D.S. Berger

Leptin suppresses anti-Mullerian hormone gene expression through the JAK2/STAT3 pathway in luteinized granulosa cells of women undergoing IVF

STUDY QUESTION Do the adipocytokines, leptin and adiponectin affect the granulosa cell expression of anti-Mullerian hormone (AMH) and its receptor (AMHR-II)? SUMMARY ANSWER Leptin suppresses AMH mRNA levels in human luteinized granulosa cells through the JAK2/STAT3 pathway, while adiponectin has no such effect. WHAT IS KNOWN ALREADY AMH is one of the most reliable markers of ovarian reserve. Serum AMH levels decline with obesity. Obesity is associated with elevated leptin and reduced adiponectin levels. STUDY DESIGN, SIZE AND DURATION This prospective study included 60 infertile women undergoing fresh IVF and ICSI cycles utilizing autologous oocytes at Montefiores Institute for Reproductive Medicine and Health between July 2010 and April 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from small (SFs; <14 mm) and large follicles (LFs; ≥14 mm) from 38 participants. Total RNA was extracted separately from mural and cumulus granulosa cells and mRNA levels were measured by RT-PCR. In an additional group of participants (N = 22), primary cumulus and mural granulosa cells (pooled SFs and LFs) were cultured in media alone or with addition of either leptin (N = 7), adiponectin (N = 8) or JAK2/STAT3 inhibitor + leptin (N = 7), and AMH and AMHR-II mRNA levels measured. Levels of AMH, leptin and adiponectin protein were measured in follicular fluid. MAIN RESULTS AND THE ROLE OF CHANCE AMH and AMHR-II mRNA and follicular fluid AMH protein levels were inversely correlated with age. AMH mRNA expression was six times higher in cumulus compared with mural granulosa cells in SFs (P< 0.05) and eight times higher in cumulus compared with mural granulosa cells in LFs (P < 0.001). In follicular fluid, leptin protein level positively correlated (r = 0.7, P = 0.03), while adiponectin protein level inversely correlated (r = -0.46, P = 0.02) with BMI. Leptin treatment suppressed AMH and AMHR-II mRNA in both cumulus and mural granulosa cells (all P < 0.05). In the presence of JAK2/STAT3 inhibitor, leptin treatment did not alter AMH but continued to suppress AMHR-II mRNA in cumulus cells (P = 0.02). Adiponectin treatment did not alter AMH or AMHR-II mRNA levels. LIMITATIONS, REASONS FOR CAUTION This study included a luteinized granulosa cell model as these cells were collected from women who were hyperstimulated with gonadotrophins. The results obtained may not fully extrapolate to non-luteinized granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS Leptin may program abnormal AMH signaling, thereby resulting in ovarian dysfunction. This study opens a new perspective for understanding the low ovarian reserve seen in obese women and provides new insights into potential mechanisms that explain the lower AMH seen in obese women. Whether our findings explain the worse response to ovulation induction observed in obese women needs to be further elucidated.

Comprehensive analysis of HLA-G: implications for recurrent spontaneous abortion.

Miscarriage is one of the most common pregnancy complications. Recurrent spontaneous abortion is defined as 2 or more pregnancy losses and may be associated with genetic variation. Human leukocyte antigen-G (HLA-G) is a ligand for natural killer (NK) cell receptors and has the ability to block NK cell activity, which if not blocked can potentially harm a fetus. Consequently a deletion or mutation of the HLA-G gene could lead to miscarriage. Our cases (n = 238) include Caucasian women experiencing 2 or more spontaneous abortions, and controls (n = 233) include women with at least 1 live birth and no history of SA. We sequenced approximately 1400 base pairs (bp) of the HLA-G promoter region, genotyped the 14 bp exon 8 insertion/deletion and single nucleotide polymorphism (SNP) in the coding region of HLA-G. Promoter haplotypes were constructed from sequence information. Twenty-three SNPs were observed in the promoter region with minor allele frequency >0.02. Twelve SNPs differed significantly in frequency between cases and controls. Two haplotypes incorporating these 12 SNPs accounted for 90% of haplotypes and differed significantly in frequency between the 2 populations. Cases were more likely to carry haplotype 2 (P = .0078) and controls to have haplotype 6 (P = .0004). Cases also had a higher frequency of individuals homozygous for the 14 bp insertion. Among the 12 alleles carried on haplotype 2, 5 are predicted to disrupt transcription factor binding sites. The HLA-G promoter is highly associated with the risk of spontaneous abortion, but high linkage disequilibrium in the promoter prevents assignment of the causal variant.

