OBJECTIVE: We aimed to identify the genetic causes of hypospadias in children using targeted gene panel sequencing for disorders of sex development (DSD).
METHODS: This study included 18 twin boys with hypospadias: seven and two pairs were monozygotic and dizygotic twins, respectively, and six were discordant and three were concordant twins. Targeted gene panel sequencing for 67 known DSD genes was performed. Sequence variants were classified into five different categories, pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign, following the American College of Medical Genetics and Genomics Standards and Guidelines.
RESULTS: The mean gestational age and birth weight were 35.3±2.0 weeks and 1.96±0.61 kg, respectively, with seven patients being small for gestational age. Hypospadias was present in 12 patients, with posterior type in 33.3% and anterior type in 66.7%. In three families with twins, both siblings had hypospadias. In addition, cryptorchidism was observed in one subject. Surgical correction of hypospadias was performed at a mean age of 22.1 months. Molecular analysis identified 12 different genetic variants, including two pathogenic mutations in the AMH (p.E389*) and SRD5A2 (p.R246Q) genes, found in subjects with hypospadias, respectively. However, only heterozygous mutations were detected.
CONCLUSIONS: This study did not identify a definitive genetic component contributing to the development of hypospadias; however, the findings suggest that intrauterine growth retardation may play a significant role.

Pregnancies ending before 26 weeks contribute 1% of births but 40% of infant deaths in the United States. The rate of these \"periviable\" births to non-Hispanic (NH) Black women exceeds four times that for NH whites. Small male periviable infants remain most likely to die. NH white periviable males weigh more than their NH Black counterparts. We argue that male infants born from twin gestations, in which one fetus died in utero (i.e., the vanishing twin syndrome), contribute to the disparity. We cannot directly test our argument because \"vanishing\" typically occurs before clinical recognition of pregnancy. We, however, describe and find associations that would emerge in vital statistics were our argument correct. Among male periviable singleton births from 288 monthly conception cohorts (January 1995 through December 2018), we found an average NH white advantage of 30 grams (759 grams versus 729 grams). Consistent with our argument, however, cohorts signaling relatively few survivors of the vanishing twin syndrome showed no disparity.

Previous studies have shown that mothers of naturally conceived dizygotic (DZ) twins tend to be taller, older, and smoke more than mothers of naturally conceived monozygotic (MZ) twin and mothers of singletons. Here, we investigate whether mothers of naturally conceived DZ twins differ from mothers who conceived their DZ twins after medically assisted reproduction (MAR) in eight maternal traits related to fertility based on observational survey data. We include data from 33,648 mothers from the Netherlands Twin Register (NTR) and 1660 mothers of twins from the Norwegian Mother, Father and Child Cohort Study (MoBA). We contrast mothers of naturally conceived DZ twins with mothers of MAR DZ twins. Next, we further segment the MAR group into mothers who underwent hormonal induction of ovulation but not in vitro fertilization (IVF) and those who IVF twins, comparing them both to each other and against the mothers of naturally conceived DZ twins. Mothers of naturally conceived DZ twins smoke more often, differ in body composition, have a higher maternal age and have more offspring before the twins than mothers of MZ twins. Compared to MAR DZ twin mothers, mothers of naturally conceived DZ twins have fewer miscarriages, lower maternal age and increased height, more offspring and are more often smokers. BMI before the twin pregnancy is similar in both natural and MAR DZ twin mothers. Mothers who received hormonal induction of ovulation (OI) have a lower maternal age, fewer miscarriages, and a higher number of offspring before their twin pregnancy than twin mothers who received IVF and/or intracytoplasmic sperm injection (ICSI) treatments. Our study shows that twin mothers are a heterogenous group and the differences between twin mothers should be taken into account in epidemiological and genetic research that includes twins.

