Discovery and validation of peritoneal endometriosis biomarkers in peritoneal fluid and serum.

RESEARCH QUESTION: What are the potential biomarkers for peritoneal endometriosis in peritoneal fluid and serum? DESIGN: Case-control studies composed of independent discovery and validation sets were conducted. In the discovery set, untargeted liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics, multivariable and univariable analyses were conducted to generate global metabolomic profiles of peritoneal fluid for endometriosis and to identify potential metabolites that could distinguish peritoneal endometriosis (n = 10) from controls (n = 31). The identified metabolites from the discovery set were validated in independent peritoneal fluid (n =19 peritoneal endometriosis and n = 20 controls) and serum samples (n = 16 peritoneal endometriosis and n = 19 controls) using targeted metabolomics. The area under the receiver-operating characteristics curve (AUC) analysis was used to evaluate the diagnostic performance of peritoneal endometriosis metabolites. RESULTS: In the discovery set, peritoneal fluid phosphatidylcholine (34:3) and phenylalanyl-isoleucine were significantly increased in peritoneal endometriosis groups compared with control groups, with AUC 0.77 (95% CI 0.61 to 0.92; P = 0.018) and AUC 0.98 (95% CI 0.95 to 1.02; P < 0.001), respectively. In the validation set, phenylalanyl-isoleucine retained discriminatory performance to distinguish peritoneal endometriosis from controls in both peritoneal fluid (AUC 0.77, 95% CI 0.61 to 0.92; P = 0.006) and serum samples (AUC 0.81, 95% CI 0.64 to 0.99; P = 0.004), with notably stronger discrimination between peritoneal endometriosis and controls in proliferative phase. CONCLUSION: Our preliminary results propose phenylalanyl-isoleucine as a potential biomarker of peritoneal endometriosis, which may be used as a minimally invasive diagnostic biomarker of peritoneal endometriosis.

Fecundability in reproductive aged women at risk of sexual dysfunction and associated risk factors: a prospective preconception cohort study.

BACKGROUND: Female sexual dysfunction (FSD) is a prevalent problem, affecting up to 41% of reproductive aged women worldwide. However, the association between female sexual function (FSF) and fecundability in women attempting to conceive remains unclear. We aimed 1) to examine the association between FSF in reproductive-aged preconception Asian women and fecundability, as measured by time-to-pregnancy in menstrual cycles, and 2) to examine lifestyle and behavioral factors associated with FSF. METHODS: From the Singapore PREconception Study of long-Term maternal and child Outcomes (S-PRESTO) prospective cohort, we evaluated FSF using the 6-item Female Sexual Function Index (FSFI-6) and ascertained time-to-pregnancy within a year of baseline assessment. We estimated fecundability ratio (FR) and 95% confidence interval (CI) using the discrete-time proportional hazards model, accounting for left-truncation and right censoring. We used multivariable logistic and linear regression models to identify potential factors related to FSF. RESULTS: Among 513 participants, 58.9% had low FSF as defined by a total FSFI-6 score at or below the median value of 22. Compared to women with high FSF, those with low FSF had a 27% reduction in fecundability (FR 0.73; 95% CI 0.54, 0.99), with adjustment for age, ethnicity, education, parity and body mass index. Overall, the FRs generally reduced with decreasing FSFI-6 scores. Physical activity, obesity, absence of probable depression and anxiety were independently associated with reduced odds of low FSF and increased FSFI-6 scores, after adjusting for sociodemographic characteristics. CONCLUSIONS: Low FSF is associated with a longer time-to-pregnancy. Early evaluation and optimization of FSF through increased physical activity and optimal mental health may help to improve female fecundity. The finding of obese women having improved FSF remains uncertain which warrants further investigations on plausibly mechanisms. In general, the current finding highlights the importance of addressing FSF in preconception care service for general women, which is currently lacking as part of the fertility promotion effort in the country.

Peritoneal Fluid Cytokines Reveal New Insights of Endometriosis Subphenotypes.

