Conservative management of stage IIB ovarian carcinoma with favorable oncology and fertility outcomes.

•Fertility treatment prior to definitive cancer therapy in stage IIB EOC.•Both fertility and oncologic outcomes were successful.•The role of Multidisciplinary team is critical.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation.

The establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Reanalysis of human blastocysts with different molecular genetic screening platforms reveals significant discordance in ploidy status.

OBJECTIVE: The objective of this study is to determine mosaicism and its effect on blastocysts; abnormal blastocysts determined by molecular testing were sequentially biopsied and retested. MATERIAL AND METHOD: We re-biopsied 37 blastocyst-stage abnormal embryos from eight patients, which were reanalyzed to determine the level of concordance between biopsies and inter-laboratory congruence between reputable commercial PGS laboratories. RESULTS: The main outcome measures were intra-embryo variation between sequential embryo biopsies and inter-laboratory variation between two PGS laboratories. The compatibility between both aCGH and NGS was found to be 11 % (3/27). Importantly, 9/27 (33 %) of embryos originally reported to be aneuploid, upon repeat assessment, were found to be euploid. The concurrence for SNP array and NGS was 50 % (3/6), and 17 % (1/6) of these abnormal embryos tested normal upon re-evaluation with NGS. NGS resulted 41 % (11/27) normal results when 27 of CGH abnormal embryos were retested. Concordance between aCGH and NGS was 4 % (1/27) whereas in three instances, gender discrepancy was observed with NGS when aCGH abnormal embryos were reanalyzed. CONCLUSIONS: The results of these studies reinforce the prevalence of inconsistencies during PGS evaluation of trophectoderm biopsies possibly due to variations in platform sensitivity and heightening concerns over the clinical tractability of such technology in human ARTs..

Vitrification of human embryos subjected to blastomere biopsy for pre-implantation genetic screening produces higher survival and pregnancy rates than slow freezing.

PURPOSE: Cryopreservation of blastocysts, especially those subjected to the trauma due to blastomere biopsy for the purposes of pre-implantation genetic screening (PGS), requires significant optimization. Laboratory and clinical outcomes were compared to determine the effect of two different cryopreservation techniques on the development of human pre-implantation embryos that underwent blastomere biopsy and blastocoel drainage prior to cryopreservation. DESIGN: Retrospective clinical study. PATIENT(S): Women who requested cryotransfer of supernumerary blastocysts were analyzed by FISH. RESULTS: The main outcome measures were post-thaw survival (SR), pregnancy (PR), and implantation (IR). The SR of slowly frozen blastocysts was 83% compared to 97% for vitrified blastocysts. In 160 cases where biopsied embryos were cryotransferred, the results for slowly frozen versus vitrified blastocysts were: SR (71% vs. 95%), PR (23% vs. 37%), and IR (26% vs. 36%, P < 0.05), respectively. CONCLUSION: The results revealed that vitrified blastocysts provided higher SR, PR and IR as compared to slowly frozen counterparts.

Genetic analysis of human embryos by metaphase comparative genomic hybridization (mCGH) improves efficiency of IVF by increasing embryo implantation rate and reducing multiple pregnancies and spontaneous miscarriages.

OBJECTIVE: To assess the benefit of selecting blastocysts for cryotransfer based upon prior comparative genomic hybridization (CGH) karyotyping of blastomeres derived from their cleaved embryos of origin. Implantation and birth rates per transfer of previtrified CGH-tested blastocysts were compared with those following the transfer of nonCGH-tested fresh and warmed embryos. DESIGN: In vitro studies. SETTING: Private infertility clinic. PATIENT(S): Women undergoing infertility treatment. INTERVENTION(S): Three groups of women with similar clinical and demographic characteristics were compared. Group A underwent transfer of warmed blastocysts derived from CGH-normal day 3 embryos. Group B underwent embryo transfer of warmed blastocysts derived from nonkaryotyped vitrified embryos. Group C underwent fresh transfers with non-CGH-tested blastocysts. MAIN OUTCOME MEASURE(S): Implantation and birth rates per embryo after the cryotransfer of CGH-tested blastocysts. RESULT(S): The birth rate per transferred blastocyst in group A was 48%, versus 15% for group B and 19% for group C. The birth rate per embryo transfer was 60% for group A, and 33% for group B and 36% for group C. The miscarriage rate was 4% in group A, 8% in group B, and 12% in group C. CONCLUSION(S): The transfer of previously vitrified blastocysts derived from CGH-normal embryos significantly improves implantation and birth rates per embryo transferred and reduces the miscarriage rate. Vitrification does not compromise this enhancement.

