Luca Pagliardini

Vitamin D Status in Women With Uterine Leiomyomas

CONTEXT Recent in vitro and in vivo experimental evidence supports a role of vitamin D insufficiency as an important factor in the development of uterine leiomyomas. However, epidemiological data supporting this possibility are scanty. OBJECTIVE Our objective was to investigate vitamin D status in women with and without uterine leiomyomas. DESIGN This was a case-control study of women referring to 2 infertility units in Italy. Women were eligible as cases if they were diagnosed with at least 1 uterine leiomyoma with a mean diameter ≥10 mm at transvaginal ultrasound. Each of them was matched to the 2 subsequent women of the same age (±1 year) whose uterus resulted unremarkable at ultrasound. Selected women provided a blood sample for the quantitative detection of 25-hydroxyvitamin D₃ levels. MAIN OUTCOME MEASURE We measured serum concentration of 25-hydroxyvitamin D₃. RESULTS A total of 128 women with leiomyomas and 256 controls were selected. The mean ± SD serum concentration of 25-hydroxyvitamin D3 was significantly lower in affected women compared with controls (18.0 ± 7.7 vs 20.8 ± 11.1 ng/mL respectively, P = .010). The number (proportion) of women with 25-hydroxyvitamin D3 deficiency (ie, <10 ng/mL) in cases and controls was 19 (15%) and 19 (7%), respectively (P = .022). The adjusted odds ratio for the presence of leiomyomas in women with serum levels of 25-hydroxyvitamin D₃ deficiency was 2.4 (95% confidence interval = 1.2-4.9) (P = .016). CONCLUSIONS Vitamin D is an emerging regulator of uterine leiomyoma development. Cohort and interventional studies are pressingly needed to confirm a causal relationship and to investigate the potential therapeutic benefits of vitamin D supplementation.

Vitamin D Deficiency and Infertility: Insights From in vitro Fertilization Cycles

CONTEXT Vitamin D deficiency has been proven to affect fertility in mammals, but data in human is less convincing. In particular, data on in vitro fertilization (IVF), an attractive model to draw information on this topic, are sparse and conflicting. OBJECTIVE Our objective was to investigate IVF outcome in women with deficient 25-hydroxy-vitamin D [25(OH)D] serum levels (<20 ng/mL). DESIGN AND SETTING This prospective cross-sectional study was conducted at the infertility unit of an academic setting. PATIENTS The main inclusion criteria were as follows: (1) indication to IVF, (2) age 18-42 years, (3) BMI 18-25 kg/m(2), (4) adequate ovarian reserve according to Bologna criteria. Eligible women provided a serum sample for 25(OH)D measurement at the time of cycle preparation. Subjects were subsequently excluded if the cycle was cancelled or if the attempt was excessively delayed. INTERVENTION Quantitative detection of serum 25(OH)D. MAIN OUTCOME MEASURE Clinical pregnancy rate. RESULTS The number of recruited women with serum 25(OH)D <20 ng/mL and ≥ 20 ng/mL was 154 and 181, respectively. The clinical pregnancy rates were 20% (30/154) and 31% (56/181), respectively (P = .02); the adjusted odds ratio for clinical pregnancy in women with vitamin D ≥ 20 ng/mL was 2.15 (95% CI: 1.23-3.77). Subgroup analyses showed that the group of women with the highest serum levels (>30 ng/mL) had the highest chances of pregnancy. CONCLUSIONS Vitamin D is an emerging factor influencing female fertility and IVF outcome. Additional studies are pressingly needed to confirm a causal relationship and to investigate the potential therapeutic benefits of vitamin D supplementation.

An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis

Background Although endometriosis may benefit from primary prevention measures, the epidemiological risk factors identified are equivocal. Two genome-wide association studies (GWAS) have been conducted for endometriosis in two different ethnic populations but results are still to be replicated consistently and across various ethnicities. To confirm the association of GWAS-derived susceptibility loci, we conducted a replication Italian case-control study and a meta-analysis. Methods An independent set of 305 laparoscopically-proven endometriosis patients and 2710 controls were recruited. Four SNPs—CDKN2BAS rs1333049, rs7521902 close to WNT4, rs12700667 in an inter-genic region on 7p15.2 and fibronectin 1 rs1250248—were selected for this association study. Results Rs1333049 risk allele G frequency resulted significantly higher in endometriosis patients compared with controls (OR 1.32, 95% CI 1.11 to 1.57), confirming the role of this locus also in the Caucasian population. The meta-analysis showed that rs7521902 was associated with endometriosis at a genome-wide significance (pmeta=2.23×10−9) while for rs1250248, a genome-wide significant pmeta value of 3.89×10−9 was detected only in association with severe forms. An epistatic interaction between rs7521902 and rs1250248 (OR 1.56, p=1.19×10−2) was found especially in presence of ovarian disease (OR=2.15, p=3.12×10−4). Conclusions We confirm WNT4, CDKN2BAS and FN1 as the first identified common loci for endometriosis.

Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law.

