Follicular flushing increases the number of oocytes retrieved: a randomized controlled trial.

STUDY QUESTION: Does follicular flushing increase the number of cumulus-oocyte complexes (COCs) retrieved compared to single aspiration? SUMMARY ANSWER: Follicular flushing significantly increases the number of COCs retrieved compared to single aspiration. WHAT IS KNOWN ALREADY: On the basis of published meta-analyses, follicular flushing does not seem to increase the number of oocytes retrieved, the probability of clinical pregnancy, or that of live birth and has been associated with an increase in the duration of oocyte retrieval. It should be noted, however, that all the eligible randomized controlled trials (RCTs) in these meta-analyses have randomized patients into either single aspiration or follicular flushing. This study design might not allow the detection of the true effect of follicular flushing. Despite randomization, this might still be obscured, to an extent, by heterogeneity in patients, stimulation characteristics, and differences in the oocyte retrieval procedure. STUDY DESIGN, SIZE, DURATION: A prospective, single centre, RCT, including 105 patients was performed between July and December 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible patients were those undergoing oocyte retrieval for ICSI, aged <43 years, with BMI 18-35 kg/m2. Patients with all types of ovarian response (low-normal-high), as assessed on the day of triggering final oocyte maturation, were included. Random allocation of the ovaries of each patient to either single aspiration or follicular flushing was performed on the day of oocyte retrieval, using a computer-generated randomization list. Patients could enter the study only once. All follicles from ovaries allocated to either follicular flushing or single aspiration, were aspirated by the same 16G double lumen needle, with a constant aspiration pressure of 190 mmHg, resulting in flow rate of 0.42 ml/s. In the ovaries allocated to the follicular flushing group, if a COC was not recovered in the initial aspirate of each follicle, follicular flushing was performed until a COC was retrieved, up to a maximum of five times. The primary outcome measure was the number of COCs retrieved. Secondary outcomes were oocyte recovery rate, oocyte maturation rate, fertilization rate, and rate of good quality embryos on Day 2. Values are expressed as a median (inter-quartile range). MAIN RESULTS AND THE ROLE OF CHANCE: Significantly more COCs were retrieved in the follicular flushing as compared to the single aspiration group in all patients [5 (7) vs 2 (3), P < 0.001, respectively], as well as in patients with high [9 (3) vs 5 (4), P < 0.001, respectively], normal [5 (2) vs 2 (3), P < 0.001, respectively] and low [1 (1) vs 1 (1), P < 0.001, respectively] ovarian response. In patients with low ovarian response, no COCs were retrieved in 5.7% of the ovaries in the flushing group vs 42.8% of the ovaries in the single aspiration group (P < 0.001). The oocyte retrieval rate was significantly higher in the follicular flushing vs the single aspiration group, in all patients [88.9% (25.0) vs 45.5% (37.5), P < 0.001, respectively], as well as in patients with high [81.8% (15.9) vs 45.5% (22.2), P < 0.001, respectively], normal [85.7% (28.6) vs 40.0% (30.0), P < 0.001, respectively], and low [100% (0) vs 50.0% (100), P < 0.001, respectively] ovarian response. No significant difference was observed regarding maturation rate [85.2% (30.8) vs 100% (33.3), P = 0.78], fertilization rate [76.4% (50) vs 83.3% (50) P = 0.42], and the proportion of good quality embryos on Day 2 [83.3% (40) vs 100% (50), P = 0.62]. Similarly, no differences in the above variables were observed in patients with different types of ovarian response. Follicular flushing as compared to single aspiration was associated with a significant increase in the duration of oocyte retrieval in all patients [248 s (332) vs 135 s (164), respectively], as well as in patients with high [464 s (225) vs 237 s (89), P < 0.001, respectively], normal [248 s (108) vs 141 s (95), P < 0.001, respectively], and low [64 s (59) vs 48 s (10), P < 0.001, respectively] ovarian response. LIMITATIONS, REASONS FOR CAUTION: Although the current study design allows for a more accurate evaluation of the true effect of follicular flushing on the number of COCs retrieved, it does not permit the evaluation of its role on the probability of pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT to suggest that follicular flushing increases the number of COCs retrieved compared to single aspiration, independently of ovarian response. This implies that follicular flushing plays an important role in the optimization of oocyte retrieval. These results, however, need to be confirmed in future studies, in which an equal flow rate should be used during oocyte retrieval. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NCT05473455. TRIAL REGISTRATION DATE: 15 July 2022. DATE OF FIRST PATIENT'S ENROLMENT: 27 July 2022.