Increasing adiposity in normal ovulatory women affects adipocytokine expression in subcutaneous and visceral abdominal fat.

OBJECTIVE: To determine which adipocytokines are differentially expressed as a function of body mass index (BMI), to compare expression of adipocytokines in abdominal subcutaneous and omental fat, and to correlate these findings with serum levels, BMI, and parameters of insulin resistance. METHODS: Serum and subcutaneous (sc) and omental (om) tissue were obtained from lean and obese ovulatory women undergoing gynecologic surgery. We determined adipocytokine expression in sc versus om abdominal fat and related this to increasing BMI. RESULTS: Serum leptin was higher and adiponectin lower in overweight subjects. Adipocytokines had higher expression in sc abdominal versus om adipose tissue with the most significant difference observed for leptin (P=0.01). As BMI increased, sc leptin expression increased and adiponectin expression decreased. The leptin/adiponectin ratio correlated significantly with BMI (R=0.84, P=0.0001). CONCLUSION: Increased adipocytokine expression correlates with BMI. Abdominal sc tissue has greater adipocytokine expression overall. The serum leptin/adiponectin ratio is highly correlated with BMI. These data may help in our understanding of how obesity affects women in a variety of ways.

Evidence for a Müllerian-inhibiting substance autocrine/paracrine system in adult human endometrium.

OBJECTIVE: To determine if adult human endometrium possesses an intact müllerian-inhibiting substance (MIS) signal transduction system and, if so, whether MIS can modulate endometrial cell viability. DESIGN: Adult human endometrial tissue was subjected to reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. In addition, cultured human endometrial stromal cells were treated with recombinant MIS or transiently transfected with MIS and/or MIS type II receptor (MISRII) expression plasmids to assess for effects upon endometrial cell viability and apoptosis. The MIS in conditioned media was quantified by specific ELISA. SETTING: Academically affiliated medical center. PATIENT(S): Reproductive-age women undergoing routine infertility evaluation. INTERVENTION(S): Endometrial sampling. MAIN OUTCOME MEASURE(S): Assessment of MIS gene transcription and protein translation in human endometrial tissue in vivo and in vitro. RESULT(S): 1) The RT-PCR revealed that adult human endometrium expresses the genes for MIS, MISRII, ALK3, and Smads 1 and 9. 2) Immunohistochemistry reveals that MIS and MISRII protein are expressed in human endometrium. 3) Immunocytochemistry of cultured human endometrial cells reveals that MIS and MISRII protein are primarily restricted to mitosing cells. 4) ELISA reveals that MIS is secreted by human endometrial cells in vitro and that this process is increased by sex steroids. 5) Increasing local MIS concentration in cultured human endometrial stromal cells either by exogenous administration or transient transfection significantly decreases cell viability and increases apoptosis. CONCLUSION(S): Adult human endometrium possesses a functional MIS signal transduction system which negatively regulates cellular viability.

Dietary curcumin significantly improves obesity-associated inflammation and diabetes in mouse models of diabesity.

