Clinical outcomes of uninterrupted embryo culture with or without time-lapse-based embryo selection versus interrupted standard culture (SelecTIMO): a three-armed, multicentre, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system. METHODS: In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants. FINDINGS: 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures. INTERPRETATION: Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned. FUNDING: Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck.

Does endometrial scratching increase the rate of spontaneous conception in couples with unexplained infertility and a good prognosis (Hunault > 30%)? Study protocol of the SCRaTCH-OFO trial: a randomized controlled trial.

BACKGROUND: In the Netherlands, couples with unexplained infertility and a good prognosis to conceive spontaneously (i.e. Hunault > 30%) are advised to perform timed intercourse for at least another 6 months. If couples fail to conceive within this period, they will usually start assisted reproductive technology (ART). However, treatment of unexplained infertility by ART is empirical and can involve significant burdens. Intentional endometrial injury, also called 'endometrial scratching', has been proposed to positively affect the chance of embryo implantation in patients undergoing in vitro fertilization (IVF). It might also be beneficial for couples with unexplained infertility as defective endometrial receptivity may play a role in these women. The primary aim of this study is to determine whether endometrial scratching increases live birth rates in women with unexplained infertility. METHOD: A multicentre randomized controlled trial will be conducted in Dutch academic and non-academic hospitals starting from November 2017. A total of 792 women with unexplained infertility and a good prognosis for spontaneous conception < 12 months (Hunault > 30%) will be included, of whom half will undergo endometrial scratching in the luteal phase of the natural cycle. The women in the control group will not undergo endometrial scratching. According to Dutch guidelines, both groups will subsequently perform timed intercourse for at least 6 months. The primary endpoint is cumulative live birth rate. Secondary endpoints are clinical and ongoing pregnancy rate; miscarriage rate; biochemical pregnancy loss; multiple pregnancy rate; time to pregnancy; progression to intrauterine insemination (IUI) or IVF; pregnancy complications; complications of endometrial scratching; costs and endometrial tissue parameters associated with reproductive success or failure. The follow-up duration is 12 months. DISCUSSION: Several small studies show a possible beneficial effect of endometrial scratching in women with unexplained infertility trying to conceive naturally or through IUI. However, the quality of this evidence is very low, making it unclear whether these women will truly benefit from this procedure. The SCRaTCH-OFO trial aims to investigate the effect of endometrial scratching on live birth rate in women with unexplained infertility and a good prognosis for spontaneous conception < 12 months. TRIAL REGISTRATION: NTR6687 , registered August 31st, 2017. PROTOCOL VERSION: Version 2.6, November 14th, 2018.

A randomized controlled, non-inferiority trial of modified natural versus artificial cycle for cryo-thawed embryo transfer.

