Study protocol for a Developmental Epidemiological Study of Children born through Reproductive Technologies (DESCRT).

STUDY QUESTIONS: The primary objective of this study is to determine what parental factors or specific ART may influence the risk for adverse cardiometabolic outcomes among children so conceived and their parents. The secondary objective of this study is to prospectively examine the effects of infertility or ART on the intrauterine environment, obstetric and neonatal outcomes. WHAT IS KNOWN ALREADY: Pregnancies conceived with ART are at an increased risk of being affected by adverse obstetric and neonatal outcomes when compared to spontaneously conceived (SC) pregnancies among fertile women. Small cohort studies have suggested ART-conceived children may have a higher risk of long-term cardiometabolic disturbances as well. Currently, few studies have compared long-term cardiometabolic outcomes among ART-conceived children and non-IVF treated (NIFT) children, to children conceived spontaneously to parents with infertility (subfertile parents). STUDY DESIGN SIZE DURATION: The Developmental Epidemiological Study of Children born through Reproductive Technologies (DESCRT) is a prospective cohort study that aims to: establish a biobank and epidemiological cohort of children born to subfertile or infertile parents who either conceived spontaneously (without assistance) or used reproductive technologies to conceive (all offspring were from couples assessed and/or treated in the same institute); prospectively examine the effects of infertility or ART on the intrauterine environment, obstetric and neonatal outcomes; and determine what parental factors or ART may influence the cardiometabolic risk of children so conceived. Pregnancies and resultant children will be compared by mode of conception, namely offspring that were conceived without medical assistance or SC or following NIFT, IVF with fresh embryo transfer or frozen embryo transfer (FET), and by fertilization method (conventional versus ICSI). DESCRT has a Child group evaluating long-term outcomes of children as well as a Pregnancy group that will compare obstetric and neonatal outcomes of children conceived since the commencement of the study. Recruitment started in May of 2017 and is ongoing. When the study began, we estimated that ∼4000 children would be eligible for enrollment. PARTICIPANTS/MATERIALS SETTING METHODS: Eligible participants are first-trimester pregnancies (Pregnancy group) or children (Child group) born to parents who were evaluated at an infertility center in the University of California, San Francisco, CA, USA who were SC or conceived after reproductive treatments (NIFT, IVF ± ICSI, FET). Children in the Child group were conceived at UCSF and born from 2001 onwards. In the Pregnancy group, enrollment began in November of 2017.The primary outcome is the cardiometabolic health of offspring in the Child group, as measured by blood pressure and laboratory data (homeostatic model assessment for insulin resistance (HOMA-IR), oral glucose disposition). There are several secondary outcome measures, including: outcomes from parental survey response (assessing parent/child medical history since delivery-incidence of cardiometabolic adverse events), anthropomorphic measurements (BMI, waist circumference, skinfold thickness), and laboratory data (liver enzymes, lipid panel, metabolomic profiles). In the Pregnancy group, outcomes include laboratory assessments (bhCG, maternal serum analytes, soluble fms-like tyrosine kinase-1 (sFLT-1), and placental growth factor (PlGF)) and placental assessments (placental volume in the second and third trimester and placental weight at delivery). Importantly, aliquots of blood and urine are stored from parents and offspring as part of a biobank. The DESCRT cohort is unique in two ways. First, there is an extensive amount of clinical and laboratory treatment data: parental medical history and physical examination at the time of treatment, along with ovarian reserve and infertility diagnosis; and treatment specifics: for example, fertilization method, culture O2 status, embryo quality linked to each participant. These reproductive data will aid in identifying explanatory variables that may influence the primary cardiometabolic outcomes of the offspring-and their parents. Second, the DESCRT control group includes pregnancies and children SC from parents with subfertility, which may help to assess when infertility, as opposed to reproductive treatments, may be affecting offspring cardiometabolic health. STUDY FUNDING/COMPETING INTERESTS: This study is funded by the National Institutes of Health NICHD (1R01HD084380-01A1). A.J.A. is a shareholder in Carrot and consultant for Flo Health. The other authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: NCT03799107. TRIAL REGISTRATION DATE: 10 January 2019. DATE OF FIRST PATIENT'S ENROLLMENT: 10 May 2017.

