Genome-wide analyses identify 21 infertility loci and over 400 reproductive hormone loci across the allele frequency spectrum.

Genome-wide association studies (GWASs) may help inform treatments for infertility, whose causes remain unknown in many cases. Here we present GWAS meta-analyses across six cohorts for male and female infertility in up to 41,200 cases and 687,005 controls. We identified 21 genetic risk loci for infertility (P≤5E-08), of which 12 have not been reported for any reproductive condition. We found positive genetic correlations between endometriosis and all-cause female infertility (rg=0.585, P=8.98E-14), and between polycystic ovary syndrome and anovulatory infertility (rg=0.403, P=2.16E-03). The evolutionary persistence of female infertility-risk alleles in EBAG9 may be explained by recent directional selection. We additionally identified up to 269 genetic loci associated with follicle-stimulating hormone (FSH), luteinising hormone, oestradiol, and testosterone through sex-specific GWAS meta-analyses (N=6,095-246,862). While hormone-associated variants near FSHB and ARL14EP colocalised with signals for anovulatory infertility, we found no rg between female infertility and reproductive hormones (P>0.05). Exome sequencing analyses in the UK Biobank (N=197,340) revealed that women carrying testosterone-lowering rare variants in GPC2 were at higher risk of infertility (OR=2.63, P=1.25E-03). Taken together, our results suggest that while individual genes associated with hormone regulation may be relevant for fertility, there is limited genetic evidence for correlation between reproductive hormones and infertility at the population level. We provide the first comprehensive view of the genetic architecture of infertility across multiple diagnostic criteria in men and women, and characterise its relationship to other health conditions.

Binucleated embryos at the two-cell stage show higher blastocyst formation rates and higher pregnancy and live birth rates compared to non-multinucleated embryos.

STUDY QUESTION: How does nucleus status at the two-cell stage predict blastocysts formation and clinical outcome after single blastocyst transfer? SUMMARY ANSWER: Binucleated embryos at the two-cell stage (2BI) show higher rates of good quality blastocyst formation, pregnancy and live birth compared to those with one nucleus in each blastomere (2MONO), whereas true multinucleated embryos at the two-cell stage (2MULTI) show lower rates of good quality blastocyst formation and pregnancy compared to 2MONO embryos. WHAT IS KNOWN ALREADY: The introduction of time-lapse culture has made it possible to study nucleus status at the two-cell stage more consistently and it shows that multinucleation at the two-cell stage (2MN) is a common event. The effect of 2MN is still unclear. High numbers of 2MN with the potential to develop to blastocysts that become clinical pregnancies and result in birth of healthy babies with no impaired perinatal outcome have been reported. However, some studies have found 2MN to be associated with impaired implantation and live birth. Furthermore, knowledge on how the different subgroups of multinucleation affects the IVF outcome is limited. STUDY DESIGN SIZE DURATION: A non-interventional retrospective study was performed in a public fertility clinic. Blastocyst formation data from 223 women attending their first IVF cycle between May 2016 and December 2018, and clinical outcome data from 1314 single blastocyst transfers between May 2014 and December 2018 were used for the study. Fresh and frozen-thawed embryo transfers were included. PARTICIPANTS/MATERIALS SETTING METHODS: Embryos were cultured until the blastocyst stage in a time-lapse incubator and nucleus status at the two-cell stage, the Gardner score and other morphokinetic parameters were annotated. We compared blastocyst development and clinical outcome, including positive hCG, ongoing pregnancy and live birth, of embryos with 2BI and/or 2MULTI blastomeres to 2MONO embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos with 2BI in one blastomere (2BI1) were twice as likely to develop to good quality blastocysts (odds ratio (OR) 2.54, 95% CI 1.30-4.95, P = 0.006) compared to 2MONO embryos. Embryos with 2MULTI in both blastomeres (2MULTI2) were significantly less able to develop to good quality blastocysts (OR 0.38, 95% CI 0.23-0.63, P < 0.001) compared to 2MONO embryos. Embryos with 2BI in both blastomeres (2BI2) had a significantly better chance of resulting in a positive hCG (OR 2.40, 95% CI 1.11-5.20, P = 0.027), ongoing pregnancy (OR 2.79, 95% CI 1.29-6.04, P = 0.009) and live birth (OR 3.16, 95% CI 1.43-6.95, P = 0.004) compared to 2MONO blastocysts after single blastocyst transfer. In contrast, 2MULTI2 embryos were significantly less likely to result in a positive hCG (OR 0.58, 95% CI 0.35-0.97, P = 0.036) and ongoing pregnancy (OR 0.51, 95% CI 0.28-0.94, P = 0.030) compared to 2MONO blastocysts. LIMITATIONS REASONS FOR CAUTION: Discrepancies among the existing studies regarding the definition of multinucleation may lead to different conclusions. Even though the distinction between binucleation and true multinucleation was a strength in our study design, a further distinction between true multinucleated and micronucleated embryos could be interesting to investigate in future studies. Also, we included any anucleated embryos in the 2MONO group. For the study of clinical outcomes, the patients were allowed to be included with more than one transfer cycle. Both fresh and thawed transfers were included. WIDER IMPLICATIONS OF THE FINDINGS: We find it important to discriminate between binucleation and true multinucleation when evaluating embryo nucleus status at the two-cell stage. Embryos displaying 2BI1 and 2BI2 have significantly better good quality blastocyst formation rates and clinical outcome after single blastocyst transfers, respectively. 2MULTI2 embryos have impaired blastocyst development potential and poorer clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: H.S.N. received an unrestricted grant from Merck for 3 months' normal salary for a medical Doctor (A.L.T.) to write the manuscript. Merck was not involved in the study design, analysis, interpretation of data, writing the paper or the decision to submit the manuscript for publication. H.S.N. has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S, Astra Zeneca, Cook Medical and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). The other authors did not report any potential conflicts of interest. All authors declared no conflicts of interest regarding this work. TRIAL REGISTRATION NUMBER: N/A.

