Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis.

BACKGROUND: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/ß-HSDs and 5α/ß-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17ß-HSD type 2 (HSD17B2). Also, 3α-HSD and 3ß-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5ß-reductase (AKR1D1) are responsible for the formation of 5α- and 5ß-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/ß-HSD activities of AKR1C1, AKR1C2 and AKR1C3.

The outcome of singleton pregnancies after IVF/ICSI in women before and after hysteroscopic resection of a uterine septum compared to normal controls.

OBJECTIVE(S): To evaluate the effect of hysteroscopic resection of a large uterine septum (Class V according to the American Fertility Society (AFS) classification) and of a small partial uterine septum (Class VI according to AFS classification or arcuate uterus) on the abortion rate in pregnancies after IVF and ICSI. STUDY DESIGN: The retrospective matched control study included 31 women who conceived following IVF or ICSI before hysteroscopic resection of a large (12 women) or small partial (19 women) uterine septum and 106 women who conceived following IVF or ICSI after hysteroscopic resection of a large (49 women) or small partial (57 women) uterine septum. For each pregnancy in the study group, we found two consecutive pregnant control women from the IVF/ICSI registry who had a normal uterus and were matched for age, BMI, stimulation protocol and the use of IVF or ICSI and for various infertility causes. The abortion/pregnancy rate was the main outcome measure. Data on the septum length were obtained during hysteroscopic resection by comparing the length of the 1.4 cm long yellow tip of the electric knife to the length of the resected septum. RESULTS: The abortion rate before hysteroscopic metroplasty was significantly higher, both in women with a small partial septum (78.9% before resection vs. 23.7% in the normal controls, OR 12.08) and a large septum (83.3% before resection vs. 16.7% in normal controls, OR 25.00) compared to women with a normal uterus. After the surgery, the abortion rate was comparable to the abortion rate in women with normal uterus: in both women with a small partial and women with a larger septum. CONCLUSION(S): Similar to a large uterine septum, a small partial uterine septum is an important and hysteroscopically preventable risk factor for spontaneous abortion in pregnancies after IVF and ICSI.

Small uterine septum is an important risk variable for preterm birth.

OBJECTIVE(S): To evaluate whether a uterus with a small septum (arcuate uterus or class VI according to the American Fertility Society (AFS) classification) behaves similarly to a uterus with a larger septum (septate or subseptate uterus or AFS class V). STUDY DESIGN: Observational study included 826 singleton deliveries to 730 women with a history of hysteroscopic resection of the uterine septum. Data on deliveries were obtained from the National Perinatal Registry of Slovenia (NPIS). Multiple gestations were excluded. We analysed and compared perinatal outcomes before and after hysteroscopic resection in two groups of women: in women with a small uterine septum (Group A) and in those with a larger uterine septum (Group B). Data on the septum length were obtained during hysteroscopic resection by comparing the length of the 1.4-cm long yellow tip of the electric knife to the length of the resected septum. A small uterine septum was defined as having a length of 1.3-1.5 cm. RESULTS: The preterm birth rate in Group A (n=420) was 33.9% before and 7.2% after hysteroscopic resection (P<0.001); the preterm birth rate in Group B (n=406) was 36.5% before and 8.0% after hysteroscopic resection (P<0.001). The very preterm birth rate in Group A was 12.5% before and 3.1% after hysteroscopic resection (P<0.001); the very preterm birth rate in Group B was 15.0% before and 2.9% after hysteroscopic resection (P<0.001). After surgery, we registered a decreased need for neonatal intensive care, as well as a significant decrease in stillbirth and neonatal death rates in both groups of patients. CONCLUSION(S): Similarly to a large uterine septum, a small uterine septum or arcuate uterus is an important hysteroscopically preventable risk variable for preterm birth.

Pretreatment evaluation of adnexal tumors predicting ovarian cancer.