OBJECTIVE To retrospectively evaluate whether fresh day-5 embryo transfer could overcomes the detrimental effect of subtle progesterone elevations at hCG administration on pregnancy outcomes in women undergoing ovarian hyperstimulation for IVF/ICSI cycles. STUDY DESIGN Retrospective study of 204 infertile patients aged 23-44 years who underwent IVF/ICSI treatment and fresh blastocyst transfer under the Italian law (embryos cryopreservation cannot be planned in advance). Women were divided into those with a progesterone level <1.5 ng/ml and those with a progesterone concentration ≥ 1.5 ng/ml at hCG triggering. The clinical pregnancy rate (CPR) after blastocyst transfer was the primary outcome. RESULTS Age, body mass index (BMI), antral follicle count, anti-Mullerian hormone (AMH) and FSH values, mean number of stimulation days, ratio of GnRH agonist and antagonist cycles and total dose of gonadotrophins administered did not differ between the two groups. Serum estradiol and number of retrieved oocytes were significantly increased in the group with elevated progesterone and a significantly higher number of oocytes was used in this group. Fertilization rate, percentage of top quality embryos, and number of transferred blastocysts were similar in the two groups. The CPR was significantly higher in women with progesterone levels <1.5 ng/ml at hCG (50%) compared with women with progesterone concentration ≥ 1.5 ng/ml (33.3%) (odds ratio = 2.00, 95% confidence interval 1.07-3.75). CONCLUSIONS A fresh blastocyst transfer does not completely overcome the detrimental effect of progesterone rise at hCG on IVF/ICSI pregnancy outcomes.

The WNT/β-catenin signaling pathway and expression of survival promoting genes in luteinized granulosa cells: endometriosis as a paradigm for a dysregulated apoptosis pathway

OBJECTIVE To analyze the WNT/β-catenin signaling pathway in luteinized granulosa cells from women with and without endometriosis in relation to cellular apoptosis. DESIGN Basic. SETTING University hospital. PATIENT(S) Patients with a laparoscopic diagnosis of endometriosis (n = 30) and women undergoing intracytoplasmic sperm injection for male infertility (control group n = 39). INTERVENTION(S) Isolation of luteinized granulosa cells. MAIN OUTCOME MEASURE(S) Gene expression analysis of components of the WNT/β-catenin pathway, protein expression levels of β-catenin, and cell cycle studies in luteinized granulosa cells. RESULT(S) Compared with luteinized granulosa cells from control women, cells derived from endometriosis patients had significantly higher transcript levels of the β-catenin-independent molecules WNT4 and WNT5a and lower levels of the β-catenin-dependent molecule WNT1. A decrease of total β-catenin as well as of its dephosphorylated active form, together with an aberrant gene expression of the downstream targets survivin and BMP4, was detected in cells from affected women. Flow cytometry analysis confirmed an enhanced apoptosis of luteinized granulosa cells from patients with endometriosis. CONCLUSION(S) The concomitant dysregulation of specific members of the WNT pathway and of its pivot molecule β-catenin in granulosa cells characterized by an increased apoptosis suggests that the WNT/β-catenin signaling pathway might be involved in leading to granulosa cell atresia.

Endometriosis as a detrimental condition for granulosa cell steroidogenesis and development: From molecular alterations to clinical impact

Endometriosis is an estrogen-dependent chronic inflammatory condition that affects women in their reproductive period. Alterations in ovarian follicle morphology and function have been documented in affected women. The local intrafollicular environment has been as well examined by various groups. In the present review, we aimed to summarize the molecular evidence supporting the idea that endometriosis can negatively influence growth, steroidogenesis and the function of the granulosa cells (GCs). Reduced P450 aromatase expression, increased intracellular ROS generation and altered WNT signaling characterize the GCs of women with endometriosis. Clear evidence for an increased level of GC apoptosis has been provided in association with the downregulation of pro-survival factors. Other potentially negative effects include decreased progesterone production, locally decreased AMH production and lower inflammatory cytokine expression, although these have been only partially clarified. The possibility that endometriosis per se may influence IVF clinical results as a consequence of the detrimental impact on the local intrafollicular environment is also discussed.

Replication and meta-analysis of previous genome-wide association studies confirm vezatin as the locus with the strongest evidence for association with endometriosis

