

Recurrent pregnancy loss (RPL) is defined as three or more consecutive pregnancy losses, and it affects 2-3% of couples trying to conceive. RPL is a multifactorial disorder, and only few evidence-based treatments are available. It is associated with an increased prevalence of stress and major depression, and also with immunogenetic markers, autoimmunity and an increase of the risk of cardiovascular disease in later life. Immunology seems to be a key element in RPL, and further research is needed to understand the pathogenesis of this heterogeneous condition in order to develop personalized treatment.