STUDY QUESTION: Are live birth rates (LBRs) after artificial cycle frozen-thawed embryo transfer (AC-FET) non-inferior to LBRs after modified natural cycle frozen-thawed embryo transfer (mNC-FET)? SUMMARY ANSWER: AC-FET is non-inferior to mNC-FET with regard to LBRs, clinical and ongoing pregnancy rates (OPRs) but AC-FET does result in higher cancellation rates. WHAT IS ALREADY KNOWN: Pooling prior retrospective studies of AC-FET and mNC-FET results in comparable pregnancy and LBRs. However, these results have not yet been confirmed by a prospective randomized trial. STUDY DESIGN, SIZE AND DURATION: In this non-inferiority prospective randomized controlled trial (acronym 'ANTARCTICA' trial), conducted from February 2009 to April 2014, 1032 patients were included of which 959 were available for analysis. The primary outcome of the study was live birth. Secondary outcomes were clinical and ongoing pregnancy, cycle cancellation and endometrium thickness. A cost-efficiency analysis was performed. PARTICIPANT/MATERIALS, SETTING, METHODS: This study was conducted in both secondary and tertiary fertility centres in the Netherlands. Patients included in this study had to be 18-40 years old, had to have a regular menstruation cycle between 26 and 35 days and frozen-thawed embryos to be transferred had to derive from one of the first three IVF or IVF-ICSI treatment cycles. Patients with a uterine anomaly, a contraindication for one of the prescribed medications in this study or patients undergoing a donor gamete procedure were excluded from participation. Patients were randomized based on a 1:1 allocation to either one cycle of mNC-FET or AC-FET. All embryos were cryopreserved using a slow-freeze technique. MAIN RESULTS AND THE ROLE OF CHANCE: LBR after mNC-FET was 11.5% (57/495) versus 8.8% in AC-FET (41/464) resulting in an absolute difference in LBR of -0.027 in favour of mNC-FET (95% confidence interval (CI) -0.065-0.012; P = 0.171). Clinical pregnancy occurred in 94/495 (19.0%) patients in mNC-FET versus 75/464 (16.0%) patients in AC-FET (odds ratio (OR) 0.8, 95% CI 0.6-1.1, P = 0.25). 57/495 (11.5%) mNC-FET resulted in ongoing pregnancy versus 45/464 (9.6%) AC-FET (OR 0.7, 95% CI 0.5-1.1, P = 0.15). χ(2) test confirmed the lack of superiority. Significantly more cycles were cancelled in AC-FET (124/464 versus 101/495, OR 1.4, 95% CI 1.1-1.9, P = 0.02). The costs of each of the endometrial preparation methods were comparable (€617.50 per cycle in NC-FET versus €625.73 per cycle in AC-FET, P = 0.54). LIMITATIONS, REASONS FOR CAUTION: The minimum of 1150 patients required for adequate statistical power was not achieved. Moreover, LBRs were lower than anticipated in the sample size calculation. WIDER IMPLICATIONS OF THE FINDINGS: LBRs after AC-FET were not inferior to those achieved by mNC-FET. No significant differences in clinical and OPR were observed. The costs of both treatment approaches were comparable. STUDY FUNDING/COMPETING INTERESTS: An educational grant was received during the conduct of this study. Merck Sharpe Dohme had no influence on the design, execution and analyses of this study. E.R.G. received an education grant by Merck Sharpe Dohme (MSD) during the conduct of the present study. B.J.C. reports grants from MSD during the conduct of the study. A.H. reports grants from MSD and Ferring BV the Netherlands and personal fees from MSD. Grants from ZonMW, the Dutch Organization for Health Research and Development. J.S.E.L. reports grants from Ferring, MSD, Organon, Merck Serono and Schering-Plough during the conduct of the study. F.J.M.B. receives monetary compensation as member of the external advisory board for Merck Serono, consultancy work for Gedeon Richter, educational activities for Ferring BV, research cooperation with Ansh Labs and a strategic cooperation with Roche on automated anti Mullerian hormone assay development. N.S.M. reports receiving monetary compensations for external advisory and speaking work for Ferring BV, MSD, Anecova and Merck Serono during the conduct of the study. All reported competing interests are outside the submitted work. No other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: Netherlands trial register, number NTR 1586. TRIAL REGISTRATION DATE: 13 January 2009. FIRST PATIENT INCLUDED: 20 April 2009.

Integration of patient characteristics and the results of Chlamydia antibody testing and hysterosalpingography in the diagnosis of tubal pathology: an individual patient data meta-analysis.

BACKGROUND: Tubal patency tests are routinely performed in the diagnostic work-up of subfertile patients, but it is unknown whether these diagnostic tests add value beyond the information obtained by medical history taking and findings at physical examination. We used individual patient data meta-analysis to assess this question. METHODS: We approached authors of primary studies for data sets containing information on patient characteristics and results from tubal patency tests, such as Chlamydia antibody test (CAT), hysterosalpingography (HSG) and laparoscopy. We used logistic regression to create models that predict tubal pathology from medical history and physical examination alone, as well as models in which the results of tubal patency tests are integrated in the patient characteristics model. Laparoscopy was considered to be the reference test. RESULTS: We obtained data from four studies reporting on 4883 women. The duration of subfertility, number of previous pregnancies and a history of previous pelvic inflammatory disease (PID), pelvic surgery or Chlamydia infection qualified for the patient characteristics model. This model showed an area under the receiver operating characteristic curve (AUC) of 0.63 [95% confidence interval (CI) 0.61-0.65]. For any tubal pathology, the addition of HSG significantly improved the predictive performance to an AUC of 0.74 (95% CI 0.73-0.76) (P < 0.001). For bilateral tubal pathology, the addition of both CAT and HSG increased the predictive performance to an AUC of 0.76 (95% CI 0.74-0.79). CONCLUSIONS: In the work-up for subfertile couples, the combination of patient characteristics with CAT and HSG results gives the best diagnostic performance for the diagnosis of bilateral tubal pathology.