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.

Mental health indicators in pregnant women compared with women in the general population during the coronavirus disease 2019 pandemic in Denmark.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic and the associated regulations issued to minimize risk of disease transmission seem to have had an impact on general mental health in most populations, but it may have affected pregnant women even more because of pregnancy-related uncertainties, limited access to healthcare resources, and lack of social support. We aimed to compare the mental health response among pregnant women with that in similarly aged women from the general population during the first wave of the COVID-19 pandemic. MATERIAL AND METHODS: From April 14 to July 3, 2020, 647 pregnant women in their second trimester were enrolled in this study. For comparison, 858 women from the general Danish population (20-46 years) were sampled from an ongoing observational study. Participants responded to a questionnaire including six mental health indicators (concern level, perceived social isolation, quality of life, anxiety, mental health, and loneliness). Loneliness was measured using the UCLA Three-item Loneliness Scale and anxiety by the Common Mental Health Disorder Questionnaire 4-item Anxiety Subscale. RESULTS: The pregnant women had better scores during the entire study period for all mental health indicators, and except for concerns, social isolation, and mental health, the differences were also statistically significant. Pregnant women were more concerned about becoming seriously ill (40.2% vs. 29.5%, p < 0.001), whereas the general population was more concerned about economic consequences and prospects. Many pregnant women reported negative feelings associated with being pregnant during the COVID-19 pandemic and concerns regarding social isolation and regulation-imposed partner absence during hospital appointments and childbirth. All mental health indicators improved as Denmark began to reopen after the first wave of the pandemic. CONCLUSIONS: Pregnant women exhibited lower rates of poor mental health compared with the general population. However, they were more concerned about becoming seriously ill, expressed negative feelings about being pregnant during the pandemic, and were worried about the absence of their partner due to imposed regulations. These finding may be taken into account by policy-makers during pandemics to balance specific preventive measures over the potential mental health deterioration of pregnant women.

The impact of early pregnancy complications on completed family size-A nationwide, registry-based cohort study with 40 years of data.