Kobal B, Rakar S, Ribic-Pucelj M, Tomazevie T, Zaletel-Kragelj L. Pretreatment evaluation of adnexal tumors predicting ovarian cancer. The objective of this study was to determine the ability of tumor marker assessment, gray-scale transvaginal with color Doppler ultrasonography to predict ovarian malignancy. One hundred thirty-four subjects with ovarian masses who entered the study prospectively underwent pelvic examination, tumor marker assessment and gray-scale transvaginal with color flow Doppler ultrasonography preoperatively. Malignancy predictors were statistically evaluated with stepwise multiple logistic regression, and the scores from the model were transformed to probability for having a malignant disease. The presence of neovascularization, intracystic papillary projections, elevated serum CA 125, and age over 45 years were significant predictors for malignancy. Positive predictive value (PPV) for the regression model was 89.0%, and negative predictive value (NPV) was 96.8%. Probability for malignancy ranged from 0.004 to 0.991 depending on which covariates were included. Logistic regression analysis of pretreatment diagnostic gray-scale and color Doppler ultrasonographic characteristics, together with CA 125 enabled a creation of probability assessment scale for individual estimation of ovarian mass, which may contribute to final clinical decision.

Safe and effective fluid management by automated gravitation during hysteroscopy.

OBJECTIVE: The automated gravitational Vario Flow system with weighing-based electronic fluid deficit indicator was used in order to reduce the risk of fluid intravasation during continuous flow hysteroscopic procedures. Early experiences are reported. METHODS: Between August 1996 and July 1997, the Vario Flow with fluid deficit indicator and alarm system was used in 203 hysteroscopic operations. Between January 1994 and August 1996 the Vario Flow without fluid deficit indicator was used in 240 hysteroscopic operations. In all, there were 443 hysteroscopic operations: 301 metroplasties, 20 endometrial ablations, 10 cases of lysis of synechiae, 58 myomectomies and 54 polypectomies. The data on fluid deficit before and after the introduction of the electronic fluid deficit indicator were similar. RESULTS: Fluid deficit indicator was proved highly efficient in 203 operations. It provided the information on fluid deficit at any moment during hysteroscopic operations. Besides intrauterine pressure, the actual fluid deficit has become one of the leading parameters during our continuous flow hysteroscopic procedures. CONCLUSIONS: We therefore conclude that by using an automated gravitational system with fluid deficit indicator and alarm system, the safety for patients during hysteroscopic procedures has been increased.

Microsurgery and in-vitro fertilization and embryo transfer for infertility resulting from pathological proximal tubal blockage.

The aim of this study was to evaluate the prognosis for the patients after the treatment of infertility resulting from proximal tubal blockage using microsurgical tubocornual anastomosis and in-vitro fertilization (IVF) and embryo transfer complementarily. A total of 59 microsurgical operations (1986-1992) for infertility resulting from pathological proximal tubal lesions were analysed. The cumulative live birth rate was 52% for tubocornual anastomosis, 58% for bilateral operations and 28% for two-site operations. In all, 35 singleton babies were born. Of the 32 operated patients who did not deliver within 2 years of surgery, 21 were treated by 66 IVF cycles; 12 babies were born. The live birth rate was 18% per cycle and 57% per patient. Combining both treatment methods the cumulative live birth rate was improved up to 69% in the group of tubocornual anastomoses, up to 75% in the group of bilateral operations, and up to 57% in the group of two-site operations. Complementary use of microsurgery and IVF and embryo transfer improves the prognosis for selected infertile patients with pathological proximal tubal blockage. In the absence of pregnancy, IVF and embryo transfer should be commenced 1 year after surgery.

Risk factors for ectopic pregnancy after in vitro fertilization and embryo transfer.

PURPOSE: To find the incidence of ectopic pregnancy (EP) in patients who conceived in the IVF-ET program, and risk factors affecting the occurrence of EP. METHODS: We analyzed the effects of the indication for IVF, type of ovarian stimulation (hMG + hCG vs. GnRH + hMG + hCG), type of embryo transfer (transcervical intratubal, intrauterine in chest-knee position and intrauterine in lithotomy position) and number of embryos transferred on the occurrence of EP. EP was treated by laparotomy, prostaglandin E2 or laparoscopic surgery. RESULTS: In 7991 stimulated and 92 natural cycles treated in the Ljubljana IVF-ET program between May 1983 and June 1994 we achieved 1059 pregnancies, of which 44 were ectopic (incidence 4.1%), the main risk being tubal factor infertility. There were 38 (86.3%) tubal, 3 (6.8%) heterotopic, 1 (2.4%) ovarian, and 2 (4.5%) cornual EP. In two patients multiple tubal EP occurred (1 twin, 1 triplet). Forty-two patients (95.4%) had tubal factor infertility, 1 (2.3%) unexplained, and 1 (2.3%) patient had male factor. The incidence of EP in patients with tubal infertility was 5.4%, in patients with unexplained infertility 2.0% and in those with male factor 0.9%. There appeared to be no correlation between the two superovulatory methods. With transcervical intrauterine ET the incidence of EP was 0 of 5 clinical pregnancies (CP); with intrauterine in chest-knee position it was 26 (3.5%) of 738 CP; with intrauterine in lithotomy position it was 17 (5.4%) of 316 CP. The difference between the two types of intrauterine ET is not statistically significant. The incidence of EP did not correlate with the number of embryos transferred. The average initial values of beta hCG performed 17 days after ET were significantly lower in patients with EP than in those with normal singleton pregnancy (157 +/- 143 mIU/ml vs. 408 +/- 148 mIU/ml). CONCLUSIONS: EP can complicate the IVF procedure. The main risk factor is tubal infertility with or without previous tubal surgery. The low initial value of beta hCG has a strong predictive value in the diagnosis of EP.

