Clinical utility of combined preimplantation genetic testing methods in couples at risk of passing on beta thalassemia/hemoglobin E disease: A retrospective review from a single center.

Thalassemia and hemoglobinopathy is a group of hereditary blood disorder with diverse clinical manifestation inherited by autosomal recessive manner. The Beta thalassemia/Hemoglobin E disease (HbE/ßthal) causes a variable degree of hemolysis and the most severe form of HbE/ßthal disease develop a lifelong transfusion-dependent anemia. Preimplantation genetic testing (PGT) is an established procedure of embryo genetic analysis to avoid the risk of passing on this particular condition from the carrier parents to their offspring. Preimplantation genetic testing for chromosomal aneuploidy (PGT-A) also facilitates the selection of embryos without chromosomal aberration resulting in the successful embryo implantation rate. Herein, we study the clinical outcome of using combined PGT-M and PGT-A in couples at risk of passing on HbE/ßthal disease. The study was performed from January 2016 to December 2017. PGT-M was developed using short tandem repeat linkage analysis around the beta globin gene cluster and direct mutation testing using primer extension-based mini-sequencing. Thereafter, we recruited 15 couples at risk of passing on HbE/ßthal disease who underwent a combined total of 22 IVF cycles. PGT was performed in 106 embryos with a 3.89% allele drop-out rate. Using combined PGT-M and PGT-A methods, 80% of women obtained satisfactory genetic testing results and were able to undergo embryo transfer within the first two cycles. The successful implantation rate was 64.29%. PGT accuracy was evaluated by prenatal and postnatal genetic confirmation and 100% had a genetic status consistent with PGT results. The overall clinical outcome of successful live birth for couples at risk of producing offspring with HbE/ßthal was 53.33%. Conclusively, combined PGT-M and PGT-A is a useful technology to prevent HbE/ßthal disease in the offspring of recessive carriers.

Luteinizing Hormone Receptor Gene and Regulator of G-protein Signaling 2 Gene Expression Level and Association with Oocyte Maturity in In vitro Fertilization/Intracytoplasmic Sperm Injection Cycle.

AIMS: The aim is to study the relation and distribution in gene expression level of the luteinizing hormone receptor (LHR) gene and regulator of G-protein signaling 2 (RGS2) gene expression with oocyte maturation. SETTING AND DESIGN: This cross-sectional study was undertaken in an instruction-based tertiary care infertility unit, department of obstetrics and gynecology. MATERIALS AND METHODS: After controlled ovarian hyperstimulation, cumulus granulosa cells (CCs) from 59 oocytes among 18 women being treated by in vitro fertilization/intracytoplasmic sperm injection cycle technique from November 2015 to January 2016 were collected on the day of oocyte retrieval. Total RNA was extracted and converted to cDNA in individual oocytes. LHR and RGS2 gene levels were measured and analyzed using digital droplet polymerase chain reaction. STATISTICAL ANALYSIS: Gene expression level was analyzed using software STATA, version 14.0 (College Station, TX: StataCorp LP, USA). RESULTS: CCs were obtained from 59 cumulus-oocyte complexes (COC), 46 COC from metaphase II (CCMII), 13 COC from metaphase I, and GV oocyte (CCMI + GV). The RGS2 gene expression level, when compared with the housekeeping gene in CCMII and CCMI + GV, was 0.15 (0.05-0.52) and 0.08 (0.02-0.27), respectively. The LHR gene expression when compared with the housekeeping gene in CCMII and CCMI + GV did not differ and was quite in the same value that was 0.02 (0.00-0.11) and 0.02 (0.00-0.06), respectively. CONCLUSION: This study showed that LHR gene expression did not differ in between oocyte groups. Even though the median of RGS2 gene expression was more in the mature oocyte group, the result was inconclusive due to scattering and overlapping of gene expression data between oocyte groups.

First successful trial of preimplantation genetic diagnosis for pantothenate kinase-associated neurodegeneration.

