Pre-implantation genetic testing for Marfan syndrome using mini-sequencing.

Marfan syndrome (MFS1) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. This study aimed to perform clinical PGT-M in a family with a history of MFS1 for two generations. A family with two members affected by MFS1 approached the hospital for PGT-M. The couple decided to join the project following extensive counselling and informed consent was obtained. The mutation contributory to MFS1 was identified using whole-exome sequencing (WES). A novel PGT-M protocol using multiplex fluorescent PCR and mini-sequencing was developed and tested. Ten blastocysts were subjected to PGT-M in two clinical PGT cycles. Mini-sequencing revealed four normal and six affected embryos. Microsatellite-based linkage analysis confirmed mutation analysis results in all samples. The embryos diagnosed as normal (non-MFS1) were chosen for transfer. A pregnancy was obtained in the third embryo transfer. Invasive prenatal diagnosis confirmed the normal genotype of the baby. This study demonstrated comprehensive management using the application of clinical-based diagnosis, WES for mutation identification within the MFS1 gene, mini-sequencing for embryo selection and microsatellite-based linkage analysis for backup of PGT-M results and contamination detection to assist couples in having a healthy child when there was a family history of Marfan syndrome.Impact StatementWhat is already known on this subject? Marfan syndrome (MFS1, OMIM#154700) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. Pre-implantation genetic testing (PGT) is an alternative to traditional invasive prenatal diagnosis (PND) giving the couples the chance of starting pregnancy with the confidence that the baby will be unaffected. Most of the previous PGT reports employed microsatellite-based linkage analysis. A few PGT studies used sequencing, mini-sequencing and mutation analysis; however, the details of the techniques were not described.What do the results of this study add? Single-cell PCR protocol using multiplex fluorescent PCR and mini-sequencing was developed and validated. Two clinical PGTs cycles for Marfan syndrome were performed. A healthy baby was resulted. The details of multiplex fluorescent PCR and mini-sequencing protocols are described in this study so that the procedures can be reproduced.What are the implications of these findings for clinical practice and/or further research? Embryo selection can help the family suffering from Marfan syndrome for two generations to start a pregnancy with confidence that their child will be unaffected. This study also shows the use of a mini-sequencing protocol for PGT, which can be a universal protocol for other mutations by changing the PCR primers and mini-sequencing primers.

Early prenatal detection of anterior uterine sacculation resulting from previous cesarean sections.

Anterior uterine sacculation was diagnosed at 15 weeks of gestation in a woman with two previous cesarean sections, based on hourglass appearance of two distinct uterine segments, namely the empty upper segment and the large thinned wall lower segment containing a fetus with posteriorly attached placenta. The pregnancy developed through the bulging weakened anterior wall instead of growing toward the upper segment. Urgent hysterectomy was performed. The operative and pathological findings confirmed the prenatal ultrasound findings. This is the first report of prenatal diagnosis of sacculation due to cesarean section, which prevented the catastrophic event of uterine rupture.

Intrauterine Adhesions: Causes and Treatment Outcomes among Thai Women

Objective: To explore the causes and treatment outcomes of intrauterine adhesions among Thai women. Material and Method: This retrospective cross-sectional study recorded and analyzed the demographic data, causes, and treatment outcomes of 75 patients who underwent hysteroscopic adhesiolysis between January 2004 and June 2015 at Maharaj Nakhon Chiang Mai Hospital, Chiang Mai, Thailand. Results: The most common etiology of intrauterine adhesions was a history of early pregnancy termination by curettage (68%). Sixty-nine percent of the patients had menstrual abnormalities. Twenty-two percent of the patients had severe adhesions. Tuberculous endometritis caused the most severe case. Sharp hysteroscopic adhesiolysis improved the condition in 77.7% of the patients. The outcomes of the patients with mild to moderate IUA were better than those with severe intrauterine adhesions (88.2% vs. 43.8%, respectively) (p = 0.001). Fifty-one patients planned to conceive, and 49% succeeded after hysteroscopic surgery. Conclusion: Menstrual abnormalities were the most common presentation of intrauterine adhesions. The most common cause of intrauterine adhesions was early pregnancy termination by curettage. However, tuberculosis endometritis caused the most severe case. Hysteroscopic adhesiolysis significantly improves menstrual function and fertility outcomes; mild to moderate cases have a better prognosis.