Physiological, metabolic and transcriptional postnatal phenotypes of in vitro fertilization (IVF) in the mouse.

Approximately 1-4% of children today are conceived using assisted reproductive technologies (ARTs), including in vitro fertilization (IVF). IVF is considered safe and the great majority of these children are healthy, yet there is increasing physiological and molecular evidence from animal models that ART is associated with postnatal metabolic and cardiovascular alterations. Understanding the mechanisms underlying these changes and determining whether they have biological significance is of paramount importance for optimizing the design of culture conditions and improving the health of ART children across the life course. In this review, we examine the evidence of molecular changes present in adult tissues of rodent offspring generated by preimplantation manipulation of gametes and embryos. Although embryo manipulation in vitro can induce common transcriptional effects in the blastocyst, transcriptional and metabolomic signatures in adult IVF tissues are largely tissue-specific. However, there is pervasive evidence of oxidative stress and metabolic dysfunction, indicating a lasting effect of IVF on molecular physiology.

Adult male mice conceived by in vitro fertilization exhibit increased glucocorticoid receptor expression in fat tissue.

Prenatal development is highly plastic and readily influenced by the environment. Adverse conditions have been shown to alter organ development and predispose offspring to chronic diseases, including diabetes and hypertension. Notably, it appears that the changes in glucocorticoid hormones or glucocorticoid receptor (GR) levels in peripheral tissues could play a role in the development of chronic diseases. We have previously demonstrated that in vitro fertilization (IVF) and preimplantation embryo culture is associated with growth alterations and glucose intolerance in mice. However, it is unknown if GR signaling is affected in adult IVF offspring. Here we show that GR expression is increased in inbred (C57Bl6/J) and outbred (CF-1× B6D2F1/J) blastocysts following in vitro culture and elevated levels are also present in the adipose tissue of adult male mice. Importantly, genes involved in lipolysis and triglyceride synthesis and responsive to GR were also increased in adipose tissue, indicating that increased GR activates downstream gene pathways. The promoter region of GR, previously reported to be epigenetically modified by perinatal manipulation, showed no changes in DNA methylation status. Our findings demonstrate that IVF results in a long-term change in GR gene expression in a sex- and tissue-specific manner. These changes in adipose tissues may well contribute to the metabolic phenotype in mice conceived by IVF.

ART and health: clinical outcomes and insights on molecular mechanisms from rodent studies.

Since the birth of the first IVF-conceived child in 1978, the use of assisted reproductive technologies (ART) has grown dramatically, contributing to the successful birth of 5 million individuals worldwide. However, there are several reported associations of ART with pregnancy complications, such as low birthweight (LBW), preterm birth, birth defects, epigenetic disorders, cancer and poor metabolic health. Whether this is attributed to ART procedures or to the subset of the population seeking ART remains a controversy, but the most relevant question today concerns the potential long-term implications of assisted conception. Recent evidence has emerged suggesting that ART-conceived children have distinct metabolic profiles that may predispose to cardiovascular pathologies in adulthood. Because the eldest IVF individuals are still too young to exhibit components of chronic middle-aged syndromes, the use of animal models has become particularly useful in describing the effects of unusual or stressful preimplantation experiences on adult fitness. Elucidating the molecular mechanisms by which embryos integrate environmental signals into development and metabolic gene expression programs will be essential for optimizing ART procedures such as in vitro culture conditions, embryo selection and transfer. In the future, additional animal studies to identify mechanisms underlying unfavorable ART outcomes, as well as more epidemiological reviews to monitor the long-term health of ART children are required, given that ART procedures have become routine medical practice.

In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm.