It has been shown that vertical transmission of the SARS-CoV-2 strain is relatively rare, and there is still limited information on the specific impact of maternal SARS-CoV-2 infection on vertical transmission. The current study focuses on a transcriptomics analysis aimed at examining differences in gene expression between placentas from mother-newborn pairs affected by COVID-19 and those from unaffected controls. Additionally, it investigates the in situ expression of molecules involved in placental inflammation. The Papa Giovanni XXIII Hospital in Bergamo, Italy, has recorded three instances of intrauterine transmission of SARS-CoV-2. The first two cases occurred early in the pandemic and involved pregnant women in their third trimester who were diagnosed with SARS-CoV-2. The third case involved an asymptomatic woman in her second trimester with a twin pregnancy, who unfortunately delivered two stillborn fetuses due to the premature rupture of membranes. Transcriptomic analysis revealed significant differences in gene expression between the placentae of COVID-19-affected mother/newborn pairs and two matched controls. The infected and control placentae were matched for gestational age. According to the Benjamani-Hochberg method, 305 genes met the criterion of an adjusted p-value of less than 0.05, and 219 genes met the criterion of less than 0.01. Up-regulated genes involved in cell signaling (e.g., CCL20, C3, MARCO) and immune response (e.g., LILRA3, CXCL10, CD48, CD86, IL1RN, IL-18R1) suggest their potential role in the inflammatory response to SARS-CoV-2. RNAscope® technology, coupled with image analysis, was utilized to quantify the surface area covered by SARS-CoV-2, ACE2, IL-1β, IL-6, IL-8, IL-10, and TNF-α on both the maternal and fetal sides of the placenta. A non-statistically significant gradient for SARS-CoV-2 was observed, with a higher surface coverage on the fetal side (2.42 ± 3.71%) compared to the maternal side (0.74 ± 1.19%) of the placenta. Although not statistically significant, the surface area covered by ACE2 mRNA was higher on the maternal side (0.02 ± 0.04%) compared to the fetal side (0.01 ± 0.01%) of the placenta. IL-6 and IL-8 were more prevalent on the fetal side (0.03 ± 0.04% and 0.06 ± 0.08%, respectively) compared to the maternal side (0.02 ± 0.01% and 0.02 ± 0.02%, respectively). The mean surface areas of IL-1β and IL-10 were found to be equal on both the fetal (0.04 ± 0.04% and 0.01 ± 0.01%, respectively) and maternal sides of the placenta (0.04 ± 0.05% and 0.01 ± 0.01%, respectively). The mean surface area of TNF-α was found to be equal on both the fetal and maternal sides of the placenta (0.02 ± 0.02% and 0.02 ± 0.02%, respectively). On the maternal side, ACE-2 and all examined interleukins, but not TNF-α, exhibited an inverse mRNA amount compared to SARS-CoV-2. On the fetal side, ACE-2, IL-6 and IL-8 were inversely correlated with SARS-CoV-2 (r = -0.3, r = -0.1 and r = -0.4, respectively), while IL-1β and IL-10 showed positive correlations (r = 0.9, p = 0.005 and r = 0.5, respectively). TNF-α exhibited a positive correlation with SARS-CoV-2 on both maternal (r = 0.4) and fetal sides (r = 0.9) of the placenta. Further research is needed to evaluate the correlation between cell signaling and immune response genes in the placenta and the vertical transmission of SARS-CoV-2. Nonetheless, the current study extends our comprehension of the molecular and immunological factors involved in SARS-CoV-2 placental infection underlying maternal-fetal transmission.

Optimal seatbelt practices for pregnant women with twins at different gestational ages remain uncertain. To offer recommendations for a comfortable seatbelt system, this cross-sectional observational study explored seatbelt usage and driving habits among women with twins across various pregnancy stages through an online survey that explored driving conditions and comfortable seatbelts at different stages of pregnancy. Women who drove daily before their pregnancy with twins decreased their driving frequency as the pregnancy progressed. Correct seatbelt usage was lower and no seatbelt usage was higher among pregnant women with twins than those with singleton pregnancies. They adapted their seatbelt-wearing techniques to minimise pressure on the chest in the first and third trimesters and the abdomen from the second trimester onwards. The comfortable seatbelts were those that could alleviate belt pressure, featuring waist belts to reduce pressure, wider belts to avoid localised pressure, and shoulder belts resembling a backpack type. When wearing a seatbelt, avoiding pressure on the thorax and abdomen is key for pregnant women with twins. This study suggests that the suitability of driving for pregnant women with twins in their last trimester and the reliability of seatbelts designed for such women should be further examined and validated.