Obesity is a major risk factor for the development of type 2 diabetes, and both conditions are now recognized to possess significant inflammatory components underlying their pathophysiologies. We tested the hypothesis that the plant polyphenolic compound curcumin, which is known to exert potent antiinflammatory and antioxidant effects, would ameliorate diabetes and inflammation in murine models of insulin-resistant obesity. We found that dietary curcumin admixture ameliorated diabetes in high-fat diet-induced obese and leptin-deficient ob/ob male C57BL/6J mice as determined by glucose and insulin tolerance testing and hemoglobin A1c percentages. Curcumin treatment also significantly reduced macrophage infiltration of white adipose tissue, increased adipose tissue adiponectin production, and decreased hepatic nuclear factor-kappaB activity, hepatomegaly, and markers of hepatic inflammation. We therefore conclude that orally ingested curcumin reverses many of the inflammatory and metabolic derangements associated with obesity and improves glycemic control in mouse models of type 2 diabetes. This or related compounds warrant further investigation as novel adjunctive therapies for type 2 diabetes in man.

Subcutaneous and omental fat expression of adiponectin and leptin in women with polycystic ovary syndrome.

OBJECTIVE: To assess message expression of adiponectin and leptin in visceral and SC fat in women with polycystic ovary syndrome (PCOS) and in control women. DESIGN: Prospective clinical trial. SETTING: Academic medical centers in Mexico City, Mexico and New York, New York. PATIENT(S): Women with PCOS and control women. INTERVENTION(S): Surgical biopsies of visceral (omental) and subcutaneous (SC) adipose tissue, fasting blood samples, and ultrasound measurements of visceral and SC fat. MAIN OUTCOME MEASURE(S): Messenger RNA assessment of adiponectin and leptin in adipose tissue samples; serum measurements of adiponectin, leptin, glucose, insulin, and hormone levels; measurements of fat quantity by ultrasound. Correlative analyses as well as comparisons between women with PCOS and control women were performed. RESULT(S): Confirming previous data, women with PCOS had more insulin resistance, similar serum leptin, but lower serum adiponectin compared with control women. When control women were divided into quartiles by body mass index (BMI), messenger RNA expression of leptin and adiponectin decreased with increasing BMI. Adiponectin and leptin expression was significantly lower in women with PCOS; in weight-matched patients and control women, leptin and adiponectin expression was statistically significantly lower in SC tissue, and adiponectin expression was statistically significantly lower in omental tissue in women with PCOS. In control women, there was greater expression in SC tissue compared with in visceral tissue. There were significant negative correlations between visceral and SC fat mass by both ultrasound as well as adiponectin and leptin expression in women with PCOS. Serum adiponectin correlated statistically significantly with visceral adiponectin expression (r = 0.64) in women with PCOS, and there was a statistically significant correlation between SC adiponectin expression and the Quantitative Insulin-Sensitivity Check Index as a marker of insulin resistance (r = 0.43). CONCLUSION(S): Adipocytokine expression in fat tissue appears to be down-regulated by an increased fat mass; this is particularly evident in the case of adiponectin expression in women with PCOS. It is probable that insulin resistance is a factor that may contribute, in part, to these findings.

Dietary-induced obesity and hypothalamic infertility in female DBA/2J mice.

The effects of diet and adiposity have been implicated in disturbances of female reproductive function. In an effort to better elucidate the relationship between obesity and female fertility, we analyzed the effect of increasing dietary fat content on body composition, insulin sensitivity, and pregnancy rates in two common inbred mouse strains, DBA/2J and C57BL/6J. After 16 wk, females of both strains on the high fat diet developed glucose intolerance and insulin resistance, but only the female DBA/2J mice developed dietary-induced obesity and hyperleptinemia. The high fat diet was associated with more than a 60% decrease in natural pregnancy rates of female DBA/2J mice, whereas the fertility of female C57BL/6J mice was unaffected. Despite developing a similar degree of obesity, insulin resistance, and hyperleptinemia, male DBA/2J mice did not manifest diminished fertility. Obese female DBA/2J mice achieved normal ovulatory responses and pregnancy rates after exogenous gonadotropin stimulation, suggesting their fertility defect to be central in origin. Real-time PCR quantification of hypothalamic cDNA revealed a 100% up-regulation of neuropeptide Y and a 50% suppression of GnRH expression accompanied by a 95% attenuation of leptin receptor type B expression in obese female DBA/2J mice. These findings suggest that obesity-associated hyperleptinemia, and not insulin resistance or increased dietary fat per se, gradually induces central leptin resistance, increases hypothalamic neuropeptide Y-ergic tone, and ultimately causes hypothalamic hypogonadism. The data establish high fat-fed female DBA/2J mice as a wild-type murine model of obesity-related infertility.