[Intravascular migration of a contraceptive implant]. / Intravasculaire migratie van een hormoonimplantaat.

BACKGROUND: The Implanon NXT is a commonly used contraceptive. Incorrect localization of the implant can cause complications. CASE DESCRIPTION: A 41-year-old woman is seen in the gynaecology outpatient clinic with a request to remove a recently placed Implanon NXT because of worsening mood symptoms. The implant can't be found on physical and ultrasound examination. Duringsurgicalexplorationthe implant is not found at theinsertion site' By means of X-ray scanning the implant becomes visible around the humeral head. The implant appears to be located in the cephalic vein and is subsequently removed. CONCLUSION: In case of a referral due to because of worsening mood symptoms after an Implanon NXT exchange, it is possible that the implant is localized incorrectly. It is recommended to use additional imaging before performing surgical exploration. Furthermore, it is important to insert the Implanon NXT according to the supplied instructions to prevent this complication.

Is home-based monitoring of ovulation to time frozen embryo transfer a cost-effective alternative for hospital-based monitoring of ovulation? Study protocol of the multicentre, non-inferiority Antarctica-2 randomised controlled trial.

STUDY QUESTION: The objective of this trial is to compare the effectiveness and costs of true natural cycle (true NC-) frozen embryo transfer (FET) using urinary LH tests to modified NC-FET using repeated ultrasound monitoring and ovulation trigger to time FET in the NC. Secondary outcomes are the cancellation rates of FET (ovulation before hCG or no dominant follicle, no ovulation by LH urine test, poor embryo survival), pregnancy outcomes (miscarriage rate, clinical pregnancy rates, multiple ongoing pregnancy rates, live birth rates, costs) and neonatal outcomes (including gestational age, birthweight and sex, congenital abnormalities or diseases of babies born). WHAT IS KNOWN ALREADY: FET is at the heart of modern IVF. To allow implantation of the thawed embryo, the endometrium must be prepared either by exogenous oestrogen and progesterone supplementation (artificial cycle (AC)-FET) or by using the NC to produce endogenous oestradiol before and progesterone after ovulation to time the transfer of the thawed embryo (NC-FET). During an NC-FET, women visit the hospital repeatedly and receive an ovulation trigger to time FET (i.e. modified (m)NC-FET or hospital-based monitoring). From the woman's point of view, a more natural approach using home-based monitoring of the ovulation with LH urine tests to allow a natural ovulation to time FET may be desired (true NC-FET or home-based monitoring). STUDY DESIGN SIZE DURATION: This is a multicentre, non-inferiority prospective randomised controlled trial design. Consenting women will undergo one FET cycle using either true NC-FET or mNC-FET based on randomisation. PARTICIPANTS/MATERIALS SETTING METHODS: Based on our sample size calculation, the study group will consist of 1464 women between 18 and 45 years old who are scheduled for FET. Women with anovulatory cycles, women who need ovulation induction and women with a contra indication for pregnancy will be excluded. The primary outcome is ongoing pregnancy. Secondary outcomes are cancellation rates of FET, pregnancy outcomes (including miscarriage rate, clinical pregnancy, multiple pregnancy rate and live birth rate). Costs will be estimated by counting resource use and calculating unit prices. STUDY FUNDING/COMPETING INTERESTS: The study received a grant from the Dutch Organisation for Health Research and Development (ZonMw 843002807; www.zonmw.nl). ZonMw has no role in the design of the study, collection, analysis, and interpretation of data or writing of the manuscript. F.B. reports personal fees from member of the external advisory board for Merck Serono, grants from Research support grant Merck Serono, outside the submitted work. A.E.P.C. reports and Unrestricted grant of Ferring B.V. to the Center for Reproductive medicine, no personal fee. Author up-to-date on Hyperthecosis. Congress meetings 2019 with Ferring B.V. and Theramex B.V. M.G. reports Department research and educational grants from Guerbet, Merck and Ferring (location VUMC) outside the submitted work. E.R.G. reports personal fees from Titus Health Care, outside the submitted work. C.B.L. reports grants from Ferring, grants from Merck, from Guerbet, outside the submitted work. The other authors have none to declare. TRIAL REGISTRATION NUMBER: Dutch Trial Register (Trial NL6414 (NTR6590), https://www.trialregister.nl/). TRIAL REGISTRATION DATE: 23 July 2017. DATE OF FIRST PATIENT'S ENROLMENT: 10 April 2018.