Ectopic pregnancy following the treatment of tubal infertility.

To evaluate the prognosis for the patient who becomes pregnant after infertility treatment, we analyzed the occurrence of ectopic pregnancy following reconstructive surgery and in vitro fertilization/embryo transfer (IVF/ET) for tubal infertility. The results of 474 microsurgical operations and the results of 2,119 stimulated IVF/ET cycles for tubal infertility in the Reproduction Unit of Ljubljana University Department of Obstetrics and Gynecology are presented. The ratio of patients who subsequently had only ectopic pregnancies to the number of operations was 12%. Ectopic pregnancies represented 28% of all pregnancies after surgery. In IVF/ET cycles for tubal infertility, ectopic pregnancy represented 2.8% of all pregnancies and 3 permiles of all transfers. There was one (0.5%) heterotopic pregnancy. The likelihood of live births (30%, one or more times) after surgery compensates the high risk for ectopic pregnancy. While the risk for ectopic pregnancy after IVF/ET is much lower than the risk after tubal surgery, it is still rather high compared with the risk in the normal population. In the cases with severe tubal lesions IVF/ET is preferable to tubal surgery. The results show the importance of considering ectopics when deciding upon treatment and in patients who become pregnant after treatment for tubal infertility.

Microsurgery and in vitro fertilization/embryo transfer for infertility resulting from distal tubal lesions.

The live birth rates were analyzed after 688 microsurgical operations for infertility resulting from distal tubal lesions and after 885 in vitro fertilization cycles for tubal factor infertility at the Ljubljana University Department of Obstetrics and Gynecology. The cumulative live birth rate five years after surgery was 31% (17% cases lost to follow-up). The cumulative live birth rate after four in vitro fertilization cycles for tubal infertility was 40% (8% per treated cycle). With two options for treatment, operable cases had a better prognosis than did the inoperable ones. If pregnancy did not occur two to three years after surgery, the patients underwent in vitro fertilization. The complementary use of microsurgery and in vitro fertilization has substantially improved the prognosis for infertility resulting from distal tubal lesions.

[Surgical treatment of symmetrically developed uterine abnormalities]. / Kirursko lijecenje simetricnih razvojnih nepravilnosti maternice.

Retrospectively 75 patients having undergone metroplasty for symmetric uterine anomalies were analysed. The main indication for operation was recurrent abortions and preterm deliveries (63 women) and primary sterility (12). Adnexal pathology which required microsurgical repair was present in 52 (57%) patients. The most frequent uterine malformations were uterus septus and subseptus (51), uterus bicornis (23) and uterus arcuatus (1). All metroplasties were performed according to the Bret-Palmer technique modified by authors. Indication for metroplasty was based on hysterosalpingography, laparoscopy and hysteroscopy in doubtful cases. Prior to metroplasty, 63 patients had 189 spontaneous abortions and 6 preterm deliveries without a living child, while 12 patients were primarily sterile. After operation 68 (90.4%) patients became pregnant and 65 (86.6%) of them delivered 92 healthy children. Pregnancy in 3 (4.0%) patients ended with repeated spontaneous abortions, while 7 (9.4%) remained sterile.

[The importance of ultrasound in the diagnosis of ectopic pregnancy]. / Znacenje ultrazvuka u dijagnostici izvanmatericne trudnoce.