INTRODUCTION: The impact of early pregnancy complications on completed family size is unknown. Here, we hypothesize that early pregnancy complications and adverse outcomes may influence family size. MATERIAL AND METHODS: In this nationwide, registry-based study we included all 458 475 women born 1957-1972 who lived in Denmark from age 20-45 years with at least one registered pregnancy. The main outcome of the study was number of children per woman by age 45, estimated using a Generalized Linear Mixed Model. Exposures were: (a) total number of pregnancy losses experienced (0, 1, 2, ≥3); (b) highest number of consecutive pregnancy losses (0, 1, 2, ≥3); (c) sex of firstborn child; (d) outcome of first pregnancy (live birth, stillbirth, pregnancy loss, ectopic pregnancy, or molar pregnancy). RESULTS: Number of live births was negatively influenced by maternal age and adverse first pregnancy outcomes, especially ectopic pregnancies. A 30-year-old woman with a first ectopic pregnancy was expected to have 1.16 children (95% CI 1.11-1.22) compared with 1.95 children (95% CI 1.86-2.03) with a first live birth. Three or more consecutive losses also decreased number of live births significantly: 1.57 (95% CI 1.50-1.65) compared with 1.92 (95% CI 1.84-2.0) with only live births. The total number of pregnancy losses had no effect before the age of 35 years. Sex of firstborn had no effect. CONCLUSIONS: Previous pregnancy history has a significant effect on number of children per woman, which is important at both individual and societal levels. Pathophysiological research of adverse pregnancy outcomes should be an urgent priority as the causes remain poorly understood.

Pregnancy loss is associated with type 2 diabetes: a nationwide case-control study.

AIMS/HYPOTHESIS: Type 2 diabetes is killing more people than ever, and early-life predictors remain critical for the development of effective preventive strategies. Pregnancy loss is a common event associated with later atherosclerotic disease and ischaemic heart failure and might constitute a predictor for type 2 diabetes. The objective of this study was to investigate whether pregnancy loss is associated with later development of type 2 diabetes. METHODS: Using a Danish nationwide cohort, we identified all women born from 1957 through to 1997 and who had a diagnosis of type 2 diabetes during the period 1977 to 2017. The women were matched 1:10 on year of birth and educational level to women without diabetes in the general Danish population. Conditional logistic regression models provided odds ratios for type 2 diabetes with different numbers of pregnancy losses. RESULTS: We identified 24,774 women with type 2 diabetes and selected 247,740 controls without diabetes. Women who had ever been pregnant (ever-pregnant women) with 1, 2 and ≥ 3 pregnancy losses had ORs of type 2 diabetes of 1.18 (95% CI 1.13, 1.23), 1.38 (95% CI 1.27, 1.49) and 1.71 (95% CI 1.53, 1.92) compared with ever-pregnant women with no pregnancy losses, respectively. Women who never achieved a pregnancy had an OR of type 2 diabetes of 1.56 (95% CI 1.51, 1.61) compared with ever-pregnant women with any number of losses. Similar results were found after adjustment for obesity and gestational diabetes. CONCLUSIONS/INTERPRETATION: We found a significant and consistent association between pregnancy loss and later type 2 diabetes that increased with increasing number of losses. Thus, pregnancy loss and recurrent pregnancy loss are significant risk factors for later type 2 diabetes. Future studies should explore whether this association is due to common background factors or whether prediabetic metabolic conditions are responsible for this association. Graphical abstract.

Sex of the first-born and obstetric complications in the subsequent birth. A study of 2.3 million second births from Denmark, Finland, Norway, and Sweden.

INTRODUCTION: Studies have shown associations between a first-born boy and increased risks of pregnancy loss, stillbirth, decreased birthweight, and preterm birth in subsequent pregnancies, but with limited precision. MATERIAL AND METHODS: We examined associations between sex of the first-born and obstetric complications in second births. We calculated the relative risks (RR)s of preeclampsia/eclampsia, placental abruption, stillbirth, and preterm birth in approximately 2.3 million second births comparing women with a preceding first-born boy to those with a first-born girl using the Medical Birth Registries of Denmark, Finland, Norway, and Sweden 1980-2008. RESULTS: In second births following a first-born boy rather than a girl, the RR was 4% higher for preeclampsia/eclampsia (RR = 1.04, 95% CI 1.02-1.06), 9% higher for placental abruption (RR = 1.09, 95% CI 1.05-1.13), 9% higher for stillbirth (RR = 1.09, 95% CI 1.04-1.14), and 8% higher for preterm birth (RR = 1.08, 95% CI 1.07-1.09). The population attributable risks ranged from 2% to 4.5%. CONCLUSIONS: Male sex of the first-born is associated with small increases in risks of obstetric complications in the second birth. Exploration of the underlying mechanisms is needed to increase our knowledge and treatment options for these serious obstetric complications.