Effects of moderate acute isovolaemic haemodilution on myocardial function in patients undergoing coronary surgery under volatile inhalational anaesthesia.

When myocardial oxygen demand is increased by elevated heart rate in patients undergoing coronary artery surgery under total intravenous anaesthesia, acute isovolaemic haemodilution may be associated with a deterioration of cardiac function. We investigated the effects of acute isovolaemic haemodilution during volatile inhalational anaesthesia. Forty patients undergoing coronary surgery were randomly assigned to two groups according to the rate of atrioventricular pacing (Group 70 at 70.min(-1) and Group 90 at 90.min(-1)). While paced at the fixed heart rate, acute isovolaemic haemodilution was performed before the start of cardiopulmonary bypass. In both groups mean (SD) stroke volume increased with haemodilution (from 65 (9) to 83 (10) ml.min(-1) (p < 0.01) in Group 70 and from 65 (9) to 81 (9) ml.min(-1) (p < 0.01) in Group 90) as a result of a decrease in systemic vascular resistance (from 1175 (231) to 869 (164) dynes.s.cm(-5) (p < 0.01) and from 1060 (185) to 849 (146) dynes.s.cm(-5) (p < 0.01), respectively) and an increase in end-diastolic volume (from 1049 (234) to 1405 (211) ml (p < 0.01) and from 1078 (106) to 1438 (246) ml (p < 0.01), respectively). Left ventricular pressure-derived data remained unchanged with acute isovolaemic haemodilution in both groups.

Pregnancy after Caesarean section: fewer or later?

BACKGROUND: It is unclear whether having a Caesarean section results in fewer subsequent pregnancies with longer intervals between pregnancies, an effect which may impact on the reproductive performance of a population. Our aim was to determine the implications of a Caesarean section on the subsequent fecundity and interpregnancy interval. METHODS: This is a cohort study. The obstetric follow-up of primiparous women who delivered by a Caesarean section of a singleton infant in breech presentation is compared with the follow-up of women who delivered vaginally of a singleton infant after a physiological, uncomplicated pregnancy. RESULTS: A total of 279 women delivered a singleton infant in breech presentation at term. From these women, 165 (59.1%) had a Caesarean section. In this group, 131 (79.4%) women had a subsequent pregnancy. In the reference group of 268 women who delivered vaginally, 208 (77.6%) became pregnant again. The median interval between birth of the first child and the beginning of the next pregnancy was 20 months for the Caesarean section group and 18 months for the reference group. No significant difference in interpregnancy interval between the different groups was found. CONCLUSIONS: Women who delivered by Caesarean section at term in their first pregnancy do not have fewer second pregnancies compared with women who delivered vaginally. The interpregnancy interval between first and second pregnancy was not prolonged.

Cardiac surgery with cardiopulmonary bypass: does aprotinin affect outcome?

BACKGROUND: Aprotinin, a non-specific serine protease inhibitor, has been used for two decades to reduce perioperative blood loss and the risk for allogeneic transfusion in cardiac surgery. This study evaluated the effects of aprotinin on outcome (mortality, cardiac events, renal failure, and cerebrovascular events) in such patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: Data were obtained in patients who received a strict blood conservation protocol: no antifibrinolytic therapy when at low risk (n = 854) and aprotinin (n = 1210) when at high risk for blood transfusion. Relative risk of different pre- and intra-operative variables was calculated for the different outcome variables. Backward stepwise logistic regression analysis was used to identify the independent risk factors associated with the different outcome variables. Statistical significance was accepted at P < 0.01. RESULTS: Postoperative mortality and morbidity were higher in the aprotinin group but this was related to an increased incidence of perioperative risk factors. Mortality was similar to that predicted by the Euroscore. Complex surgery was the only independent variable associated with postoperative cardiac events. Preoperative heart failure, preoperative creatinine > 1.5 mg dl(-1), urgent, and redo surgery were the independent variables associated with postoperative haemodialysis. Age > 70 yr was identified as the only independent variable associated with neurologic dysfunction. CONCLUSIONS: In the present study, patients receiving aprotinin as part of a strict blood conservation strategy represent a population at high risk for postoperative complications. For the outcome variables studied, aprotinin administration was not identified as an independent risk factor.

[A levonorgestrel-containing IUD for the treatment of endometriosis]. / Levonorgestrelhoudend IUD voor de behandeling van endometriose.