A decision-making algorithm for performing or cancelling embryo transfer in patients at high risk for ovarian hyperstimulation syndrome after triggering final oocyte maturation with hCG.

STUDY QUESTION: Can the grade of ascites, haematocrit (Ht), white blood cell (WBC) count and maximal ovarian diameter (MOD) measured on Day 3 be used to construct a decision-making algorithm for performing or cancelling embryo transfer in patients at high risk for severe ovarian hyperstimulation syndrome (OHSS) after an hCG trigger? SUMMARY ANSWER: Using cut-offs of ascites grade>2, Ht>39.2%, WBC>12 900/mm3 and MOD>85 mm on Day 3, a decision-making algorithm was constructed that could predict subsequent development of severe OHSS on Day 5 with an AUC of 0.93, a sensitivity of 88.5% and a specificity of 84.2% in high-risk patients triggered with hCG. WHAT IS KNOWN ALREADY: Despite the increasing popularity of GnRH agonist trigger for final oocyte maturation as a way to prevent OHSS, ≥75% of IVF cycles still involve an hCG trigger. Numerous risk factors and predictive models of OHSS have been proposed, but the measurement of these early predictors is restricted either prior to or during the controlled ovarian stimulation. In high-risk patients triggered with hCG, the identification of luteal-phase predictors assessed post-oocyte retrieval, which reflect the pathophysiological changes leading to severe early OHSS, is currently lacking. STUDY DESIGN SIZE DURATION: A retrospective study of 321 patients at high risk for severe OHSS following hCG triggering of final oocyte maturation. High risk for OHSS was defined as the presence of at least 19 follicles ≥11 mm on the day of triggering of final oocyte maturation. PARTICIPANTS/MATERIALS SETTING METHODS: The study includes IVF/ICSI patients at high risk for developing severe OHSS, who administered hCG to trigger final oocyte maturation. Ascites grade, MOD, Ht and WBC were assessed in the luteal phase starting from the day of oocyte retrieval. Outcome measures were the optimal thresholds of ascites grade, MOD, Ht and WBC measured on Day 3 post-oocyte retrieval to predict subsequent severe OHSS development on Day 5. These criteria were used to construct a decision-making algorithm for embryo transfer, based on the estimated probability of severe OHSS development on Day 5. MAIN RESULTS AND THE ROLE OF CHANCE: The optimal Day 3 cutoffs for severe OHSS prediction on Day 5 were ascites grade>2, Ht>39.2%, WBC>12 900/mm3 and MOD>85 mm. The probability of severe OHSS with no criteria fulfilled on Day 3 is 0% (95% CI: 0-5.5); with one criterion, 0.8% (95% CI: 0.15-4.6); with two criteria, 13.3% (95% CI: 7.4-22.8); with three criteria, 37.2% (95% CI: 24.4-52.1); and with four criteria, 88.9% (95% CI, 67.2-98.1). The predictive model of severe OHSS had an AUC of 0.93 with a sensitivity of 88.5% and a specificity of 84.2%. LIMITATIONS REASONS FOR CAUTION: This is a retrospective study, and therefore, it cannot be excluded that non-apparent sources of bias might be present. In addition, we acknowledge the lack of external validation of our model. We have created a web-based calculator (http://ohsspredict.org), for wider access and usage of our tool. By inserting the values of ascites grade, MOD, Ht and WBC of high-risk patients on Day 3 after oocyte retrieval, the clinician instantly receives the predicted probability of severe OHSS development on Day 5. WIDER IMPLICATIONS OF THE FINDINGS: The present study describes a novel decision-making algorithm for embryo transfer based on ascites, Ht, WBC and MOD measurements on Day 3. The algorithm may be useful for the management of high-risk patients triggered with hCG and for helping the clinician's decision to proceed with, or to cancel, embryo transfer. It must be emphasized that the availability of the present decision-making algorithm should in no way encourage the use of hCG trigger in patients at high risk for OHSS. In these patients, the recommended approach is the use of GnRH antagonist protocols, GnRH agonist trigger and elective embryo cryopreservation. In addition, in patients triggered with hCG, freezing all embryos and luteal-phase GnRH antagonist administration should be considered for the outpatient management of severe early OHSS and prevention of late OHSS. STUDY FUNDING/COMPETING INTERESTS: NHMRC Early Career Fellowship (GNT1147154) to C.A.V. No conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Serum vascular endothelial growth factor levels following luteal gonadotrophin-releasing hormone antagonist administration in women with severe early ovarian hyperstimulation syndrome.