Differences in gene expression and imprinting have been reported, comparing in vivo versus in vitro generated preimplantation embryos. Furthermore, mouse studies have shown that placenta development is altered following in vitro culture. However, the molecular mechanisms underlying these findings are unknown. We therefore isolated trophectoderm (TE) and inner cell mass (ICM) cells from in vivo and in vitro fertilization (IVF) embryos and evaluated their transcriptome using microarrays. We found that the transcriptomes of in vitro produced ICM and TE cells showed remarkably few differences compared to ICM and TE cells of in vivo generated embryos. In vitro fertilization embryos showed a reduced number of TE cells compared to in vivo embryos. In addition, TE of IVF embryos showed significant downregulation of solute transporter genes and of genes involved in placenta formation (Eomesodermin, Socs3) or implantation (Hbegf). In summary, IVF and embryo culture significantly affects the transcriptome of ICM and TE cells.

Effect of ICSI on gene expression and development of mouse preimplantation embryos.

BACKGROUND: In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS: We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS: Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)). CONCLUSIONS: Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model.

BACKGROUND: Abnormal placentation is a potential mechanism to explain the increased incidence of low birthweight observed after IVF. This study evaluates, in a mouse model, whether the method of conception and embryo transfer affect placentation and fetal development. METHODS: IVF blastocysts (CF1 x B6D2F1/J) were cultured in Whitten's medium (IVF(WM), n = 55) or K modified simplex optimized medium with amino acids (IVF(KAA), n = 56). Embryos were transferred to the uteri of pseudo-pregnant recipients. Two control groups were created: unmanipulated embryos produced by natural mating (in vivo group, n = 64) and embryos produced by natural mating that were flushed from uterus and immediately transferred to pseudo-pregnant recipients (flushed blastocysts, FB group, n = 57). At gestation age 12.5 days, implantation sites were collected and fixed; fetuses and placentas were weighed and their developmental stage (DS) evaluated. Placental areas and vascular volume fractions were calculated; parametric statistics were applied as appropriate. RESULTS: IVF fetuses showed a modest but significant delay in development compared with FB mice (P < 0.05). In addition, IVF conceptuses were consistently smaller than FB (P < 0.05). Importantly, these differences persisted when analyzing fetuses of similar DS. The placenta/fetus ratio was larger in the IVF group (IVF(WM) 0.95; IVF(KAA) = 0.90) than the FB group (0.72) (P < 0.05 for all comparisons). Gross morphology of the placenta and ratio labyrinth/fetal area were equivalent in the IVF and FB groups, as were percentage of fetal blood vessels, maternal blood spaces and trophoblastic components. CONCLUSIONS: In vitro embryo culture affects fetal and placental development; this could explain the lower birthweight in IVF offspring.

Methylenetetrahydrofolate reductase C677T and A1298C variants do not affect ongoing pregnancy rates following IVF.

BACKGROUND: There is concern that IVF could compromise normal imprinting and methylation of DNA. Methylenetetrahydrofolate reductase (MTHFR) regulates the flow of folic acid-derived, one-carbon moieties for methylation and is critical to early embryonic development. Therefore, we hypothesized that common polymorphisms in MTHFR could associate with IVF outcome. METHODS: MTHFR C677T and A1298C polymorphism genotyping was performed on 374 subjects for this study, representing 197 couples undergoing IVF in a university setting from July 2005 to January 2006. Analysis of variance (ANOVA), chi-square and/or multivariate analyses were used to assess whether these polymorphisms are associated with embryo quality or with ongoing pregnancy or spontaneous abortion rates. RESULTS: Allele frequencies for C677T ( p=0.67, q=0.33) and A1298C ( p=0.71, q=0.29) were in Hardy-Weinberg equilibrium. The C677T and A1298C variants, either alone or in combination, did not associate with embryo quality or short-term pregnancy outcome. CONCLUSIONS: The common polymorphisms in MTHFR are not associated with embryo quality, as defined by cell number or fragmentation score, or with short-term pregnancy outcomes. Therefore, in our population in which women receive adequate folic acid, MTHFR genotypes are not informative in explaining IVF failure. Further studies, however, examining birth outcomes and the other enzymes in the folic acid pathway are warranted.

Premenstrual syndrome as a predictor of menopausal symptoms.

