Ovarian biomarkers predict controlled ovarian stimulation for in vitro fertilisation treatment in Singapore.

INTRODUCTION: Ovarian biomarkers have been shown to predict responses to controlled ovarian hyperstimulation (COH) during in vitro fertilisation (IVF) in predominantly Caucasian populations, with limited studies performed in Southeast Asian women in Singapore. METHODS: We evaluated the performance of serum anti-Müllerian hormone (AMH), follicle-stimulating hormone and oestradiol levels, antral follicle count (AFC), body mass index, ovarian volume, and age to establish thresholds for the prediction of poor (< 4 oocytes retrieved) and excessive responses (> 19 oocytes retrieved) in 263 women undergoing COH. Univariate and multivariate logistic regression analysis and receiver operating characteristic curves were used to calculate probabilities for poor and excessive responders to COH. RESULTS: 36 (13.7%) and 50 (19.0%) women had poor and excessive response to COH, respectively. An AMH value of 0.69 ng/mL predicted poor ovarian response with positive likelihood ratio (LR) of 2.94, compared to an AFC of ≤ 5 when the positive LR is 2.36. Conversely, an AMH value of ≥ 3.06 ng/mL predicted excessive ovarian response with positive LR of 2.24, compared to an AFC cut-off of ≥ 12 with positive LR of 1.93. CONCLUSION: AMH levels and AFC are equivalent in the prediction of both poor and excessive ovarian response in women undergoing IVF. Our study highlights the importance of establishing population-specific cut-off biomarker values so that protocols can be tailored to optimise IVF treatment.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

Estimating cumulative live-birth rates after IVF treatment with Kaplan-Meier and competing risk methods.

OBJECTIVE(S): To explore the use of competing risk (CR) as compared to the commonly used Kaplan-Meier (KM) methodology in estimating the cumulative live-birth rate (CLBR) after IVF Treatment in a context of high dropout rates and informative censoring. STUDY DESIGN: We compare the KM and CR methodologies for estimating 2-year CLBR in a retrospective cohort of 2779 patients undergoing 5002 embryo transfers over a period of 9 years, from 2000 to 2008, at KKIVF Centre. RESULTS: We observed a total of 1105 LB (39.8%), and a dropout rate of 44.2% (1228 patients). The overall CLBR is lower with CR compared with KM method (39% vs 52%) after up to nine embryo-transfer cycles over a period of two years. The highest CLBR was achieved for ovulation disorders (57% vs 49%, KM vs CR) followed by male factors (54% vs 43%, KM vs CR), with poorer outcomes from patients with decreased ovarian reserve (37% vs 16%, KM vs CR) and endometriosis (36% vs 25%, KM vs CR). As dropouts in our cohort are generally older and more likely to have poorer ovarian reserves, the CR method, which accounted for these dropouts, is likely to give more meaningful estimation of IVF success rates. CONCLUSION(S): The CR method should be considered as a useful alternative in deriving CLBR for IVF treatment where dropout rates are high and when informative censoring is involved.

Dysregulated sphingolipid metabolism in endometriosis.

BACKGROUND: In endometriosis, the establishment and subsistence of ectopic lesions outside the endometrium suggest an altered cellular state for pathological hyperplasia. Sphingolipids are bioactive compounds, and their biosynthesis and metabolism modulate a range of cellular processes including proliferation, migration and apoptosis. We demonstrate that aberrations in sphingolipid metabolism occur in women with endometriosis. METHODS: Targeted mass spectrometry on >120 sphingolipids were measured in the sera (n = 62), peritoneal fluid (n = 63), and endometrial tissue (n = 14) of women with and without endometriosis. Quantitative RT-PCR and immunohistochemistry were performed on endometrial tissues determine the expression levels of sphingolipid enzymes. RESULTS: Sphingolipidomics identified the in vivo accumulation of numerous sphingolipids, including the functionally antagonistic glucosylceramides and ceramides in the serum and PF of women with endometriosis. We found upregulation of specific sphingolipid enzymes, namely sphingomyelin synthase 1 (SMS1), sphingomyelinase 3 (SMPD3), and glucosylceramide synthase (GCS) in the endometrium of endometriotic women with corresponding increased GlcCer, decreased sphingomyelin levels, and decreased apoptosis in the endometrium. CONCLUSIONS: Our sphingolipidomics approach provided evidence of altered sphingolipid metabolism flux in serum, peritoneal fluid, and endometrial tissue in women with endometriosis. The results provide new information on how sphingolipids and eutopic endometrium may contribute to the pathophysiology of endometriosis. The results also have implications for the use of sphingolipids as potential biomarkers.

Younger women with ovulation disorders and unexplained infertility predict a higher success rate in superovulation (SO) intrauterine insemination (IUI).