Recurrence of Endometrioma Following Conservative Ovarian Endometrioma Cystectomy: Laparoscopy versus Laparotomy.

OBJECTIVE: To investigate the recurrence rate and disease-free interval between laparoscopy versus laparotomy for the conservative surgery of endometrioma. MATERIAL AND METHOD: A retrospective cohort study was conducted. The medical records of reproductive women who underwent conservative ovarian cystectomy surgery (laparoscopy or laparotomy) for endometrioma at Thammasat University Hospital were retrieved. The patients were followed through 24 months to evaluate the recurrence of endometrioma. Propensity scoring was used to adjust for confounding by indication and confounding by contraindication. Model for competing time to event was used in analysis. RESULTS: One hundred and twenty-eight and 114 patients were enrolled in laparoscopy and laparotomy groups, respectively. Mean age and body weight in laparotomy group were statistically higher than those in the other group were. Mean height and body mass index were, however not statistically different in either groups. In addition, the stage of disease and bilaterality in both groups were comparable. Diameter ofendometrioma in laparotomy group was significantly larger than that in laparoscopy group (7.0 ± 2.5 vs. 6.2 ± 1.8 cm, respectively; p = 0.004). After adjusting for propensity scoring, the endometrioma recurrence rate was significantly higher in laparoscopy group as compared to laparotomy group (27.3% vs. 14.9%, respectively; p = 0.02). However, the cumulative rate of pregnancy after surgery was not statistically different (4.7% vs. 4.4%, respectively; p = 1.0). CONCLUSION: The present study has demonstrated that the surgical technique has a strong impact on the recurrence or disease-free interval. Laparoscopy might not eradicate the disease pathology as effectively as open laparotomy in some situations, such as in cases with complexity of disease.

Impact of hemostasis methods, electrocoagulation versus suture, in laparoscopic endometriotic cystectomy on the ovarian reserve: a randomized controlled trial.

OBJECTIVE: To evaluate the impact on ovarian reserve between two different methods ofhemostasis after laparoscopic ovarian endometrioma excision. MATERIAL AND METHOD: A randomized controlled study was conducted from January to December 2013 in Thammasat University Hospital, Thailand. Reproductive women, age 18-45years who underwent laparoscopic ovarian cystectomy were randomized in electrocoagulation and suture groups. Clinical baseline data and ovarian reserve outcome (anti-Mullerian hormone (AMH)) were evaluated. RESULTS: Fifty participants were recruited and randomized in two groups. Electrocoagulation and suture groups consisted of 25 participants. Baseline characteristics between 2 groups (age, weight, BMI, height, cyst diameter, duration and estimated blood loss) were not statistically different. There were no significant difference of AMIH between electrocoagulation and suture group atpre-operative (2.90±2.26 vs. 2.52±2.37 ng/ml), 1 week (1.78±1.51 vs. 1.99±1.71 ng/ml), 1 month (1.76±1.50 vs. 2.09±1.62 ng/ml), 3 months (2.09±1.66 vs. 1.96±1.68 ng/ml) and 6 months (2.11±1.84 vs 1.72±1.68 ng/ml), respectively. However mean AMH ofboth groups significantly decreased since the first week of operation. Effect oflaparoscopic ovarian surgery had significantly declined and sustained AMH level until 6 months. CONCLUSION: Laparoscopic cystectomy of ovarian endometrioma has negative impact to ovarian reserve. Either electroco- agulation or suture method had no different effects.

Factors affecting pregnancy rates after microsurgical reversal of tubal sterilization.

The aim of this study was to determine the prognostic factors and pregnancy rates after microsurgical reversal of tubal sterilization. Patients undergoing tubal anastomosis from 2001 to 2008 were included. Relevant data were extracted from their medical records. Pregnancy outcomes were ascertained by responses to mailed questionnaires and telephone contact. A total of 98 patients were identified. We found that the mean duration of follow-up was 67 ± 28 months. Fifty-five patients conceived (pregnancy rate 62.5%; 95% confidence interval [CI] 52 to 72.8%). Of these, 50 were intrauterine and 5 were tubal pregnancies. Life-table analysis estimated cumulative pregnancy rates to be 30.7%, 39.8%, 49%, and 53.7% at 6, 12, 18, and 24 months after reversal, respectively. Age at the time of reversal was the only significant prognostic factor multivariate model. We concluded that age of the patient at the operation is the most important prognostic factor.