BACKGROUND: Cardiovascular diseases (CVDs) are major causes of mortality and morbidity worldwide; yet the understanding of their molecular basis is incomplete. Multi-omics studies have significant potential to uncover these mechanisms, but such studies are challenged by genetic and environmental confounding-a problem that can be effectively reduced by investigating intrapair differences in twins. Here, we linked data on all diagnoses of the circulatory system from the nationwide Danish Patient Registry (spanning 1977-2022) to a study population of 835 twins holding genome-wide DNA methylation and gene expression data. CVD diagnoses were divided into prevalent or incident cases (i.e., occurring before or after blood sample collection (2007-2011)). The diagnoses were classified into four groups: cerebrovascular diseases, coronary artery disease (CAD), arterial and other cardiovascular diseases (AOCDs), and diseases of the veins and lymphatic system. Statistical analyses were performed by linear (prevalent cases) or cox (incident cases) regression analyses at both the individual-level and twin pair-level. Significant genes (p < 0.05) in both types of biological data and at both levels were inspected by bioinformatic analyses, including gene set enrichment analysis and interaction network analysis.
RESULTS: In general, more genes were found for prevalent than for incident cases, and bioinformatic analyses primarily found pathways of the immune system, signal transduction and diseases for prevalent cases, and pathways of cell-cell communication, metabolisms of proteins and RNA, gene expression, and chromatin organization groups for incident cases. This potentially reflects biology related to response to CVD (prevalent cases) and mechanisms related to regulation and development of disease (incident cases). Of specific genes, Myosin 1E was found to be central for CAD, and DEAD-Box Helicase 5 for AOCD. These genes were observed in both the prevalent and the incident analyses, potentially reflecting that their DNA methylation and gene transcription levels change both because of disease (prevalent cases) and prior disease (incident cases).
CONCLUSIONS: We present novel biomarkers for CVD by performing multi-omics analysis in twins, hereby lowering the confounding due to shared genetics and early life environment-a study design that is surprisingly rare in the field of CVD, and where additional studies are highly needed.

BACKGROUND: The incidence of twin pregnancies is on the rise worldwide due to assisted reproductive technologies. Cervical dilatation is a frequent complication and can be considered a cause of premature births in twin pregnancies. In vitro fertilization (IVF) twins are more prone to preterm delivery. Routine cervical cerclage with normal cervical length is not recommended in twins, but studies were not done on IVF twins. So, this study aims to evaluate the effectiveness of routine transvaginal cerclage in twin IVF pregnancies with normal cervical length on maternofetal outcome.
METHODS: A retrospective case-control study was conducted at a maternity hospital in eastern India from January 2016 to December 2019 with over 21800 deliveries. Two groups were taken, cases were those IVF twins with normal cervical length in whom cervical cerclage was done as they have referred to our hospital from the IVF centers for cerclage and in control no-cervical cerclage as they are referred from other IVF centers where cerclage was not a routine protocol. We have excluded cerclage done for history, ultrasound indicated, uterine anomalies, and monochorionic twins. Fifteen participants were recruited in both cases and controls.
RESULTS: In our study 2 (6.66%), 4 (13%), and 26 (86%) were IVF twins delivered at a gestational age of <28 weeks, 28-32 weeks, and 32-37 weeks, respectively. The mean age of participants, mean gestation age at delivery, and birth weight in cases and control were 35.27 ± 5.98 years (min: 23; max: 45), 32.40 ± 5.54 years (min: 25; max: 44); 34 weeks 2 days ± 3.28 (min: 31; max: 37), 33 weeks 5 days ± 1.66 (min: 25; max: 37); 1961.33 ± 340 gram, 1899.33 ± 437.48 gram, respectively with no statistical significant difference (p = 0.186, p = 1, p = 0.668, respectively) Conclusion: Routine transvaginal cerclage is not effective in twin IVF pregnancies with normal cervical length for preventing preterm births. IVF twin women usually present at a late age and their birth weight are also low.