OBJECTIVE: To investigate the kinetics of serum vascular endothelial growth factor (VEGF) following gonadotrophin-releasing hormone (GnRH) antagonist administration in the luteal phase in women with established severe early ovarian hyperstimulation syndrome (OHSS). DESIGN: Pilot observational cohort study. SETTING: Private in vitro fertilisation (IVF) Unit. POPULATION: Twelve IVF women diagnosed with established severe early OHSS 5 days post oocyte retrieval (POR). METHODS: Women undergoing IVF diagnosed with severe early OHSS 5 days POR were given 0.25 mg GnRH antagonist for 4 days, from day 5 until and including day 8 POR, combined with elective blastocyst cryopreservation. Serum VEGF was measured from the day of oocyte retrieval until day 11 POR. Ovarian volume, ascites, serum estradiol and progesterone, haematocrit and white blood cells were monitored during the same period. MAIN OUTCOME MEASURES: Kinetics of VEGF following luteal GnRH antagonist administration in women with established severe early OHSS. RESULTS: The concentration of VEGF was highest (390.9 ± 137.4 pg/ml) 5 days POR, coinciding with the day of diagnosis of severe OHSS. There was a significant decline of VEGF on day 7 (302.8 ± 104.9 pg/ml; P = 0.026), day 9 (303.3 ± 148.3 pg/ml; P = 0.007), and day 11 (252.6 ± 182.7 pg/ml; P = 0.010) compared with day 5 POR. This decline was associated with an improvement of ultrasound and laboratory parameters, indicating regression of severe OHSS. All women were managed at an outpatient level. CONCLUSIONS: GnRH antagonist administration in the luteal phase is associated with a significant decline of VEGF and with regression of established severe early OHSS.

Pregnancy and neonatal outcomes following luteal GnRH antagonist administration in patients with severe early OHSS.

