Vascular health of fathers with history of intracytoplasmic sperm injection.

OBJECTIVE: Studies suggest that men who undergo assisted reproductive technologies (ART) may have a higher risk of cardiovascular disease; however, limited data on this matter is available. This observational pilot study aimed to investigate the overall vascular health of fathers with history of intracytoplasmic sperm injection (ICSI) compared to fathers whose partners conceived spontaneously. METHODS: Diet quality, physical activity, sedentary behavior as well as overall vascular function including the assessment of pulse wave analysis, intima-media thickness (cIMT), arterial stiffness of the common carotid artery (CCA) and blood lipids, were evaluated. RESULTS: A total of 34 fathers with history of ICSI and 29 controls (48.49 [46.32 - 57.09] years vs. 47.19 [40.62 - 55.18] years, p = 0.061) were included. After adjusting for age, no significantly increased cardiovascular risk was detected regarding vascular function. CONCLUSIONS: The results suggest an unaltered cardiovascular risk profile in fathers with history of ICSI. In the future, prospective multicenter studies are required to validate these preliminary results.

Vascular Health in Adults Born After Using Assisted Reproductive Technologies.

An increasing number of children are conceived by assisted reproductive technologies (ART). Several studies indicated vascular alterations in ART children. However, limited data is available within the adult ART population. Therefore, this study investigated the overall vascular health of young ART adults in comparison to spontaneously conceived peers. In total, 16 ART subjects and 22 spontaneously conceived peers (22.06 ± 2.21 years vs. 22.00 ± 2.14 years, p = 0.194) were enrolled for the assessment of endothelial function, brachial blood pressure, central blood pressure, pulse wave velocity, carotid intima-media thickness, and blood lipids. No significant differences in vascular function were detected between the in vitro fertilization subgroup (n = 9), the intracytoplasmic sperm injection subgroup (n = 7) and spontaneously conceived peers. This pilot study suggests an unimpaired vascular function in young ART adults. In the future, multi-centric studies with a greater sample size are required to confirm the results of the current study and enable precise cardiovascular risk stratification of the adult ART population.

Seasonal dynamic of cholecalciferol (D3) and anti-Muellerian hormone (AMH) with impact on ovarian response and IVF/ICSI.

OBJECTIVE: Recent studies revealed intriguing associations between cholecalciferol (D3) and reproductive functions. Seasonal changes of D3 concentrations are well known; however, they are not always considered in the context of reproductive functions. In this study, we analyzed D3 serum concentration in IVF/ICSI patients with respect to seasonal 3-month quartiles and anti-Muellerian hormone (AMH) referring to the impact on Assisted Reproductive Technologies (ART) outcome. MATERIALS AND RESEARCH METHODS: We studied 469 female patients, presenting between 2012 and 2018 for ART treatment in our fertility center. D3 as well as the AMH serum concentrations were measured at the beginning of the follicle stimulation (days 3-5 of menstrual cycles). Results were evaluated with respect to seasonal quartiles and outcome of the ART cycles. RESULTS: D3 concentrations showed significant fluctuations within annual quartiles with a pronounced peak in August-October and a minimum in February-April (26.0 vs. 20.5 mg/dl; p < 0.0001). Similar seasonal dynamics were found for AMH (2.98 vs. 1.78 ng/ml; p = 0.010) and these were associated with significantly shorter stimulation periods during August-October (11.29 vs. 12.12 days; p = 0.042), higher number of fertilized oocytes between August and October (6.23 vs. 4.97; p = 0.05) along with a trend towards higher numbers of cumulus-oocyte complexes. However, no such differences were found for the numbers of MII oocytes or pregnancy rates. CONCLUSION: Our data indicate seasonal 3-month quartile variations of AMH concentrations and characteristics of ART, such as days of ovarian stimulation and number of fertilized oocytes. Highest AMH concentrations were found between August and October and this quartile was associated with highest D3 concentrations.

Maternal and paternal carriage of the annexin A5 M2 haplotype: a possible risk factor for recurrent implantation failure (RIF).