Metabolic syndrome and insulin resistance in Thai women with polycystic ovary syndrome.

OBJECTIVE: To determine the prevalence of metabolic syndrome and insulin resistance in Thai women with polycystic ovary syndrome (PCOS). MATERIAL AND METHOD: Oral glucose tolerance tests were performed in 70 PCOS women. Blood was taken for the measurement of fasting insulin, 2-hr postprandial insulin, lipid profiles, testosterone and sex hormone binding globulin RESULTS: The prevalence of metabolic syndrome and insulin resistance was 24.3% and 30.7%, respectively. Diabetes mellitus and impaired glucose tolerance was detected in 11.4% and 31.4%, respectively Homeostatic model assessment (HOMA) was the most sensitive screening test for insulin resistance. PCOS women with metabolic syndrome had significantly higher body mass index, free testosterone index and insulin levels than those without the syndrome. CONCLUSION: There was a high prevalence of metabolic syndrome, insulin resistance, diabetes mellitus and impaired glucose tolerance in Thai PCOS women. A combination of fasting glucose, a 2-hr glucose tolerance test and fasting insulin level effectively identified PCOS women who were at high risk of metabolic syndrome.

Closed-system solid surface vitrification versus slow programmable freezing of mouse 2-cell embryos.

PURPOSE: To compare closed-system solid surface vitrification with slow freezing. METHODS: Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. RESULTS: Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). CONCLUSIONS: Closed-system vitrification was more effective than conventional slow freezing.

Gender differences in infertility-related stress and the relationship between stress and social support in Thai infertile couples.

OBJECTIVE: To study infertility-related stress among men and women and to examine its relationship with the level of perceived social support. MATERIAL AND METHOD: The Fertility Problem Inventory (FPI) and the Personal Resource Questionnaire (PRQ) were translated into Thai and used to assess the level of infertility-related stress and perceived social support, respectively, in 238 infertile subjects. RESULTS: The global FPI scores for men and women were 154.2 +/- 18.3 and 154.7 +/- 22.6, respectively (p > 0.05). There was no significant difference in their perceived social support (PRQ scores = 137.8 + 14.0 and 134.0 +/- 16. 7 respectively). A significant negative correlation (r = -0.1894; p < 0.001) existed between global stress and social support in women, but not in men. CONCLUSION: Thai infertile couples experienced a high level of stress. Unlike previous studies from Western countries, there was no gender diference in infertility-related stress.

Antimullerian hormone changes after laparoscopic ovarian cystectomy for endometrioma compared with the nonovarian conditions.

Laparoscopic ovarian cystectomy is recommended for surgical procedure of endometrioma. The negative impact on ovarian reserve following removal had been documented. Little evidence had been reported for nonovarian originated effects. Objective. To evaluate the impact of laparoscopic ovarian cystectomy for endometrioma on ovarian reserve, measured by serum antimullerian hormone (AMH), compared to nonovarian pelvic surgery. Materials and Methods. A prospective study was conducted. Women who underwent laparoscopic ovarian cystectomy (LOC) and laparoscopic nonovarian pelvic surgery (NOS) were recruited and followed up through 6 months. Clinical baseline data and AMH were evaluated. Results. 39 and 38 participants were enrolled in LOC and NOS groups, respectively. Baseline characteristics (age, weight, BMI, and height) and preoperative AMH level between 2 groups were not statistically different. After surgery, AMH of both groups decreased since the first week, at 1 month and at 3 months. However, as compared to the LOC group at 6 months after operation, the mean AMH of the NOS group had regained its value with a highly significant difference. Conclusion. This study demonstrated the negative impact of nonovarian or indirect effects of laparoscopic surgery to ovarian reserve. The possible mechanisms are necessary for more investigations.

Cervical pregnancy with massive bleeding after treatment with methotrexate.