Ultrasonography was applied on 305 patients where ectopic pregnancy was suspected. In 65 (21.3%) the diagnosis was later confirmed and 240 (78.7%) patients did not conceive outside of the uterus. Ultrasonographic findings were classified as positive, suspect, negative, false positive, and false negative. A positive predictive value (79%) and a negative predictive value (91%) of ultrasonography were determined. The false positive rate was 2.9% and the false negative rate 29.2%. A combined use of ultrasonography and beta HCG determinations offers new possibilities for a more reliable diagnosis of ectopic pregnancy.

Treatment of ectopic pregnancy with prostaglandin E2.

In 10 patients the treatment of ectopic pregnancy consisted of the use of 0.75 to 2.25 mg of prostaglandin E2 (Prostin E2, 1 mg/ml, Upjohn). In 8 patients PGE2 was injected in the tubal wall under laparoscopic control, in 2 under the control of vaginal ultrasound probe. The effect of the treatment was controlled by serial determinations of serum beta HCG. In 8 patients with PGE2 injected laparoscopically, negativization of beta HCG took place in 14 +/- 8 days which meant successful treatment of ectopic pregnancy. For persistently elevated beta HCG concentrations in both patients with PGE2 applied under the ultrasound control, laparotomy had to be performed.

Transvaginal ultrasound or laparoscopy for oocyte retrieval (experiences in the Ljubljana IVF program).

At present, the less invasive ultrasound-directed techniques are the methods of choice for oocyte retrieval in most in vitro fertilization and embryo transfer (FIVET) centers. Among the ultrasound-direct techniques, the transvaginal follicle aspiration guided by transvaginal ultrasound for oocyte recovery is gaining popularity in many FIVET centers. This study compare cycles outcome following transvaginal ultrasound oocyte retrieval (105 cycles) to laparoscopic oocyte retrieval (218 cycles); no statistically significant difference could be demonstrated between the groups in all parameters evaluated but better clinical results have been obtained in transvaginal ultrasound group. The Authors conclude that transvaginal oocyte recovery represents an improvement and a simplification of the FIVET procedure.

In vitro fertilization and embryo transfer programme in Ljubljana.

Results of the IVF-ET programme from June 1983 to July 1984 are presented. The procedure was performed in 98 women in 127 cycles. Six normally ongoing clinical pregnancies, three early spontaneous abortions and six biochemical pregnancies were achieved. The laparoscopic techniques of oocyte retrieval were used. Ovulation was first stimulated by clomiphene citrate and later on by gonadotropins only. Comparison of these two regimens showed gonadotropin stimulation to be more effective than clomiphene stimulation, considering the respective transfer rate of 73.3% (gonadotropins) and 18.8% (clomiphene).

[Laparoscopic aspiration of oocytes for in vitro fertilization and embryo transfer]. / Laparoskopska aspiracija oocita kod postupka fertilizacije in vitro i transfera embrija.

Within the in vitro fertilization and embryo transfer procedure, 127 laparoscopies were performed in 89 women. The only indication was tubal infertility. There were 116 gonadotropin stimulated cycles and 11 clomiphene citrate stimulated cycles. Because of a limited ovarian access laparoscopic adhesiolysis was necessary in 30%. Egg recovery amounted to 440 oocytes in 92% successful laparoscopies, which represents 4.1 oocytes per laparoscopy. This resulted in 15 pregnancies of which 3 ended with spontaneous abortion, 6 biochemical and 6 ongoing pregnancies. Two pregnancies resulted in the live birth of normal twins, and one in the birth of a healthy girl.

[Optimal terms for sterilization]. / Optimalni termin za sterilizaciju.

Sterilization can be performed in the immediate postpartum, post abortion, or as an interval procedure. Traditionally, postpartum sterilization should be performed within the first two days after delivery, thereafter the risk for infection is believed to increase. Sterilization and artificial abortion are performed as concomitant procedures, while interval sterilization is carried out at least six weeks after the termination of pregnancy. Though many authors consider the sterilization in the immediate postpartum and postabortion period safe enough, the comparative studies show that interval sterilization has less complications. Postpartum (postabortion) sterilization has much more disadvantages than advantages in comparison to interval sterilization. The main advantage of postpartum sterilization is only one hospitalization while disadvantages are the increased risk of hemorrhage, uterine perforation, thromboembolic disease, infection, technical difficulties, and psychological problems. For these reasons interval sterilization is recommended.