Pregnancy loss: A 40-year nationwide assessment.

INTRODUCTION: Pregnancy loss is frequent. We aimed to assess the frequency and trends in pregnancy losses according to female age and mode of conception over a 40-year follow-up period. MATERIAL AND METHODS: In a national historical prospective cohort study, we followed all Danish women 10-49 years over the 40-year study period 1978-2017. Data on pregnancies and their outcomes were obtained from the National Health Registry, the Medical Birth Registry and the National Fertility Registry. Incidence rates per 100 pregnancies and per 1,000 women-years as well as lifetime risks per 100 women were calculated. Women included in the lifetime analysis were followed from age 12 to age 49. Pregnancy loss included spontaneous abortion, missed abortion and anembryonic pregnancy. RESULTS: In 3 519 455 recorded pregnancies, 337 008, or 9.6%, were diagnosed with a pregnancy loss. The proportion increased from 7.5% in 1978-1979, peaked at 10.7% in 2000 and thereafter decreased to 9.1% in 2015-2017. Pregnancy loss rate in women 10-14 years was 3.9%, increasing gradually with age to 26.9% in pregnant women 45-49 years, a 6.9-fold increase. Loss rates were slightly lower in naturally conceived pregnancies than in assisted pregnancies except for women above 45 years, where the risk of loss was higher in the spontaneously conceived group. Lifetime risk of specific numbers of losses were: 0: 76.9%, 1: 17.9%, 2: 3.9%, 3: 0.87%, and 4+: 0.35%. CONCLUSIONS: The proportion of women experiencing pregnancy loss has changed little throughout four decades and is still primarily influenced by female age. More than 75% of pregnant women are never recorded with a pregnancy loss, and <1.5% will experience three or more losses.

Pregnancy Loss and Cancer Risk: A Nationwide Observational Study.

BACKGROUND: Cancer is the second leading cause of death worldwide. Few studies have investigated if recurrent pregnancy loss is associated with an increased risk of cancer. We aimed to assess whether pregnancy loss is associated with later cancer development. METHODS: We identified all invasive cancers after age 40, among all Danish women born between January 1957 and December 1972, ensuring a full reproductive history. Cases were matched by birth year 1:10 to cancer-free controls. Women were followed until the end of 2017. The number of pregnancy losses (miscarriages or still births) was correlated to long-term cancer risk using conditional logistic regression, providing odds ratios for specific cancers with different numbers of pregnancy losses, all adjusted for age, education, and other potential confounders. FINDINGS: The study included 28,785 women with cancer (mean age 48.7 [SD 5.0]) and 283,294 matched controls (mean age 48.6 [SD 5.0]). We found no overall association between pregnancy loss and later development of 11 site-specific types of cancer or cancer overall. Taking the sequence of pregnancy losses into account, primary recurrent pregnancy loss (three consecutive pregnancy losses without prior live birth) was associated with later overall cancer by an odds ratio of 1.27 (1.04-1.56). Secondary recurrent pregnancy loss showed no association to cancer. INTERPRETATION: Pregnancy loss was not associated with later cancer development. Women with primary recurrent pregnancy loss had a borderline significant association to later cancer overall, this may be a chance finding. FUNDING: Ole Kirk's Foundation and Copenhagen University Hospital Rigshospitalet's Research Grant.

Treatment with intravenous immunoglobulin in patients with recurrent pregnancy loss: An update.

Intravenous immunoglobulin (IVIg) has a documented clinical effect in many autoimmune diseases and has so far been tested in >10 randomised controlled trials (RCTs) in women with recurrent pregnancy loss (RPL). The results of the RCTs have, however, been very divergent. In meta-analyses of all trials, no significant impact on live birth rate has been reported. In contrast, in sensitivity analyses, IVIg significantly increased live birth rates when initiated prior to conception and it had a borderline significant therapeutic effect in women with secondary RPL. Higher dosages of IVIg and serological signs of autoimmunity in the treated patients tended to increase the success rate after treatment. A follow-up study of patients from our recent RCT also supports a significant therapeutic effect in patients who had received IVIg before conception. The lessons learned from the published trials and meta-analyses should be incorporated in the design of future RCTs of IVIg in the treatment of RPL.

The Fertility Assessment and Counseling Clinic - does the concept work? A prospective 2-year follow-up study of 519 women.