PURPOSE: We aim to present a case of a healthy infant born after intracytoplasmic sperm injection-in vitro fertilization (ICSI-IVF) with a preimplantation genetic diagnosis (PGD) for pantothenate kinase-associated neurodegeneration (PKAN) due to PANK2 mutation. METHODS: ICSI-IVF was performed on a Thai couple, 34-year-old female and 33-year-old male, with a family history of PKAN in their first child. Following fertilization, each of the embryos were biopsied in the cleavage stage and subsequently processed for whole-genome amplification. Genetic status of the embryos was diagnosed by linkage analysis and direct mutation testing using primer extension-based mini-sequencing. Comprehensive chromosomal aneuploidy screening was performed using a next-generation sequencing-based strategy. RESULTS: Only a single cycle of ICSI-IVF was processed. There were seven embryos from this couple-two were likely affected, three were likely carriers, one was likely unaffected, and one failed in target genome amplification. Aneuploidy screening was performed before making a decision on embryo transfer, and only one unaffected embryo passed the screening. That embryo was transferred in a frozen thawed cycle, and the pregnancy was successful. The diagnosis was confirmed by amniocentesis, which presented with a result consistent with PGD. At 38 weeks of gestational age, a healthy male baby was born. Postnatal genetic confirmation was also consistent with PGD and the prenatal results. At the age of 24 months, the baby presented with normal growth and development lacking any neurological symptoms. CONCLUSIONS: We report the first successful trial of PGD for PKAN in a developing country using linkage analysis and mini-sequencing in cleavage stage embryos.

Effect of estradiol valerate on endometrium thickness during clomiphene citrate-stimulated ovulation.

AIM: The aim of this study was to examine the effects of estradiol valerate (EV) on the thickness of clomiphene citrate (CC)-stimulated endometrium. MATERIAL AND METHODS: Thirty-four normal ovulatory women were randomized double-blindly into two groups to receive CC 100 mg/day on day 2-6 of the treatment cycle, and either vitamin B (placebo) or EV 6 mg/day on day 10-14 of the cycle. The endometrial thickness, endometrial pattern, numbers of mature follicles, and maximal diameters of preovulatory follicles were evaluated by transvaginal sonographic examination. RESULTS: Thirty women completed both treatment cycles. Two other participants dropped out during the treatment due to side-effects (headache). The average endometrial thickness of the group treated with CC + placebo became slightly thinner when compared to the thickness at the baseline (9.04 vs 9.52 mm; P = 0.24). The CC + placebo and the CC + EV resulted in similar endometrial pattern, ovulation day, numbers of mature follicles, and sizes of the leading follicles before ovulation. However, an addition of EV into the CC cycle significantly increased the average endometrial thickness (10.7 mm vs 9.04 mm; P < 0.001). CONCLUSIONS: We concluded that the addition of 6 mg/day EV following the CC treatment can prevent the endometrial thinning without perturbing folliculogenesis and ovulation.

A comparison of the effects of clomiphene citrate and the aromatase inhibitor letrozole on superovulation in Asian women with normal ovulatory cycles.

OBJECTIVE: To compare the effect of the aromatase inhibitor letrozole and clomiphene citrate (CC) on superovulation in women with normal ovulation. METHODS: A cross-over randomized study of 22 women with normal ovulation, divided randomly into two equal cohorts, was carried out. Each group of 11 women was randomly allocated to take letrozole or CC for one cycle. After washing out for one cycle, the alternative drug was administered in the subsequent cycle. The number and size of mature follicles, endometrial thickness, and estradiol and progesterone levels were monitored. RESULTS: The number of mature follicles and estradiol levels on ovulation day were significantly lower in the letrozole group than the CC group (p < 0.05 for both). However, no differences between the two groups in endometrial thickness and pattern were observed. Progesterone levels showed ovulation in all cycles. CONCLUSIONS: The administration of 50 mg CC on days 3-5 was superior to 2.5 mg letrozole for superovulation induction in women with normal ovulation.

Prevalence and clinical predictors of endometrial hyperplasiain anovulatory women presenting with amenorrhea.