OBJECTIVE: To estimate whether premenstrual syndrome (PMS) predicts common menopausal symptoms assessed longitudinally for 5 years among women in the transition to menopause. METHODS: Data were obtained from a structured interview questionnaire, daily symptom ratings, and standard measures of depressive symptoms and sleep quality at 7 assessment periods in a population-based cohort of 436 women. Menstrual status was determined by menstrual bleeding dates. Hormones were measured in the early follicular phase, with a maximum of 14 measures per subject. Multivariate logistic regression models for repeated measures were used to estimate the effects of study variables. RESULTS: Premenstrual syndrome significantly decreased with age (P <.001) and with changes in menstrual bleeding status (P =.003). Women with PMS at enrollment were more likely over the 5-year period to report menopausal hot flushes (odds ratio [OR] 2.09; confidence interval [CI] 1.42, 3.08; P <.001); depressed mood (OR 2.34; CI 1.60. 3.43; P <.001); poor sleep (OR 1.72; CI 1.16, 2.53; P =.007), and decreased libido (OR 1.54; CI 1.06, 2.24; P =.024) after adjusting for age, race, diagnosis of major depression, and estradiol. Subjects' fluctuations in estradiol were significantly associated with hot flushes, depressive symptoms, and poor sleep. CONCLUSION: Premenstrual syndrome decreased in the transition to menopause. Women who reported PMS at baseline were at greater risk of menopausal hot flushes, depressed mood, poor sleep, and decreased libido. Further studies of the associations of symptoms and changes in ovarian function are needed to elucidate the underlying symptom physiology and aid in the development of effective treatments for women during the menopausal transition.

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development.

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.

Microinjection of mitochondria into zygotes creates a model for studying the inheritance of mitochondrial DNA during preimplantation development.

OBJECTIVE: To determine the effect of mutant mitochondria on preimplantation embryo development and of preimplantation embryo development on the survival of mutant mitochondrial DNA. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): None. INTERVENTION(S): Mutant and wild-type mitochondria, fractionated from tissue obtained from a patient with MELAS syndrome, a mitochondrial disease, were microinjected into mouse zygotes. Control zygotes received either no injection or sham injection. MAIN OUTCOME MEASURE(S): Preimplantation embryo development and survival of mutant mitochondrial DNA as determined by polymerase chain reaction analysis. RESULT(S): After microinjection into zygotes, the MELAS mutation could be identified by polymerase chain reaction until the hatched blastocyst stage of embryo development. The survival of MELAS-injected zygotes, observed for 4 days after injection, did not differ from the survival of zygotes injected with wild-type mitochondria or from the survival of uninjected or sham-injected controls. CONCLUSION(S): It appears that preimplantation embryo development does not screen out mitochondrial DNA mutations introduced into fertilized oocytes, and low levels of mutant mitochondrial DNA do not disrupt early embryo development.

Atrial septal defect and pregnancy: a retrospective analysis of obstetrical outcome before and after surgical correction.

STUDY OBJECTIVES: To describe the obstetrical outcome and long-term cardiac follow-up of a group of patients who had surgery for atrial septal defect repair before pregnancy and a group who underwent pregnancy before surgical correction. DESIGN: Retrospective. SETTING: Public Institution. PATIENTS: Eighty women (mean age 20.15 +/- 4.5 years) complaining of atrial septal defect, 60 of which had surgical correction before pregnancy and subsequently had 115 pregnancies, and 20 of which underwent pregnancy before the correction of cardiac defect and subsequently had 48 pregnancies. INTERVENTIONS: Surgical correction of atrial septal defect: 49 by open heart technique (13 with cardiopulmonary bypass, 36 with light hypothermic circulatory arrest), 11 by closed heart techniques. RESULTS: Patients who underwent pregnancy before surgery had an increased incidence of miscarriage, pre-term delivery and cardiac symptoms during pregnancy. Patients operated before pregnancy required more frequently cesarean section and pharmacological therapy. The frequency of stillbirths, malformations in the offspring and long-term cardiac complications were similar in both groups. CONCLUSIONS: Surgical correction of atrial septal defect before pregnancy is recommended even in case of apparently well compensated hemodinamic situation for the low rate of surgical complications in spite of the higher incidence of obstetrical problems in case of medical therapy alone.