This report describes a catastrophic bleeding following methotrexate treatment of cervical pregnancy and dramatic response to Bakri surgical obstetric silicone (SOS) balloon tamponade in controlling massive bleeding. A 42-year-old woman was diagnosed for cervical pregnancy with a viable fetus at 12 weeks by transvaginal ultrasound. Conservative treatment with intrafetal potassium chloride injection and systemic methotrexate were instituted. On the sixth day of therapy, catastrophic bleeding lead to hypovolemic shock. After resuscitation and blood transfusion, we attempt to control the bleeding with evacuation and curettage but was unsuccessful. Bakri SOS balloon tamponade was applied with immediate and effective response. Nevertheless, total abdominal hysterectomy was performed. In conclusion, conservative treatment of cervical pregnancy with systemic methotrexate could be catastrophic in some patients. Control of active bleeding with Bakri SOS balloon tamponade may possibly be helpful in case of uncontrolled bleeding in selected cases.

Slow programmable and ultra-rapid freezing of human embryos.

AIM: To compare the outcomes of slow freezing with ultra-rapid freezing (URF) of cleavage-stage human embryos on aluminum foil. METHODS: Two-cell mouse embryos were used to test our method of ultra-rapid freezing. The embryos were randomly allocated to a non-frozen control (208 embryos), and slow (204 embryos) or ultra-rapid freezing groups (204 embryos). Immediate survival rate, further cleavage and blastocyst formation were compared. After validating our ultra-rapid freezing method on mouse embryos, we applied a similar ultra-rapid freezing protocol to human embryos. Consecutive human frozen/thawed embryo transfer (FET) cycles from October 1998 to June 2005 were reviewed. The survival rate, further cleavage rate and the pregnancy outcomes were compared between the URF and slow programmable freezing. RESULTS: Mouse embryos in the URF group survived the freezing/thawing process better than those in the slow freezing group (93.1% vs 82.8%, P = 0.001). Blastocyst and hatching blastocyst formation of the surviving embryos were comparable in the URF and slow freezing group (59% vs 58.6%, P = 0.944 and 32.6% vs 42%, P = 0.066, respectively). There were 146 human FET cycles in the URF group and 28 cycles in the slow freezing group. The immediate survival of embryos was higher in the URF group than in the slow freezing group (87.9% and 64.3%, P < 0.001). There was no significant difference in the mean number of embryos per transfer (3.7 +/- 1.3 and 3.3 +/- 1.2, P = 0.178), clinical pregnancy rate per transfer (28.5% and 21.4%, P = 0.444) and implantation rate per embryo (10.98% and 10.9%, P = 0.974) in the URF or slow freezing groups. CONCLUSION: Our in-house URF method gave comparable results to slow programmable freezing. Although the risk of potential contamination is a major drawback of the present ultra-rapid freezing technique, future refinement will minimize or entirely eliminate this concern.

Clinical, endocrine and ultrasonographic features of polycystic ovary syndrome in Thai women.

AIM: To study the prevalence, reproductive hormone profiles and ovarian sonographic appearance of Thai women with polycystic ovary syndrome (PCOS). METHODS: One thousand and ninety-five women were screened for oligomenorrhea/amenorrhea, and the clinical symptoms of hyperandrogenism. Ovarian morphology and volume were assessed by ultrasonography in diagnosed cases. Blood was taken for the measurement of the follicle stimulating hormone, luteinizing hormone, prolactin, testosterone, androstenedione, dehydroepiandrosterone and 17-hydroxyprogesterone. RESULTS: The prevalence of PCOS was 5.7%. The mean age of women with PCOS was less than that of non-PCOS cases (27.4 +/- 6.5 and 31.1 +/- 6.4 years, respectively; P < 0.0001). Abnormal uterine bleeding and infertility were the leading presenting symptoms. The mean ovarian volume in women with PCO appearance was 9.22 +/- 4.36 mL compared to 6.53 +/- 3.31 mL in those without this appearance (P = 0.04). Hyperandrogenemia was confirmed in 23 of the 62 cases (37.1%). CONCLUSIONS: The prevalence and clinical presentations of Thai women with PCOS were similar to those in other reports. However, hirsutism, elevated testosterone level and acanthosis nigricans were uncommon in our population. Serum androstenedione was a more sensitive indicator of hyperandrogenemia than total testosterone. Further research is needed to clarify whether there is an ethnic difference in endocrine profiles and risks of metabolic syndrome.