Follistatin-related protein and follistatin differentially neutralize endogenous vs. exogenous activin.

Follistatin-related protein (FSRP) is a new addition to the expanding follistatin (FS)-related gene family whose members contain at least one conserved 10-cysteine follistatin domain. In contrast to other members of this family, FSRP and follistatin also share a common exon/intron domain structure, substantial primary sequence homology, and an ability to irreversibly bind activin. In this study, we further explored the hypothesis that FSRP is a functional as well as structural homologue of FS. N-terminal sequencing of recombinant FSRP revealed that signal peptide cleavage occurs within exon 1, a significant structural difference from FS, in which cleavage occurs at the exon/intron boundary. Solid-phase radioligand competition assays revealed both FS and FSRP to preferentially bind activin with the next closest TGF-beta superfamily member, bone-morphogenic protein-7, being at least 500-fold less potent. Consistent with their similar activin-binding affinities, FSRP and FS both prevented exogenous (endocrine or paracrine) activin from accessing its receptor and inducing gene transcription in bioassays. However, FS was at least 100-fold more potent than FSRP in inhibiting gene transcription and FSH release mediated by endogenously produced (autocrine) activin-A or activin-B in multiple cell systems. Finally, FSRP lacks the heparin-binding sequence found in FS, and we found that it was also unable to bind cell surface heparin sulfated proteoglycans. These findings suggest that structural differences between FSRP and FS may underlie their different neutralizating capabilities with respect to exogenous vs. endogenous activin. Taken together with our previous studies showing that activin binding is essential for FS's biological activity, the differential activities of FSRP and FS further indicate that activin binding is necessary but not sufficient to account for all of FS's actions.

A comparative analysis of 57 serous borderline tumors with and without a noninvasive micropapillary component.

The literature concerning serous borderline tumors with a noninvasive micropapillary component suggests an association with invasive implants. We compared the clinicopathologic features of micropapillary serous borderline tumors (MSBTs) with typical SBTs to determine the following: 1) the importance of focal micropapillary architecture in an otherwise typical SBT, 2) the behavior of low-stage MSBTs, 3) whether high-stage MSBTs are inherently more aggressive than high-stage SBTs, and 4) whether invasive implants are prevalent in an MSBT cohort without referral selection bias. The 57 borderline tumors studied were diagnosed at a university hospital between 1981 and 1998; they included 14 MSBTs, 35 SBTs, and 8 SBTs with focal micropapillary features. None of the specimens were referrals for expert pathologic consultation, thus distinguishing our study group from most of those previously reported. Neither MSBTs nor SBTs were associated with invasive implants at diagnosis (0 of 14 and 0 of 43, respectively). They also did not differ with respect to overall stage at diagnosis, but MSBTs were more frequently bilateral than SBTs (71% versus 23%, p = 0.001). There was an increased risk of recurrence in MSBT versus SBT (3 of 14 versus 1 of 43, p = 0.035), but this was stage related; there was no difference between groups when evaluating recurrence in stage I disease (0 of 8 versus 0 of 27). There was no difference in recurrence or stage at diagnosis between SBTs with focal micropapillary features and other SBTs. There was 100% survival in all groups. We conclude that high-stage MSBTs with noninvasive implants should be considered a subtype of SBTs with an increased risk of recurrence. Stage I MSBTs demonstrate clinical features that are similar to low-stage SBTs. Focal micropapillary architecture (<5 mm) has no bearing on outcome. MSBTs in the general population are not strongly associated with invasive implants.