INTRODUCTION: The Fertility Assessment and Counseling (FAC) Clinic was initiated to provide women with information about their current fertility status to prevent infertility and smaller families than desired. The aim was to study the predictive value of a risk assessment score based on known fertility risk factors in terms of time to pregnancy. MATERIAL AND METHODS: Prospective cohort study of the first 570 women attending the FAC Clinic from 2011 to 2013 at Rigshospitalet, Denmark. A consultation included: risk assessment score sheet with items on infertility risk factors, anti-Müllerian hormone and ultrasound. The risk score was categorized as low, medium or high. After 2 years an email-based questionnaire was distributed regarding subsequent pregnancies. RESULTS: The follow-up questionnaire was answered by 519 women (91.1%). The mean age was 35 years and 38% were single at inclusion. The majority (67.8%, 352/519) tried to conceive within 2 years after attending the FAC Clinic. At follow up, 73.6% (259/352) had achieved a pregnancy, 21% (74/352) were still trying and 5.4% (19/352) had given up. Two-thirds (65%) with only low risk scores conceived spontaneously within 12 months, although this figure was only 32% for women with at least one high risk score (n = 82). Accordingly, presence of at least one high risk score reduced the odds of achieving a pregnancy within 12 months by 75% (OR 0.25, 95% CI 0.12-0.52). CONCLUSION: The new FAC Clinic concept seems usable and offers a tool for fertility experts to guide women on how to fulfill their reproductive life-plan.

Immunologic Abnormalities, Treatments, and Recurrent Pregnancy Loss: What Is Real and What Is Not?

Recurrent pregnancy loss, depending on the definition, affects 1% to 3% of women aiming to have a child. Little is known about the direct causes of recurrent pregnancy loss, and the condition is considered to have a multifactorial and complex pathogenesis. The aim of this review was to summarize the evaluation and the management of the condition with specific emphasis on immunologic biomarkers identified as risk factors as well as current immunologic treatment options. The review also highlights and discusses areas in need of further research.

Inheritance of the 8.1 ancestral haplotype in recurrent pregnancy loss.

BACKGROUND AND OBJECTIVES: The 8.1 ancestral haplotype (AH) (HLA-A1, C7, B8, C4AQ0, C4B1, DR3, DQ2) is a remarkably long and conserved haplotype in the human major histocompatibility complex. It has been associated with both beneficial and detrimental effects, consistent with antagonistic pleiotropy. It has also been proposed that the survival of long, conserved haplotypes may be due to gestational drive, i.e. selective miscarriage of fetuses who have not inherited the haplotype from a heterozygous mother. Recurrent pregnancy loss (RPL) is defined as three or more consecutive pregnancy losses. The objective was to test the gestational drive theory for the 8.1AH in women with RPL and their live born children. METHODOLOGY: We investigated the inheritance of the 8.1AH from 82 heterozygous RPL women to 110 live born children. All participants were genotyped for HLA-A, -B and -DRB1 in DNA from EDTA-treated blood or buccal swaps. Inheritance was compared with a Mendelian inheritance of 50% using a two-sided exact binomial test. RESULTS: We found that 55% of the live born children had inherited the 8.1AH, which was not significantly higher than the expected 50% (P = 0.29). Interestingly, we found a non-significant trend toward a higher inheritance of the 8.1AH in girls, 63%, P = 0.11 as opposed to boys, 50%, P = 1.00. CONCLUSIONS AND IMPLICATIONS: We did not find that the 8.1AH was significantly more often inherited by live born children of 8.1AH heterozygous RPL women. However our data suggest that there may be a sex-specific effect which would be interesting to explore further, both in RPL and in a background population.

Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe.