Endometriosis is a chronic condition that can cause severe pain and have a significant effect on the quality of life. The currently available hormonal treatments with analogues of gonadotrophin releasing hormone (GnRH) and oral progestational agents are effective in the short term, but the systemic side effects affect treatment compliance. Recurrence of the symptoms is therefore not uncommon. Recently, a few studies have assessed the effect of a levonorgestrel-releasing IUD on the pain associated with endometriosis. This treatment may have a positive effect on endometriosis, both on the symptoms, the measured extent of the lesions, and the serum levels of CA-125. However, the level of evidence of these studies is lower than that of the standard hormonal treatment options, since large randomised controlled trials are still lacking.

Endometriosis: a review on its pathogenesis.

Although peritoneal endometriosis was recognized in 1860, its pathogenesis still remains unclear. Several theories attempt to explain the pathogenesis of this condition. From these, the implantation theory maintains that peritoneal endometriosis is the result of implantation and subsequent growth of retrogradely shed viable endometrial cells. Based on a second theory, the peritoneal mesothelium transforms to an endometrium-like tissue under the influence of products of regurgitated endometrium (induction). Cell adhesion molecules could be functionally involved in the binding of the endometrial cells to the peritoneal lining. In peritoneal endometriosis, a delicate equilibrium seems to exist between attacking forces (retrograde menstruation) and the defense mechanisms. On one hand, the amount and the nature of the regurgitated menstrual debris seems important to the development of the disease. On the other hand, the active intra-abdominal milieu may be involved. This milieu probably converts the regurgitated endometrial tissue into single cells via loss of functional cell adhesion properties. Endometriosis may result form the impairment of the function of the peritoneal milieu in disposing of the regurgitated cells. Alternatively, the endometriosis may occur if the number of regurgitated cells is too large. An intact peritoneal lining may be an important additional line of defense in preventing the binding of the endometrial cells. Endometriosis is likely to develop if such defense mechanisms fail. Here, the scientific basis of the endometriosis theories is discussed.

Retrograde ejaculation: successful treatment with artificial insemination.

Retrograde ejaculation is characterized by aspermia or oligospermia and results from an incompetent bladder neck, often due to a dysfunction of the internal sphincter. In almost 3 years, eight couples who suffered from infertility due to retrograde ejaculation were treated with inseminations with spermatozoa gained from the urine. Ovulation was predicted on the basis of blood LH levels. The urine-semen sample was collected in 100 mL of Hepes medium and 5 mL 1% human albumin (pH 7.4). After centrifuging, the remaining sperm pellet was dispersed on a Percoll gradient. After centrifuging and resuspending, followed by two washing procedures with Ham's F-10 and human albumin 1%, the remaining sample was used for intrauterine insemination. Twelve pregnancies were thus achieved; two women became pregnant twice and one three times. The pregnancy rate per cycle was 44.4%. In seven couples, pregnancy was achieved within three cycles. Four pregnancies ended in spontaneous abortion and five ended in the birth of a healthy child; three pregnancies were continuing at the time of writing. Retrograde ejaculation can be treated successfully with inseminations using spermatozoa obtained from urine. It seems important to collect the urine-semen sample in a buffered medium and to time the insemination on the basis of the LH surge.

Non-Hodgkin lymphoma of the puerperal uterus.

Non-Hodgkin lymphoma was diagnosed initially in a curettage specimen of a puerperal uterus 3 weeks after normal delivery of a healthy infant. The disease, which probably existed during the last trimester of pregnancy, was classified as clinical stage IVb. Combination chemotherapy resulted in immediate improvement and complete remission after several months. Histopathologic examination of the uterus 10 months after starting therapy demonstrated complete disappearance of the malignant lymphoma.

Cyclic changes in the concentration of glucose and fructose in human cervical mucus.