STUDY QUESTION Is it possible to replicate the genetic association of single nucleotide polymorphisms (SNPs) rs13394619, rs4141819, rs7739264, rs17694933 and rs10859871 in five genetic loci previously identified as associated with endometriosis in an Italian Caucasian population? SUMMARY ANSWER SNP rs10859871 near the vezatin (VEZT) gene was found to be significantly associated with endometriosis in general while SNPs rs17694933 and rs4141819 were associated with Stage III/IV and ovarian disease, respectively. WHAT IS KNOWN ALREADY Endometriosis represents a complex disease in which the phenotypic manifestations are influenced by both genetic and environmental factors. Recent genome-wide association studies (GWASs) have allowed to identify some SNPs associated with the predisposition to the disease. A meta-analysis published in 2014 combined results from GWAS and replication studies showing that of the nine loci found to be associated with the disease in at least one of the studies, six (rs7521902, rs1270667, rs13394619, rs7739264, rs1537377 and rs10859871) remained genome-wide significant while two others (rs1250248 and rs4141819) showed borderline genome-wide significant association with more severe disease. STUDY DESIGN, SIZE, DURATION Allele frequencies of selected SNPs (rs13394619, rs4141819, rs7739264, rs17694933 and rs10859871) were investigated in 305 women with laparoscopically proven endometriosis, 285 laparoscopic controls and 2425 healthy, blood donor controls from the general population. A meta-analysis with previous data was also conducted. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 590 women who underwent endoscopic surgery were enrolled in the study and a blood sample was collected. After DNA extraction, genotype was obtained using Taq-Man pre-designed assay. Genotype data from healthy blood donor women were obtained from an existing genotype bank. MAIN RESULTS AND THE ROLE OF CHANCE A statistically significant association with endometriosis was found for SNP rs10859871, close to the VEZT gene, compared with both general population [odds ratio (OR) = 1.43, 95% confidence interval (CI): 1.20-1.71, P = 6.9 × 10(-5)] and laparoscopic controls (OR = 1.58, 95% CI: 1.24-2.02, P = 2.1 × 10(-4)). Meta-analysis with previous data confirmed the rs10859871 SNP as that with the strongest evidence for association with endometriosis (OR = 1.19, 95% CI: 1.15-1.24, P = 7.9 × 10(-20)). A further meta-analysis conducted using data from Stage III-IV endometriosis resulted in stronger genome-wide significant effect sizes for four out of the five SNPs tested. LIMITATIONS, REASONS FOR CAUTION The inability to confirm all previous demonstrated associations considering all stages of endometriosis may be due to a lack of statistical power and differences in the definition of cases included. WIDER IMPLICATIONS OF THE FINDINGS The associations with the SNPs identified so far have been obtained with a relatively small sample size supporting a limited heterogeneity across the various datasets. This represents an important advance in the identification of genetic markers of this disease. STUDY FINDING/COMPETING INTERESTS No funding to declare. The authors have no competing financial interests in relation to the content of this research paper.

How to Manage Bowel Endometriosis: The ETIC Approach

A panel of experts in the field of endometriosis expressed their opinions on management options in a 35-year-old patient desiring pregnancy with a history of previous surgery for endometrioma and bowel obstruction symptoms. Many questions that this paradigmatic patient may pose to the clinician are addressed, and various clinical scenarios are discussed. A decision algorithm derived from this discussion is proposed as well.

High Prevalence of Vitamin D Deficiency in Infertile Women Referring for Assisted Reproduction

A comprehensive analysis of the vitamin D status of infertile women is the first step in understanding hypovitaminosis impact on reproductive potential. We sought to determine vitamin D profiles of women attending an infertility center and to investigate non-dietary determinants of vitamin D status in this population. In this cross-sectional analysis, a cohort of 1072 women (mean age ± standard deviation 36.3 ± 4.4 years) attending an academic infertility center was used to examine serum 25-hydroxy-vitamin D (25(OH)D) levels in relation to demographic characteristics, seasons and general health risk factors. Both unadjusted and adjusted levels of serum 25(OH)D were examined. Median 25(OH)D concentration was below 30 ng/mL for 89% of the entire year. Over the whole year, 6.5% of patients had 25(OH)D levels ≤10 ng/mL, 40.1% ≤20 ng/mL, and 77.4% ≤30 ng/mL. Global solar radiation was weakly correlated with 25(OH)D levels. At multivariable analysis, 25(OH)D levels were inversely associated with BMI; conversely, 25(OH)D levels were positively associated with height and endometriosis history. Serum 25(OH)D levels are highly deficient in women seeking medical help for couple’s infertility. Levels are significantly associated with body composition, seasonal modifications and causes of infertility. Importantly, this deficiency status may last during pregnancy with more severe consequences.

A Comprehensive Analysis of Body Mass Index Effect on in Vitro Fertilization Outcomes

The effect of a raised body mass index (BMI) on the outcome of assisted reproduction technology (ART) still represents a controversial issue. Even less clear is whether BMI acts with a potential detrimental effect on IVF outcomes via a deleterious effect on innate quality of oocytes or on the environmental milieu within the uterus. With the aim to better understand the mechanisms underlying the potential deleterious effect of an increased BMI on IVF outcomes, we have evaluated the effects of female BMI on number and quality of retrieved oocytes, fertilization rate, embryo score and incidences of ongoing pregnancy and live births among couples undergoing IVF in an Italian population. Data from 1602 women who underwent their first IVF cycle were retrospectively analyzed. A significantly reduced percentage of mature oocytes when comparing obese (BMI ≥ 30 kg/m2) and normal-weight patients (BMI = 18.50–24.99 kg/m2) was found. After adjusting for maternal age and other confounders, odds for ongoing pregnancy rate showed no differences across different BMI categories. However, a significant increased odds ratio (OR) could be observed for miscarriage rate in patients with BMI ≥ 25 (OR = 2.5; p = 0.04). These results should be taken into account in order to define optimal strategies for overweight and obese patients referring to ART procedures.