OBJECTIVE: This study was carried out to determine the potential role of the M2/ANXA5 haplotype as a risk factor for recurrent implantation failure (RIF). Carriage of the M2/ANXA5 haplotype that induces prothrombotic changes has been implicated in failure of early pregnancies and placenta-mediated complications (preeclampsia, IUGR, preterm birth). MATERIAL AND METHODS: In the present case control study, 63 couples (females and males) with RIF presenting for IVF/ICSI to the Fertility Center of [masked] were analyzed. RIF was defined as ≥ 4 consecutive failed ART-transfers of ≥ 4 blastocysts or ≥ 8 cleavage-stage embryos of optimal quality and maternal age ≤ 41. Fertile female controls (n = 90) were recruited from the same center. Population controls (n = 533) were drafted from the PopGen biobank, UKSH Kiel. RESULTS: Couples carrying the M2/ANXA5 haplotype turned out to have a significantly increased relative risk (RR) for RIF. Compared with female fertile controls, RR was 1.81 with p = 0.037 (OR 2.1, 95%CI 1.0-4.3) and RR was 1.70, with p = 0.004 (OR 2.0, 95%CI 1.2-3.1) compared with population controls (15.4% M2 carriers). Male partners were comparable with RIF females for M2/ANXA5 haplotypes (28.6% vs. 23.8%, p = 0.54). RIF females compared with population controls had a RR of 1.55 (p = 0.09) and RIF males compared with population controls had a RR of 1.9 (p = 0.01). Couples with ≥ 7 failed transfers showed a RR of 1.82 (p = 0.02) compared with population controls. CONCLUSION: Our findings suggest that maternal as well as paternal M2/ANXA5 haplotype carriages are risk factors for RIF. These results allow new insights into the pathogenesis of RIF and might help to identify relevant risk groups.

Correlation of Vitamin D3 (Calcitriol) Serum Concentrations with Vitamin B12 and Folic Acid in Women Undergoing in vitro Fertilisation/Intracytoplasmatic Sperm Injection.

BACKGROUND: The important role of vitamin D3 in human health is well recognized. In this study, we measured serum concentrations of vitamin D3, vitamin B12 and B9 (folic acid) in 410 women undergoing in vitro fertilisation (IVF)/intracytoplasmatic sperm injection (ICSI) with dedicated focus on 3-month changes in consideration of patients' BMI. METHODS: Patients were of European origin and did not take any supplementation of D3. In preparing for pregnancy, patients took ≥4 weeks 400 µg folic acid combined with 9 µg vitamin B12 and 150 µg iodide as recommended. RESULTS: We found a significant 3-month quartile change of D3 serum concentrations (p < 0.0001) with maximum levels in autumn and lowest in spring. D3 correlated significantly with B12 (p = 0.035, ρ = 0.102) and folic acid (p < 0.0001, ρ = 0.191). BMIs however showed a negative correlation with B12 (p = 0.031, ρ = -0.105) and folic acid (p = 0.012, ρ = -0.125). CONCLUSIONS: Our results suggest a model in which the sun exposure during summer months enables storage of D3 followed by a slow release as a major factor to maintain D3 levels throughout the year. Finally, our data indicate that B12 and folic acid uptake might be influenced by vitamin D receptor and D3, where D3 and the BMI appear to have an indirect relationship - via B12 and folic acid.

Assessment of M2/ANXA5 haplotype as a risk factor in couples with placenta-mediated pregnancy complications.