OBJECTIVE: To determine a possible cyclic change in the concentration of glucose and fructose in the aqueous phase of human cervical mucus (CM). DESIGN: Concentrations of glucose and fructose were longitudinally determined in the aqueous phase of CM of normal cycling women using enzymatic techniques, modified for small quantities. SETTING: Patients visiting a fertility clinic were selected. PATIENTS: Nine healthy women with regular menstrual cycles of 28 +/- 3 days that appeared to be ovulatory, demonstrated by sonographic follicle immaging and serum progesterone (P) measurements. INTERVENTIONS: Cervical mucus samples were longitudinally collected preovulatory, postovulatory, and premenstrual in ovulatory cycles, monitored by ultrasound and blood estradiol and P measurements. MAIN OUTCOME MEASURES: The study was designed to measure glucose and fructose longitudinally on three different points during one cycle. RESULTS: The preovulatory glucose concentrations in CM were lower than postovulatory and premenstrual. The preovulatory fructose concentrations were lower than premenstrual. The glucose concentration correlated with the blood P level. CONCLUSION: There is a consistent change in the glucose concentration measured in human CM in three phases of the menstrual cycle. The preovulatory and premenstrual fructose concentrations differ significantly. Knowledge of the carbohydrate metabolism in human cervical mucus may contribute in illuminating the possible role of the carbohydrate metabolism in sperm migration at midcycle and implantation in the luteal phase.

Expression of integrins and E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, and endometriosis.

OBJECTIVE: To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle. DESIGN: An immunohistochemical study. SETTING: Tertiary care university medical center. PATIENTS: Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles. INTERVENTIONS: A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including the integrin alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 and E-cadherin, as determined by immunohistochemistry on frozen sections. RESULTS: All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins alpha 2 beta 1 and alpha 3 beta 1 was found in epithelial cells in 13 of 16 cases. Integrin alpha 5 beta 1 was detected in 11 of 16 samples, and integrins alpha 4 beta 1 and alpha 6 beta 1 were detected in 5 of 16 samples. In PF, integrin alpha 3 beta 1 was found in epithelial cells in 12 of 16 samples, integrin alpha 5 beta 1 in 5 of 16, and integrins alpha 4 beta 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin. CONCLUSION: Integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Expression of cadherins and integrins in human endometrium throughout the menstrual cycle.

OBJECTIVE: To detect cadherin and integrin expression in biopsies of endometrium in the phases of the cycle. Cell adhesion molecules may be involved in endometrial shedding during menstruation and attachment of shed endometrial tissue to the peritoneal lining in endometriosis patients. DESIGN: An immunohistochemical study on fresh frozen sections. SETTING: Tertiary-care university medical center. PATIENTS: Sixteen patients undergoing monitoring of their cycle as part of a subfertility workup. All patients had regular and ovulatory cycles. INTERVENTIONS: Endometrium samples were obtained at well-defined phases of the cycle. Simultaneously, blood samples were collected for E2 and P assay. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including E- and P-cadherin and the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and the expression of estrogen receptor (ER) and P receptor (PR). RESULTS: E- and P-cadherin expression was demonstrated in all endometrium samples. Integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were detected in samples from all cycle phases, whereas integrin alpha 2 beta 1 was not detected in midluteal samples. The serum levels of E2 were 24.7 pg/mL (range: 10.9 to 35.4 pg/mL) in the early follicular phase and 190.7 pg/mL (range: 152.5 to 256.1 pg/mL) in the preovulatory phase (conversion factor to SI unit, 3.671). Serum P was 13.7 ng/mL (range: 10.3 to 16.7 ng/mL) in the midluteal phase and 6.4 ng/mL (range: 1.2 to 13.7 ng/mL) in the premenstrual phase (conversion factor to SI unit, 3.180). The portion of cells staining for ER and PR was at a maximum during the preovulatory phase, both for epithelial and stromal cells. CONCLUSIONS: E- and P-cadherin expression was detected in all samples and did not vary throughout the menstrual cycle. If their expression is involved functionally in the cyclic menstrual shedding, the loss of expression is limited to a short period of time. Of the beta 1 integrins, only alpha 2 beta 1 expression was modulated during the menstrual cycle and found to be absent in the midluteal phase. No relation was found between the expression of cell adhesion molecules and the expression of ER and PR. Because the cadherins and beta 1 integrins could be detected in late luteal phase endometrium, these cell adhesion molecules could be involved in the attachment of endometrial fragments to the peritoneal lining as a result of retrograde menstruation. The potential function in the pathogenesis of endometriosis remains to be elucidated.

Endometrial cell adhesion in an in vitro model using intact amniotic membranes.