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

The effect of air abrasion on shear bond strength to dentin with dental adhesives.

This study compared the effects of different dentin surface treatments on the shear bond strengths of three adhesive systems. The adhesive systems included a resin-modified glass ionomer, Fuji II LC, and two dentin bonding systems, One Step and Scotchbond Multi-Purpose Plus. The surface treatments compared for each adhesive system were as follows: 1) the controls, which were conditioned, 2) air abrasion at 120 psi without conditioning, 3) air abrasion at 160 psi without conditioning, 4) air abrasion at 120 psi with conditioning, and 5) air abrasion at 160 psi with conditioning. The KCP 1000 Whisperjet was used for all air-abrasive specimens. Controls for each adhesive material (Fuji II LC, One Step, Scotchbond Multi-Purpose Plus) were bonded using manufacturers' recommendations. Results showed that air abrasion significantly lowered bond strength of the resin-modified glass ionomer, conditioned or nonconditioned (P < 0.01). Air abrasion alone significantly lowered bond strengths of the dentin bonding agent systems (P < 0.01). However, air abrasion plus conditioning of the dentin surface resulted in bond strengths that were similar to the conditioned-only specimens (P < 0.01).

Hormone induction of ascorbic acid transport in immature granulosa cells.

Ascorbic acid serves a vital role as an antioxidant, and like FSH, it inhibits apoptosis of granulosa cells in cultured follicles. In contrast, reactive oxygen species block the action of FSH and induce DNA damage in these cells. As the uptake of ascorbic acid by granulosa cells may be a site for regulation, we examined the nature of this process and whether uptake is under hormone control. Granulosa cells were isolated from immature rats pretreated with estradiol or diethylstilbestrol for 3-4 days and placed in culture. Culture of the cells with either FSH (50 ng/ml) or insulin-like growth factor I (IGF-I; 30 ng/ml) for 48 h increased ascorbic acid uptake by 2.7- and 1.9-fold (P < 0.05), respectively, and the response to FSH plus IGF-I was additive (4.5-fold; P < 0.05). The interval for maximum induction of ascorbic acid transport by FSH was between 4-8 h, whereas a significant response to IGF-I was not seen until 48 h. GnRH (1 microM), phorbol ester (phorbol 12-myristate 13-acetate; 1 microM), and 8-bromo-cAMP (8Br-cAMP; 1 mM) also induced ascorbic acid transport by 1.7-, 1.9-, and 2.3-fold (P < 0.05) within 24 h, and the response to maximal levels of phorbol ester and 8Br-cAMP was synergistic (4.8-fold; P < 0.05). Kinetic analysis showed a similar Michaelis constant (K(m); 50.8 +/- 5.3 microM) and maximum velocity (3.3 +/- 0.4 pmol/10(6) cells.min) for ascorbic acid transport in FSH-, 8Br-cAMP-, or phorbol ester-treated cells. Ouabain (100 microM) or removal of extracellular Na+ significantly inhibited ascorbic acid uptake, as did dinitrophenol (1 mM), an inhibitor of mitochondrial production of ATP. The induction of ascorbic acid transport by FSH, IGF-I, or GnRH was abolished by simultaneous incubation with tyrphostin (AG-18; 80 microM), a specific tyrosine kinase inhibitor, whereas induction was unaffected by an inactive, but chemically similar, compound (A-1; 80 microM). From these results we conclude that ascorbic acid uptake is energy and Na+ dependent and that the induction of ascorbic acid transporters in granulosa cells occurs through multiple hormones that ultimately influence tyrosine-specific protein kinases. The hormone-dependent induction of ascorbic acid accumulation in granulosa cells appears to be an essential process for the development and maintenance of a viable follicle.

Malacoplakia of testis.

A case of malacoplakia of testis with a typical clinical course, related to urogenital E. coli infection is presented. By means of electron microscopy, we have identified several forms of Michaelis-Gutmann bodies within the lesion. X-ray spectral analysis revealed that these bodies contain large amounts of calcium, phosphate, sulfur, sodium and chloride. The pathogenesis of malacoplakia and the formation of Michaelis-Gutmann bodies is shortly discussed.