PURPOSE: The aim of this study was to confirm the associated M2/ANXA5 carrier risk in women with placenta-mediated pregnancy complications (PMPC) and to test their male partners for such association. Further analysis evaluated the influence of maternal vs. paternal M2 alleles on miscarriage. METHODS: Two hundred eighty-eight couples with preeclampsia (PE), intrauterine growth restriction (IUGR), or premature birth (PB) were recruited (n = 96 of each phenotype). The prevalence of the M2 haplotype was compared to two control cohorts. They included a group of women with a history of normal pregnancy without gestational pathology (Munich controls, n = 94) and a random population sample (PopGen controls, n = 533). RESULTS: Significant association of M2 haplotype and pregnancy complications was confirmed for women and for couples, where prevalence was elevated from 15.4 to 23.8% (p < 0.001). Post hoc analyses demonstrated an association for IUGR and PB individually. A strong link between previous miscarriages and M2 carrier status was identified which may explain the predisposition to placental pregnancy complication. M2/ANXA5 appears to be a risk factor for adverse pregnancy outcomes related, but not limited to miscarriages, with similar prevalence in women and their male partners. CONCLUSION: These findings support the proposed physiological function of ANXA5 as an embryonic anticoagulant that appears deficient in contiguous specter of thrombophilia-related pregnancy complications culminating more frequently in miscarriage in a maternal M2 carrier background.

Unexplained recurrent miscarriages are associated with an aberrant sperm protamine mRNA content.

STUDY QUESTION: Are unexplained recurrent miscarriages associated with abnormal protamine-1 and protamine-2 mRNA levels in spermatozoa? SUMMARY ANSWER: Both protamine-1 and protamine-2 mRNA levels as well as the protamine-1 to protamine-2 mRNA ratio in spermatozoa from men whose female partners experienced two or more consecutive miscarriages were significantly different compared to those from both healthy control men and subfertile couples undergoing IVF/ICSI. WHAT IS KNOWN ALREADY: Aberrant sperm protamine ratios are known to be associated with male-factor infertility. Data from this study suggest that the protamine mRNA ratio may additionally affect early embryo development. STUDY DESIGN, SIZE, DURATION: The study population was recruited from men whose female partners presented with two or more consecutive unexplained miscarriages in a consultation for recurrent pregnancy loss between 2014 and 2016. At the research laboratory of the Urological Clinic of the University Giessen, spermatozoa from cases and controls were subjected to reverse transcription quantitative PCR (RTqPCR) using specific primer pairs for protamine-1 and protamine-2. PARTICIPANTS/MATERIALS, SETTING, METHODS: Protamine-1 and protamine-2 mRNA levels were analysed in semen samples from 25 men whose female partners experienced at least two consecutive idiopathic miscarriages before the 20th week of gestation. The couples were recruited during consultation at the Fertility Center of the LMU Munich, Germany, and at the Clinical Division of Gynecologic Endocrinology and Reproductive Medicine of the Medical University of Vienna, Austria. Results were compared with those from 32 healthy donors (WHO, 2010) recruited at the Department of Urology, Pediatric Urology and Andrology, Giessen, Germany, and 107 men whose partners participated in an IVF/ICSI program at the Fertility Center of the LMU Munich, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: Protamine-1 and protamine-2 mRNA levels as well as the protamine mRNA ratio and all routine semen parameters revealed significant differences between recurrent miscarriage couples and healthy volunteers (P < 0.01). When comparing recurrent miscarriage couples with couples undergoing IVF/ICSI, Ct-values of protamine-1 and protamine-2 mRNAs were significantly higher and the protamine mRNA ratio was significantly lower in RM couples (P < 0.01). When comparing protamine mRNA levels and the protamine mRNA ratio with routine semen parameters, a significant negative correlation was evident between progressive motility and the protamine-2 mRNA level (P = 0.015), as well as between non-progressive motility and the protamine mRNA ratio (P = 0.023). LIMITATIONS REASONS FOR CAUTION: Although our data demonstrate significant abnormalities in RM, larger sample sizes will be needed to confirm our results. Larger sample sizes should also balance the fact that we had to focus mainly on median protamine mRNA levels. Finally, men in the healthy control group were younger in age than those in the case group, which might have introduced some bias, at least concerning the classic semen parameters. Moreover, only protamine mRNA instead of protein levels could be measured. WIDER IMPLICATIONS OF THE FINDINGS: Although the exact mechanism remains to be elucidated, our data suggest that protamine mRNA levels in spermatozoa are not only important for successful fertilization, but also for proper development of the early embryo. STUDY FUNDING/COMPETING INTEREST(S): Grant from the University Clinic Giessen and Marburg (UKGM 29/2015GI). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.