OBJECTIVE: To study adhesion between endometrial tissue and peritoneum using intact amniotic membranes as an in vitro model. DESIGN: A cell biologic and immunohistochemical study. SETTING: Tertiary-care university medical center. PATIENTS: Ten patients with regular and ovulatory cycles. INTERVENTIONS: Mechanically dissected tissue fragments from endometrial biopsies were cultured on either side of intact amniotic membranes. Also, the carcinoma cell lines RL95-2 and AN3CA were used. MAIN OUTCOME MEASURES: The adhesion of endometrial fragments and of endometrial carcinoma cell lines to amniotic membrane was studied in vitro and evaluated using frozen sections. The composition of the extracellular matrix and the presence of intermediate filament proteins of amniotic membrane were determined using immunohistochemistry and compared with those of normal peritoneum. RESULTS: Peritoneum and amniotic membrane were similar with respect to expression of cytokeratins in epithelial lining and of extracellular matrix (ECM) components. The endometrial fragments did not adhere to the intact epithelial side of the amniotic membrane. In contrast, adhesion did occur to the nonepithelial side of the amnion. The carcinoma cell lines RL95-2 and AN3CA adhered to either side of intact amniotic membranes. CONCLUSIONS: An intact epithelial lining prevents adhesion of endometrial fragments to the ECM of amniotic membranes in vitro. An intact epithelium could be important defense mechanism in preventing initial adhesion of retrogradely shed endometrium fragments to peritoneum.

"The Flemish Giant", reflections on the defense against endometriosis, inspired by Professor Emeritus Ivo A. Brosens.

Reproductive research benefits from combining animal and clinical studies. In Leuven, rabbits have constituted an animal model for many reproductive disorders, especially for those that involved surgical treatment. Much of what has been learned from animal experiments has been applied to human clinical reproductive research soon after. In this manuscript we wish to address the problem of the constant intra-abdominal battle between the menstrual aggressor and the peritoneal defense. From all published evidence we may conclude that endometriosis appears to be a dynamic disease, especially in the early phase, with subtle, atypical lesions emerging and vanishing again. In the end however the peritoneal defense system will prevail and the disease will be contained in the majority of patients. When doing repeat laparoscopies in young patients one should be prepared to encounter more advanced histological types of lesions, which not necessarily do have to indicate more advanced stages of the disease: the classical, blue and black powderburn spots and blueberry lesions reflect the extinguishing phase of the dynamic endometriotic process, and herald its inactivated histological end-stage. The dynamic phase of the disease may involve a varying interval of each patient's life, and medical suppression of the activity of the implants during this interval may lead one to conclude erroneously that treatment has been effective. If subsequently (after the end of medical suppression of the activity of the lesions) ovarian activity resumes and the lesions are stimulated again by ovarian steroids, their productive activity returns. Recurrence of disease may be diagnosed if at that stage a laparoscopy would be performed, whereas in reality only reactivation of temporarily obscured lesions did occur. The suppressed, dormant (but never absent) lesions produce mucus again, desquamation occurs, and reaction by the surrounding tissue. The inflammatory response, the local hyperemia and the neogenesis of vessels accentuate the presence of previously invisible endometriosis lesions and make them visible again. Endometriosis resumes its temporarily halted natural course of development, tissue remodeling occurs again, the battle between the aggressor and the defense resumes and waxing and waning of the several types of lesions, red, white and black, can be found again.

Vasoactive properties of rat coronary artery: in the tissue and isolated.

Perfusion of the heart takes place mainly in diastole. It is therefore important to study the factors that affect coronary diastolic flow. One of the factors that may limit coronary artery vasoactive responses is the surrounding cardiac tissue. We have therefore studied the intramyocardial septal artery, both when still embedded in the diastolic, unstretched myocardial tissue and after complete dissection (n = 6). In situ, the average external diameter was 351 +/- 21 microns; after dissection, it was 362 +/- 21 microns. These values were not significantly different. The average response of the vessel to KCl (125 mM, receptor-independent constriction) reduced the diameter to 56.1 +/- 5.0% and 69.4 +/- 3.7% of the maximal diameter for in situ and dissected vessels, respectively. The reduction in diameter after dissection was significantly less than the reduction in situ. The response to vasopressin (1,000 microU/ml, a receptor-dependent constrictor) was a reduction to 62.6 +/- 4.7% and 70.4 +/- 4.5%, respectively. The reduction in diameter of the dissected vessel is significantly smaller than that of the in situ vessel. The average values of the ratios of the diameter reductions for vasopressin and KCl were 0.85 +/- 0.06 in the in situ condition and 0.95 +/- 0.08 after dissection and were not significantly different (paired t-test). The results show that the dilated diameter and the diameter responses of intramyocardial conduit arteries are not affected by the surrounding diastolic cardiac tissue.