The aim of the present study was to determine the prevalence and clinical predictors of endometrial hyperplasia (EH) in amenorrheic women with anovulation. Fifty-seven women were enrolled in the study. Of these, 43 were diagnosed to have polycystic ovary syndrome (PCOS) and 14 to have idiopathic anovulation. All women received transvaginal sonography to assess endometrial thickness (ET), patterns and abnormalities. At the same time, an endometrial biopsy was taken using a Pipelle instrument. The women's age, body mass index (BMI) and waist-to-hip ratio (WHR) were 32.0+/-6.0 years, 27.3+/-6.5 kg/m(2) and 0.82+/-0.06 (mean+/-standard deviation), respectively. Twenty (35.1%) and 19 (33.3%) women were classified as obese by BMI and WHR, respectively. Hypertension was found in 17 (29.8%) women. The prevalence of EH was 45.6%. Most cases were simple EH, and only one (1.75%) was simple EH with atypia. EH prevalence was 48.8% and 35.7% in PCOS and idiopathic anovulatory women, respectively. Age, BMI, WHR and ET did not predict EH, whereas the endometrial hyperechogenic pattern was a clinical predictor of EH with borderline significance. In conclusion, this study demonstrated that almost half of the anovulatory women with amenorrhea had EH and no significant predictor was found. In view of these findings, an endometrial biopsy should be performed in all women with this disorder.

Increase accuracy of visual estimation of blood loss from education programme.

OBJECTIVE: To study the increase accuracy of visual estimation of blood loss after an education program. MATERIAL AND METHOD: Seven simulated scenarios with known measured amount of blood were created by using expired packed red cell from blood bank and common surgical materials. Ninety nurses were randomized into two groups. The experimental group attended blood loss estimation course while the control group did not. The percentage of errors in blood loss estimation were calculated and compared between both groups. The main outcome of this study was percentage of nurses who had accurate estimation. We assumed that if the estimated blood volume is within twenty percentage of actual volume it is accurate. RESULTS: There were no difference in age group (p = 0.08), clinical experiences (p = 0.95) and type of work (p = 0.47) between both groups. Educational program significantly increase accuracy in blood loss estimation (p < 0.05) in all seven scenarios. CONCLUSION: Educational program increased the accuracy of visual estimation of blood loss.

Comparison of outcomes and direct cost between minimal stimulation and conventional protocols on ovarian stimulation in in vitro fertilization.

AIM: To determine whether minimal stimulation with clomiphene and gonadotropin provides outcomes and direct costs comparable with those of a conventional GnRHa-gonadotropin stimulation protocol for infertile patients undergoing in vitro fertilization. METHODS: A non-randomized clinical trial was conducted from 1 July 1996 to 31 March 2003 at the Infertility and Assisted Reproductive Unit, Ramathibodi Hospital, Faculty of Medicine, Mahidol University, Thailand. A total of 192 patients were recruited of whom 96 cases underwent ovarian stimulated cycles with minimal stimulation protocol, and 96 controls underwent ovarian stimulated cycles with GnRHa-gonadotropin protocol, with cases and controls matched for age and infertility cause. RESULTS: The median patient age was 35 years. Endometriosis was the most frequent infertility cause (28.1%). The conventional GnRHa-gonadotropin protocol could give more oocyte numbers than the minimal stimulation protocol (7.3 +/- 4.9 vs 4.5 +/- 3.3 oocytes). The fertilization rate and cleavage rate were similar (73.4 +/- 31.9 and 84.9 +/- 32.6 in minimal stimulation protocol, 69.3 +/- 29.6 and 88.4 +/- 28.0 in GnRHa-gonadotropin protocol, respectively). The pregnancy rate per oocyte retrieval cycle in the GnRHa-gonadotropin protocol was similar to the minimal stimulation protocol. (13.1%vs 13.0%, P = 1.000). However, the cost per pregnancy of minimal stimulation protocol was less than that of GnRHa-gonadotropin protocol. (6021.95 US dollars for minimal stimulation protocol per pregnancy, 10,785.65 US dollars for GnRHa-gonadotropin protocol per pregnancy, P < 0.000). CONCLUSION: Minimal stimulation was less effective than conventional GnRHa-gonadotropin on the ovarian stimulation. However, the total costs of minimal stimulation were cheaper than the conventional GnRHa-gonadotropin protocol. The decreased costs of minimal stimulation justifies further evaluation of its role in the treatment of infertility in selected cases.