STUDY QUESTION: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility? SUMMARY ANSWER: TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells. STUDY DESIGN, SIZE, DURATION: Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy). MAIN RESULTS AND THE ROLE OF CHANCE: During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006). LIMITATIONS, REASONS FOR CAUTION: Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential. WIDER IMPLICATIONS OF THE FINDINGS: The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility. STUDY FUNDING: Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1). COMPETING INTERESTS: None.

Low-molecular-weight heparin for women with unexplained recurrent pregnancy loss: a multicenter trial with a minimization randomization scheme.

BACKGROUND: A daily injection of low-molecular-weight heparin (LMWH) is often prescribed to women with unexplained recurrent pregnancy loss (RPL), although evidence suggesting a benefit is questionable. OBJECTIVE: To determine whether LMWH increases ongoing pregnancy and live-birth rates in women with unexplained RPL. DESIGN: Controlled, multicenter trial with randomization using minimization conducted from 2006 to 2013. (ClinicalTrials.gov: NCT00400387). SETTING: 14 university hospitals and perinatal care centers in Germany and Austria. PATIENTS: 449 women with at least 2 consecutive early miscarriages or 1 late miscarriage were included during 5 to 8 weeks' gestation after a viable pregnancy was confirmed by ultrasonography. INTERVENTION: Women in the control group received multivitamin pills, and the intervention group received vitamins and 5000 IU of dalteparin-sodium for up to 24 weeks' gestation. MEASUREMENTS: Primary outcome was ongoing pregnancy at 24 weeks' gestation. Secondary outcomes included the live-birth rate and late pregnancy complications. RESULTS: At 24 weeks' gestation, 191 of 220 pregnancies (86.8%) and 188 of 214 pregnancies (87.9%) were intact in the intervention and control groups, respectively (absolute difference, -1.1 percentage points [95% CI, -7.4 to 5.3 percentage points]). The live-birth rates were 86.0% (185 of 215 women) and 86.7% (183 of 211 women) in the intervention and control groups, respectively (absolute difference, -0.7 percentage point [CI, -7.3 to 5.9 percentage points]). There were 3 intrauterine fetal deaths (1 woman had used LMWH); 9 cases of preeclampsia or the hemolysis, elevated liver enzyme level, and low platelet count (HELLP) syndrome (3 women had used LMWH); and 11 cases of intrauterine growth restriction or placental insufficiency (5 women had used LMWH). LIMITATION: Placebo injections were not used, and neither trial staff nor patients were blinded. CONCLUSION: Daily LMWH injections do not increase ongoing pregnancy or live-birth rates in women with unexplained RPL. Given the burden of the injections, they are not recommended for preventing miscarriage. PRIMARY FUNDING SOURCE: Pfizer Pharma.

Prevention, management and extent of adverse pregnancy outcomes in women with hereditary antithrombin deficiency.

Antithrombin (AT) deficiency is a rare hereditary thrombophilia with a mean prevalence of 0.02 % in the general population, associated with a more than ten-fold increased risk of venous thromboembolism (VTE). Within this multicenter retrospective clinical analysis, female patients with inherited AT deficiency were evaluated concerning the type of inheritance and extent of AT deficiency, medical treatment during pregnancy and postpartally, VTE risk as well as maternal and neonatal outcome. Statistical analysis was performed with SPPS for Windows (19.0). A total of 18 pregnancies in 7 patients were evaluated, including 11 healthy newborns ≥37th gestational weeks (gw), one small for gestational age premature infant (25th gw), two late-pregnancy losses (21st and 28th gw) and four early miscarriages. Despite low molecular weight heparin (LMWH) administration, three VTE occurred during pregnancy and one postpartally. Several adverse pregnancy outcomes occurred including fetal and neonatal death, as well as severe maternal neurologic disorders occurred. Patients with substitution of AT during pregnancy in addition to LMWH showed the best maternal and neonatal outcome. Close monitoring with appropriate anticoagulant treatment including surveillance of AT levels might help to optimize maternal and fetal outcome in patients with hereditary AT deficiency.