Implications of blood type for ovarian reserve

BACKGROUND We explored the relevance of blood type to ovarian reserve, as reflected by early follicular phase FSH levels. METHODS For this cross-sectional observational study, early follicular phase serum levels of FSH (mIU/ml) and estradiol (E2, pg/ml), and information on blood type (A, B, AB and O) and patient age were procured for female patients, ≤ 45 years age (n= 544), who were undergoing fertility evaluation at one of two tertiary care facilities. Serum FSH > 10 mIU/ml was taken to reflect diminished ovarian reserve (DOR). Data distribution for FSH and age was analyzed and non-parametric tests used for comparisons across blood groups. Multivariable logistic regression analyses determined the relationship between elevated FSH and respective blood types after adjusting for age and study site. RESULTS Prevalence of blood types according to order of frequency was: O (45%), A (35%), B (16%) and AB (5%). After adjusting for age and study site, patients with blood type O were twice as likely to exhibit FSH > 10 mIU/ml compared with those with A or AB blood types [odds ratio (OR) 2.36; 95% confidence interval (CI) 1.27-4.41; P= 0.007], and three times as likely to manifest FSH > 12 m IU/ml (OR 3.48, 95% CI 1.46-7.32, P= 0.004). The B blood group antigen failed to exhibit any relationship with ovarian reserve as reflected by baseline FSH (P> 0.05). CONCLUSIONS The A blood group antigen appears to be protective for ovarian reserve, whereas blood type O appears to be associated with DOR, in a relationship that is independent of advancing age. Further studies are needed to establish causality and identify the underlying mechanisms for the association.

Male adiposity impairs clinical pregnancy rate by in vitro fertilization without affecting day 3 embryo quality.

Male adiposity is detrimental for achieving clinical pregnancy rate (CPR) following assisted reproductive technologies (ART). The hypothesis that the association of male adiposity with decreased success following ART is mediated by worse embryo quality was tested.

Reproductive Aging is Associated With Altered Gene Expression in Human Luteinized Granulosa Cells

Declining reproductive success with aging is attributable to qualitative and quantitative deterioration in oocytes, which are nurtured by granulosa cells (GCs). This prospective study assesses whether reproductive aging is accompanied by differential gene expression in luteinized GCs from in vitro fertilization (IVF) patients. Women with nonovarian infertility etiologies were categorized as younger (≤30, n = 3) or older (≥40, n = 3). During oocyte retrieval, mural GCs were isolated; messenger RNA (mRNA) was extracted and transcribed for complementary DNA (cDNA) microarray analysis. Differential gene expression was confirmed by real-time polymerase chain reaction (PCR). Analysis revealed 120 genes were differentially expressed. Three genes were upregulated and 117 were downregulated (including interleukin [IL]-1β, IL-1R2, and IL-6R) in GCs of older versus younger patients. Our data provide evidence of downregulation in IL-1 and IL-6 gene families in luteinized GCs with advancing age. Given previously recognized roles for the IL gene family in folliculogenesis and ovulation, our findings may partly explain ovulatory and luteal dysfunctions associated with reproductive aging.

Serum and follicular fluid monocyte chemotactic protein-1 levels are elevated in obese women and are associated with poorer clinical pregnancy rate after in vitro fertilization: a pilot study

OBJECTIVE To determine whether monocyte chemotactic protein-1 (MCP-1), a proinflammatory chemokine important in ovulation, is abnormally elevated in obese women undergoing IVF and whether serum and follicular fluid (FF) levels of MCP-1 are associated with IVF outcome. DESIGN Prospective pilot study. SETTING Academic center. PATIENT(S) Women undergoing IVF. INTERVENTION(S) Serum and FF were collected from women undergoing IVF. MAIN OUTCOME MEASURE(S) Correlation between MCP-1 and other inflammatory markers with adiposity and pregnancy outcome after IVF. RESULT(S) Obese women had significantly higher serum and FF MCP-1 levels compared with overweight and normal weight women. Serum MCP-1, granulocyte colony stimulating factor, catalase, and C-reactive protein (CRP) were positively correlated with body mass index (BMI). After adjusting for age and baseline FSH, these correlations remained significant for serum MCP-1, granulocyte colony stimulating factor, and CRP. In the FF, only MCP-1 was positively correlated with BMI. Women who became pregnant had significantly lower serum MCP-1 and CRP levels compared with those who did not become pregnant; this difference was more pronounced among women with diminished ovarian reserve. Receiver operating characteristic curve demonstrated that serum MCP-1 levels >373.0 pg/mL in all women and >362.6pg/mL in women with diminished ovarian reserve predicted failure to achieve a clinical pregnancy. CONCLUSION(S) Elevations in serum and FF MCP-1 levels are positively correlated with adiposity and negatively correlated with pregnancy rates (PRs) in women undergoing IVF.