Endometriosis is a common inflammatory gynecological disorder which causes pelvic scarring, pain, and infertility, characterized by the implantation of endometrial-like lesions outside the uterus. The peritoneum, ovaries, and deep soft tissues are the commonly involved sites, and endometriotic lesions can be classified into three subphenotypes: superficial peritoneal endometriosis (PE), ovarian endometrioma (OE), and deep infiltrating endometriosis (DIE). In 132 women diagnosed laparoscopically with and without endometriosis (n = 73, 59 respectively), and stratified into PE, OE, and DIE, peritoneal fluids (PF) were characterized for 48 cytokines by using multiplex immunoassays. Partial-least-squares-regression analysis revealed distinct subphenotype cytokine signatures-a six-cytokine signature distinguishing PE from OE, a seven-cytokine signature distinguishing OE from DIE, and a six-cytokine-signature distinguishing PE from DIE-each associated with different patterns of biological processes, signaling events, and immunology. These signatures describe endometriosis better than disease stages (p < 0.0001). Pathway analysis revealed the association of ERK1 and 2, AKT, MAPK, and STAT4 linked to angiogenesis, cell proliferation, migration, and inflammation in the subphenotypes. These data shed new insights on the pathophysiology of endometriosis subphenotypes, with the potential to exploit the cytokine signatures to stratify endometriosis patients for targeted therapies and biomarker discovery.

GnRH agonist trigger and ovarian hyperstimulation syndrome: relook at 'freeze-all strategy'.

A case is reported of early onset ovarian hyperstimulation syndrome (OHSS) after gonadotrophin-releasing hormone agonist (GnRHa) trigger for final oocyte maturation in a GnRH antagonist protocol. The use of GnRHa in place of HCG as a trigger for final oocyte maturation in an antagonist IVF cycle has been proposed as a method for preventing OHSS in predicted high-responders. This approach, however, did not prevent the occurrence of OHSS in our case despite a freeze-all strategy. To the best of our knowledge, this is a possible index case of severe OHSS with GnRHa trigger for oocyte maturation without any luteal HCG rescue for a high responder, despite IVF cycle segmentation.

Peritoneal autoantibody profiling identifies p53 as an autoantibody target in endometriosis.

OBJECTIVE: To map the peritoneal autoantibody (AAb) landscape in women with endometriosis. DESIGN: Case-control laboratory study. SETTING: Academic medical and research units. PATIENT(S): Women who presented with or without endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using native-conformation and citrullinated modified protein arrays, proteome-wide analysis of AAbs against 1,623 proteins were profiled in peritoneal fluids (PFs) of 25 women with endometriosis and 25 women without endometriosis. RESULT(S): In women with endometriosis, the median number of AAbs detected was 4, including AAbs that targeted autoantigens involved in implantation, B-cell activation/development, and aberrant migration and mitogenicity. Forty-six percent of women with endometriosis have ≥5 peritoneal AAbs. Conversely, in women without endometriosis, the median number of detected AAbs was 1. Autoantibodies recognizing tumor suppressor protein p53 were the most commonly detected AAbs, being present in 35% of women with endometriosis, and p53 AAb was associated with a monocyte/macrophage-like PF cytokine signature. Further investigation of the global reactivity of AAbs against citrullinated PF antigens by peptidylarginine deiminase enzymes 1, 2, and 6 revealed anticitrullinated p53 as the only AAb target elevated and citrullinated by all 3 peptidylarginine deiminase isotypes. Furthermore, unsupervised hierarchical clustering and integrative pathway analysis revealed that 60% of women with endometriosis-associated infertility were positive for AAbs, which are involved in platelet-derived growth factor, transforming growth factor-ß, RAC1/PAK1/p38/MMP2 signaling, LAT2/NTAL/LAB-mediated calcium mobilization, and integrin-mediated cell adhesion. CONCLUSION(S): Together, our data identify peritoneal autoimmunity in a significant subset of women with endometriosis, with implications on infertility and disease pathophysiology. In these patients, p53 was identified as the most frequent PF AAb target, which was present in both the native and citrullinated forms.

Outcomes and cost analysis of single-embryo transfer versus double-embryo transfer.