INTRODUCTION: Annexin A5 is an essential component of placental integrity that may potentially mediate susceptibility to phenotypes of compromised pregnancy. A promoter haplotype termed M2 of the coding gene ANXA5 has been implicated in various pregnancy complications such as preeclampsia and recurrent pregnancy loss (RPL), however with inconclusive results. STUDY SUBJECTS AND METHODS: A retrospective case-control study combining resequencing and restriction fragment length polymorphism (RFLP) analysis was undertaken in 313 women with unexplained RPL and 214 fertile women from Estonia and Denmark to estimate the RPL disease risk of the M2 haplotype in Northern Europe. Comparative prevalence of the studied ANXA5 genetic variants in human populations was estimated based on the 1000 Genomes Project (n = 675, whole-genome sequencing data) and the KORA S3 500K dataset of South German samples (n = 1644, genome-wide genotyping data). RESULTS: Minor allele frequency of common polymorphisms in ANXA5 promoter was up to two-fold lower among Estonian RPL subjects than fertile controls. The M2 haplotype was not associated with RPL and a trend for decreased prevalence was observed among RPL patients compared to controls both in Estonia (8.1% vs 15.2%, respectively) and Denmark (9.7% vs 12.6%). The high M2 prevalence in fertile controls was consistent with estimations for European and East Asian populations (9.6%-16.0%). CONCLUSIONS: This study cautions to consider the M2 haplotype as a deterministic factor in early pregnancy success because: i) no RPL disease risk was associated with the haplotype in two clinically well-characterized RPL case-control study samples, ii) high prevalence of the haplotype among fertile controls and world-wide populations is inconsistent with the previously proposed severe impact on early pregnancy success, iii) weak impact of M2 haplotype on the production of ANXA5 protein has been established by others.

Antibody response to influenza A(H1N1)pdm09 in vaccinated, serologically infected and unaffected pregnant women and their newborns.

OBJECTIVE: To evaluate the serological response in pregnant Danish women immunized during the 2009 pandemic by serologic infection or by vaccination with influenza A(H1N1) Pandemrix(®) and describe levels of passively acquired maternal antibody in their offspring. DESIGN: Observational cohort study. SETTING: Department of Obstetrics, Aarhus University Hospital, Skejby, Denmark, October to December 2009. POPULATION: Pregnant women and their offspring METHODS: Serological analysis of antibodies to influenza A(H1N1)pdm09 by hemagglutination inhibition assay in 197 women and their offspring. Blood samples were collected consecutively at delivery from the mother and the umbilical cord. In a subgroup of 124 of the 197 women, an additional blood sample from gestational weeks 9-12 was available for analysis. MAIN OUTCOME MEASURES: Seroconversion, geometric mean titer, geometric mean-fold rise and protective antibodies. RESULTS: 33 of the 124 subgroup women (27%) seroconverted during pregnancy, 79% after vaccination and 17% after serologic infection (p < 0.001). The geometric mean titer after delivery in non-vaccinated, non-serologically infected women was 17.1 (95%CI 15.7-18.6). The geometric mean titer increased significantly after serologic infection with H1N1 [76.5 (95%CI 51.3-113.9), p < 0.001] and after vaccination [589.6 (95%CI 339.3-1024.7), p < 0.001]. The geometric mean-fold rise (mother at delivery/mother early pregnancy) was significantly higher after vaccination [2.23 (1.93-2.54)] than after serologic infection [1.73 (1.59-1.87), p = 0.013]. In newborns of vaccinated mothers, 89.5% had protective antibody levels compared with 15.8% in newborns of serologically infected mothers (p < 0.001). CONCLUSIONS: Influenza vaccination during pregnancy confers passive immunity to the newborn.

Structural genomic variation as risk factor for idiopathic recurrent miscarriage.

Recurrent miscarriage (RM) is a multifactorial disorder with acknowledged genetic heritability that affects ∼3% of couples aiming at childbirth. As copy number variants (CNVs) have been shown to contribute to reproductive disease susceptibility, we aimed to describe genome-wide profile of CNVs and identify common rearrangements modulating risk to RM. Genome-wide screening of Estonian RM patients and fertile controls identified excessive cumulative burden of CNVs (5.4 and 6.1 Mb per genome) in two RM cases possibly increasing their individual disease risk. Functional profiling of all rearranged genes within RM study group revealed significant enrichment of loci related to innate immunity and immunoregulatory pathways essential for immune tolerance at fetomaternal interface. As a major finding, we report a multicopy duplication (61.6 kb) at 5p13.3 conferring increased maternal risk to RM in Estonia and Denmark (meta-analysis, n = 309/205, odds ratio = 4.82, P = 0.012). Comparison to Estonian population-based cohort (total, n = 1000) confirmed the risk for Estonian female cases (P = 7.9 × 10(-4) ). Datasets of four cohorts from the Database of Genomic Variants (total, n = 5,846 subjects) exhibited similar low duplication prevalence worldwide (0.7%-1.2%) compared to RM cases of this study (6.6%-7.5%). The CNV disrupts PDZD2 and GOLPH3 genes predominantly expressed in placenta and it may represent a novel risk factor for pregnancy complications.