INTRODUCTION: Superovulation-intrauterine insemination (SO-IUI) is the most common assisted reproductive technique (ART) in the world, with good evidence of efficacy and cost-effectiveness. However, parameters affecting its success have not been consistently reported. So in this study, we aim at determining the parameters influencing the success rate of SO-IUI. MATERIALS AND METHODS: We conducted a retrospective cohort study of 797 SO-IUI cycles from 606 patients, performed between 2007 and 2009 in a single centre. These women received clomiphene citrate (CC), recombinant FSH (rFSH) or both. RESULTS: There were 127 clinical pregnancies with a pregnancy rate (PR) of 15.9% (127/797) per treatment cycle. Factors associated with higher PR included maternal age <38 (P = 0.02), subfertility diagnoses of ovulatory disorders, unexplained infertility, sexual dysfunction and unilateral tubal obstruction (P = 0.02), an endometrial thickness ≥8 mm (P = 0.03), total number motile spermatozoa (TNMS) of ≥1 million (P = 0.03), and spermatozoa normal forms (NF) ≥4% (P <0.01) on bivariate analysis. When CC is used, the endometrial thickness is more likely to be suboptimal (<8 mm). All the above parameters remained significant except the subfertility diagnoses on multivariate analysis. CONCLUSION: Patients' selection with women <38 years old and preferably with ovulation disorders and unexplained infertility is associated with the highest PR in SO-IUI. Cycle parameters such as the use of rFSH alone, with the avoidance of CC, TNMS ≥1 million and NF ≥4% is likely to result in the best outcomes and reduce the high order multiple pregnancy risk.

Academy of Medicine-Ministry of Health Clinical Practice Guidelines: assessment and management of infertility at primary healthcare level.

The Academy of Medicine (AMS) and Ministry of Health (MOH) have developed the clinical practice guidelines on Assessment and Management of Infertility at Primary Healthcare Level to provide doctors and patients in Singapore with evidence-based treatment for infertility. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the AMS-MOH clinical practice guidelines on Assessment and Management of Infertility at Primary Healthcare Level, for the information of SMJ readers. Chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website: http://www.moh.gov.sg/content/moh_web/healthprofessionalsportal/doctors/guidelines/cpg_medical/2013/cpgmed_infertility.html. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

Preimplantation genetic diagnosis of chromosome translocations by analysis of polymorphic short tandem repeats.

INTRODUCTION: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations. METHODS: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo. RESULTS: PGD cycles were performed on five couples where one spouse carried a balanced translocation. 27 embryos were analysed, of which 12 were normal/balanced, 12 were abnormal/unbalanced and three were indeterminate. Four PGD cycles proceeded to embryo transfer, of which two led to pregnancy. The first pregnancy showed a normal male karyotype, and a healthy baby was delivered at term. A second pregnancy unexpectedly miscarried in the second trimester from unknown causes. CONCLUSION: STR analysis is a simple and suitable alternative to FISH for detecting unbalanced chromosomal states in preimplantation embryos.

Attitudes toward fertility preservation in female cancer patients.

OBJECTIVE: To survey patient attitudes toward fertility preservation techniques in the case of infertility from cancer treatment. STUDY DESIGN: A cross-sectional survey on adolescent cancer patients (ACPs) aged 15-21 years and their parents, as well as on breast cancer patients (BCPs). RESULTS: A total of 69% of ACPs were aware of the problem of infertility. However, only 31% recalled being spoken to about treatment effects on fertility. Parents were significantly more likely to have been spoken to (58% vs. 31%, p < 0.022) and tended to be more interested in fertility preservation. The groups were in agreement that cancer therapy should not be delayed. Of the BCPs, 67% expressed substantial concern regarding infertility. They were more likely to have been spoken to and tended to be younger and nulliparous, although statistical significance was not reached. Again, most were unwilling to delay therapy. CONCLUSION: There is an interest infertility preservation amongst our patients, but the medical information received may influence, to a certain extent, the attitudes of the patients. Greater emphasis should therefore be placed on counseling the patient during the treatment planning process.

Simplified PGD of common determinants of haemoglobin Bart's hydrops fetalis syndrome using multiplex-microsatellite PCR.

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.

Microsurgical reversal of sterilisation - is this still clinically relevant today?

INTRODUCTION: Women with previous tubal sterilisation seeking fertility are faced with treatment options of reconstructive tubal surgery or in vitro fertilisation (IVF) techniques. The aim was to assess the current viability of tubal anastomosis in a local clinical practice. MATERIALS AND METHODS: A retrospective cohort review of all sterilisation reversal cases from January 1998 to January 2008. The main outcome measures included fi rst pregnancy success and live birth after surgery. Subsequent live births, ectopic pregnancies, miscarriages, duration of surgery and hospitalisation within the study period were also reported. We included cases aged less than 40 years, without any known semen abnormalities, and performed by only one operator. Cases with only unilateral reversal were excluded. RESULTS: Nineteen cases with previous Filshie clip ligation (9 laparoscopic/10 open) were reviewed. Cumulative pregnancy rates with surgery were 47.4% (<6 months), 57.9% (6 to 12 months), 68.4% (12 to 48 months) and 73.7% (>48 months). Pregnancy (77.8% vs 70.0%) and live birth rates (66.7% vs 60.0%) were similar between laparoscopy and open surgery. The mean interval to pregnancy was marginally lower via laparoscopy (11.3 vs 13.6 months). Hospitalisation stay was significantly halved (1.43 vs 3.00 days) but ectopic pregnancies were increased 3-fold (3 vs 1) with laparoscopy. Compared with IVF, the estimated average cost per delivery for laparoscopic reversal was reduced for laparoscopic reversal with no multiple pregnancies. CONCLUSION: Our results favour surgical reversal after sterilisation for patients younger than 40 years old. It avoids hyperstimulation risks and the economic burdens associated with multiple pregnancies. Where expertise is available, laparoscopic reversal should be performed.