Co-twin studies are an elegant and powerful design that allows controlling for the effect of confounding variables, including genetic and a range of environmental factors. There are several approaches to carry out this design. One of the methods commonly used, when contrasting continuous variables, is to calculate difference scores between members of a twin pair on two associated variables, in order to analyse the covariation of such differences. However, information regarding whether and how the different ways of estimating within-pair difference scores may impact the results is scant. This study aimed to compare the results obtained by different methods of data transformation when performing a co-twin study and test how the magnitude of the association changes using each of those approaches. Data was simulated using a direction of causation model and by fixing the effect size of causal path to low, medium, and high values. Within-pair difference scores were calculated as relative scores for diverse within-pair ordering conditions or absolute scores. Pearson\'s correlations using relative difference scores vary across the established scenarios (how twins were ordered within pairs) and these discrepancies become larger as the within-twin correlation increases. Absolute difference scores tended to produce the lowest correlation in every condition. Our results show that both using absolute difference scores or ordering twins within pairs, may produce an artificial decrease in the magnitude of the studied association, obscuring the ability to detect patterns compatible with causation, which could lead to discrepancies across studies and erroneous conclusions.

BACKGROUND: Separating with close siblings and leaving the parental home at an early age represents a major life event for an adolescent (reflected by age at separation in a twin pair) and may predispose them to poor mental health. This study aims to examine the association of age at separation and residential mobility on depressive symptoms in late adolescence and young adulthood and to explore possible underlying genetic effects.
METHODS: Residential mobility consisted of the number and total distance of moves before age 17. Based on 3071 twins from the FinnTwin12 cohort, we used linear regression to assess the association of age at separation and residential mobility with General Behavior Inventory (GBI) scores at age 17 and in young adulthood. A higher GBI score indicated more depressive symptoms occurred. Then, the mixed model for repeated measures (MMRM) was used to visualize the scores\' trajectory and test the associations, controlling for \"baseline\" state. Twin analyses with a bivariate cross-lagged path model were performed between the difference in GBI scores, between cotwins, and separation status for the potential genetic influence.
RESULTS: Compared to twins separated before age 17, twins who separated later had significantly lower GBI scores at age 17 and in young adulthood. In MMRM, separation at a later age and a higher number of moves were associated with a higher GBI score in young adulthood. A small genetic effect was detected wherein GBI within-pair differences at age 17 were associated with separation status before age 22 (coefficient: 0.01).
CONCLUSIONS: The study provides valid evidence about the influence of siblings and family on depressive symptoms in later adolescence and young adulthood while finding some evidence for a reverse direction effect. This suggests more caution in the interpretation of results. A strong association between residential mobility and depressive symptoms was affirmed, although further detailed research is needed.

OBJECTIVE: Multifetal gestation (MFG) is much more common in pregnancies that utilize assisted reproductive technologies (ART). We assessed how these rates have changed over the previous decade and the impact on live birth rates (LBR).
METHODS: This retrospective cohort study uses the National Summary Reports of the Society for Assisted Reproductive Technology (SART) from 2014 to 2020. Data points included only autologous cycles. The data were divided into five age groups as reported in the database: < 35, 35-37, 38-40, 41-42, and > 42 years old. Descriptive statistics and a two-tailed T-test were used to determine the trends and statistical significance (p < 0.05).
RESULTS: Rates of twin births decreased substantially from 2014 to 2020 for autologous embryo transfers across all age groups and diagnoses. Surprisingly, the overall LBR for autologous IVF cycles decreased at similar rates from 2014 to 2020 in all age groups. The mean number of embryos transferred has dramatically reduced, especially across age groups < 42.
CONCLUSIONS: Rates of twin and higher-level gestations have decreased substantially over the past decade; the effect correlates with the increased utilization of eSET and PGT. The cause of infertility did not significantly impact the rate of MFG.