Maternal homozygocity for a 14 base pair insertion in exon 8 of the HLA-G gene and carriage of HLA class II alleles restricting HY immunity predispose to unexplained secondary recurrent miscarriage and low birth weight in children born to these patients.

Homozygous carriage of a 14 base pair (bp) insertion in exon 8 of the HLA-G gene may be associated with low levels of soluble HLA-G and recurrent miscarriage (RM). We investigated the G14bp insertion(ins)/deletion(del) polymorphism in 339 women with unexplained RM and 125 control women. In all patients and patients with secondary RM after a firstborn boy, 19.2% and 23.9%, respectively, were G14bp ins/ins compared with 11.2% of controls (p<0.05 and p<0.01). Among secondary RM patients with a firstborn boy, G14bp del/del and no carriage of an HLA class II (HYrHLA) allele restricting immunity against male-specific minor HY antigens was found less often than in controls (p<0.05) whereas G14bp ins/ins and carriage of HYrHLA predisposed (p<0.08) to this clinical entity. The mean birth weight of firstborn boys born to G14bp ins positive secondary RM patients was significantly lower than expected (p<0.001) but only in carriers of HYrHLA alleles (p<0.01). In conclusion, homozygosity for G14bp ins predisposes to RM. The combination of G14 ins homozygosity and carriage of HYrHLA predisposes to secondary RM in women with a firstborn boy and negatively affects birth weight in these boys.

Glucagon-like Peptide-1 receptor agonists activate rodent thyroid C-cells causing calcitonin release and C-cell proliferation.

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog developed for type 2 diabetes. Long-term liraglutide exposure in rodents was associated with thyroid C-cell hyperplasia and tumors. Here, we report data supporting a GLP-1 receptor-mediated mechanism for these changes in rodents. The GLP-1 receptor was localized to rodent C-cells. GLP-1 receptor agonists stimulated calcitonin release, up-regulation of calcitonin gene expression, and subsequently C-cell hyperplasia in rats and, to a lesser extent, in mice. In contrast, humans and/or cynomolgus monkeys had low GLP-1 receptor expression in thyroid C-cells, and GLP-1 receptor agonists did not activate adenylate cyclase or generate calcitonin release in primates. Moreover, 20 months of liraglutide treatment (at >60 times human exposure levels) did not lead to C-cell hyperplasia in monkeys. Mean calcitonin levels in patients exposed to liraglutide for 2 yr remained at the lower end of the normal range, and there was no difference in the proportion of patients with calcitonin levels increasing above the clinically relevant cutoff level of 20 pg/ml. Our findings delineate important species-specific differences in GLP-1 receptor expression and action in the thyroid. Nevertheless, the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation.

Study of the structure and impact of human leukocyte antigen (HLA)-G-A, HLA-G-B, and HLA-G-DRB1 haplotypes in families with recurrent miscarriage.

A 14-base pair (bp) long insertion (ins)/deletion (del) polymorphism in exon 8 in the 3'-untranslated region of the human leukocyte antigen (HLA)-G gene is suggested to affect transcription of the gene. Carriage of the G14bp ins is associated with low levels of soluble HLA-G and increases the risk of recurrent miscarriage (RM). Due to existence of strong linkage disequilibrium (LD) in the HLA region, the primary susceptibility genes for RM in the HLA-G region have not yet been identified. HLA-A, -B, -DRB1, and -G14bp polymorphisms were investigated in 29 Caucasian families with two or more siblings suffering unexplained RM. Strong positive LD was detected between the G14bp ins and HLA-A*01, -A*11, -A*31, -B*08, and DRB1*03, whereas strong negative LD was found between G14bp ins and HLA-A*02, -A*03, and -A*24. The frequency of haplotypes with HLA-G14bp ins inherited from the mother was significantly increased in probands with RM (p = 0.05). The increased compatibility between probands and their mothers for maternal G14 ins positive haplotypes suggests that maternal-fetal compatibility for chromosomal segments adjacent to HLA-G locus is a risk factor for female offspring to experience RM in their later reproductive life.