BACKGROUND: Studies had compared single-embryo transfer to double-embryo transfer with cleavage stage embryos and found that while single-embryo transfer was less costly, it was also associated with a lower live birth rate than double-embryo transfer. A single blastocyst transfer has been shown to improve the live birth rate per cycle compared to single-embryo transfer at cleavage stage. OBJECTIVES: To compare live birth rates and real costs of elective single-embryo transfer to double-embryo transfer and to determine the incremental cost-effectiveness ratio of these two strategies in an unselected pool of women in a single center. DESIGN: Retrospective study. METHODS: We analyzed data of 4232 women who underwent their first fresh in vitro fertilization/intra-cytoplasmic sperm injection cycles with at least two embryos available for transfer in KK Women's and Children's Hospital from 2010 to 2017. RESULTS: Five hundred and sixty-four women underwent elective single-embryo transfer and 3668 women underwent double-embryo transfer. One hundred and fifty-six women who failed to achieve a live birth in their fresh elective single-embryo transfer cycle underwent a sequential thaw single-embryo transfer cycle. Live birth rate of fresh elective single-embryo transfer was significantly higher at 41.3% than that of double-embryo transfer at 32.6%. Cumulative live birth rate for sequential elective single-embryo transfer (fresh elective single-embryo transfer + thaw single-embryo transfer) was 47.9%. After accounting for variables which may affect live birth rates such as age and stage of embryo transfer, the odds of achieving a live birth from double-embryo transfer was 24% lower than that from sequential single-embryo transfer, although not statistically significant. For every live birth gained from an elective single-embryo transfer compared to double-embryo transfer, cost savings were S$20,172 per woman. If a woman had to have a sequential single-embryo transfer after a failed single-embryo transfer in her fresh cycle, cost savings were reduced to S$1476 per woman. CONCLUSION: Single-embryo transfer is a dominant strategy in an unselected population and adopting it in assisted reproductive treatments (ART) can produce cost savings without compromising on live birth rates.

Laparoscopic Tubal Re-anastomosis or In Vitro Fertilisation in Previously Ligated Patients: A Comparison of Fertility Outcomes and Survey of Patient Attitudes

INTRODUCTION: We aim to compare live birth rates, cost analysis and a survey of patient attitudes between laparosopic tubal re-anastomosis and in vitro fertilisation (IVF). MATERIALS AND METHODS: A retrospective study was done in a single reproductive medicine and IVF unit in Singapore from January 2011 to December 2016. Previously ligated patients underwent either laparoscopic tubal re-anastomosis or IVF. The primary outcome was first live birth after treatment. Interval to first pregnancy, miscarriage and ectopic pregnancies were also reported. Patients attending the subfertility clinic completed a questionnaire on IVF and tubal re-anastomosis on preferred choice of treatment, before and after reading an information sheet. RESULTS: Twelve patients underwent tubal re-anastomosis while 31 patients underwent IVF treatment. Pregnancy (75.0% vs 35.5%) and live birth (58.3% vs 25.8%) were significantly higher in the tubal surgery group (P <0.05%) after transferring all available embryos in one stimulated IVF cycle. Cost per live birth was lower in the tubal surgery group (SGD27,109 vs SGD52,438). One hundred patients participated in the survey. A majority of patients preferred tubal surgery to IVF (68.2% vs 31.8%) before given information on the procedures, but indicated a preference for IVF (54.6%) to surgery (45.4%) after receiving information on the procedures. CONCLUSIONS: For women less than 40 years of age, desiring fertility after tubal ligation, laparoscopic tubal re-anastomosis offers better live birth rates and cost-effectiveness. Patients in Singapore are equivocal as to their preference after education regarding the choices. Thus, laparoscopic tubal re-anastomosis remains a viable alternative to IVF treatment.

Elevated peritoneal fluid ceramides in human endometriosis-associated infertility and their effects on mouse oocyte maturation.

OBJECTIVE: To characterize the peritoneal fluid (PF) sphingolipid profile in endometriosis-associated infertility (EAI), and to assess the plausible functional role(s) of ceramides in oocyte maturation potential. DESIGN: Retrospective case-control study and in vitro mouse oocyte study. SETTING: University-affiliated hospital and university laboratory. SUBJECTS: Twenty-seven infertile patients diagnosed with endometriosis and 20 infertile patients who did not have endometriosis; BALB/c female mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): PF sphingolipid concentrations. Number of metaphase II (MII) mouse oocytes. RESULT(S): Liquid chromatography-tandem mass spectrometry revealed 11 significantly elevated PF sphingolipids in infertile women with severe endometriosis compared with infertile women without endometriosis (change >50%, false discovery rate ≤10%). Logistic regression analysis identified three very-long-chain ceramides potentially associated with EAI. Functional studies revealed that very-long-chain ceramides may compromise or induce murine MII oocyte maturation. The oocyte maturation effects induced by the very long-chain ceramides were triggered by alterations in mitochondrial superoxide production in a concentration-dependent manner. Scavenging of mitochondrial superoxide reversed the maturation effects of C24:0 ceramide. CONCLUSION(S): EAI is associated with accumulation of PF very-long-chain ceramides. Mouse studies demonstrated how ceramides affect MII oocyte maturation, mediating through mitochondrial superoxide. These results provide an opportunity for direct functional readout of pathophysiology in EAI, and future therapies targeted at this sphingolipid metabolism may be harnessed for improved oocyte maturation.