STUDY QUESTION: Do high-risk patients who develop severe early ovarian hyperstimulation syndrome (OHSS) and receive low-dose GnRH antagonist in the luteal phase have lower live birth rates compared with high-risk patients who do not develop severe early OHSS and do not receive GnRH antagonist in the luteal phase? SUMMARY ANSWER: Low-dose luteal GnRH antagonist administration in women with severe early OHSS is associated with similar live birth rates to that of high-risk patients who do not develop severe early OHSS and do not receive GnRH antagonist in the luteal phase. WHAT IS KNOWN ALREADY: It has been reported that luteal GnRH antagonist administration in patients with established severe early OHSS appears to prevent patient hospitalization and results in quick regression of the syndrome on an outpatient basis. However, the effect of such treatment on pregnancy outcome has been investigated in only a small number of animal studies. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort study of 192 IVF patients who were at high risk for OHSS and who did not wish to cancel embryo transfer and have all embryos cryopreserved. The study was conducted between January 2009 and December 2011 at Eugonia Assisted Reproduction Unit. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were <40 years of age, with polycystic ovaries, at high risk for OHSS (defined by the presence of at least 20 follicles ≥11 mm on the day of triggering of final oocyte maturation) and not willing to cancel embryo transfer and cryopreserve all embryos, if severe early OHSS was diagnosed by Day 5 of embryo culture. Patients who were diagnosed with severe early OHSS on Day 5 post-oocyte retrieval were administered 0.25 mg of ganirelix for 3 days, from Day 5 until and including Day 7 (OHSS + antag group, n = 22). High-risk patients who did not develop the severe early OHSS did not receive GnRH antagonist in the luteal phase (control group, n = 172). All patients underwent embryo transfer on Day 5. MAIN RESULTS AND THE ROLE OF CHANCE: Live birth rates (40.9 versus 43.6%), ongoing pregnancy rates (45.5 versus 48.8%), clinical pregnancy rates (50 versus 65.1%), positive hCG (72.7 versus 75%), duration of gestation (36.86 ± 0.90 weeks versus 36.88 ± 2.38 weeks) and neonatal weight (2392.73 ± 427.04 versus 2646.56 ± 655.74 g) were all similar in the OHSS + antag and control groups, respectively. The incidence of major congenital malformations was 2.9% (3/103) in children born in the control group compared with no cases (0/14) in children born following luteal GnRH antagonist administration. No stillbirths or intrauterine deaths, and no cases of pregnancy-induced late OHSS were recorded in either group. None of the 22 patients with severe early OHSS required hospitalization following luteal antagonist administration. Ovarian volume, ascites, hematocrit, white blood cell count, serum estradiol and progesterone decreased significantly (P < 0.001) by the end of the monitoring period (Day 11 post-oocyte retrieval), indicating rapid resolution of the severe OHSS. LIMITATIONS, REASONS FOR CAUTION: This is a prospective cohort investigation with a very limited number of patients receiving the intervention and a larger number of control patients. Our findings suggest that low-dose luteal GnRH antagonist administration during the peri-implantation period may be safe, although larger studies with follow-up of the children born are required. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests for the first time that low-dose luteal GnRH antagonist administration in women with severe early OHSS is associated with a favourable IVF outcome, comparable to control high-risk patients without severe OHSS and not receiving the intervention. Regarding the wider implications on the concept of an OHSS-free clinic, administration of GnRH antagonist in the luteal phase may present a tertiary management level in patients with established severe OHSS, along with the use of GnRH antagonist protocols for primary prevention and the replacement of hCG with GnRH agonist for triggering final oocyte maturation for secondary prevention. However, at present, fresh embryo transfer combined with antagonist administration should only be used with caution by experienced practitioners, after carefully deciding which patients can have a fresh transfer or embryo cryopreservation, until the current data are confirmed by larger trials.

Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis.

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

Live births after management of severe OHSS by GnRH antagonist administration in the luteal phase.

Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation protocols. Currently, no curative therapy exists and the main preventive option is cycle cancellation. Gonadotrophin-releasing hormone (GnRH) antagonist administration in the luteal phase was recently proposed as a new approach for the management of patients with established severe OHSS. Three polycystic ovarian syndrome patients undergoing IVF treatment developed severe OHSS, diagnosed 6 days after oocyte retrieval. On day 6, the patients underwent blastocyst transfer and received GnRH antagonist for 4 days, combined with luteal phase support using exogenous oestradiol and progesterone. Two patients had successful pregnancies that resulted in births of healthy infants, while one patient had a biochemical pregnancy. In all patients, established severe OHSS regressed to a moderate form of the syndrome, no pregnancy-induced life-threatening OHSS was observed, while a short monitoring period was required at an outpatient level, avoiding the need for patient hospitalization. This is the first report in the literature on GnRH antagonist administration in the luteal phase, combined with embryo transfer and exogenous oestradiol and progesterone supplementation. This novel treatment was effective in the regression of established severe OHSS, and resulted in the birth of healthy infants.

The expression of receptivity markers in the fallopian tube epithelium.

Pinopodes represent the morphological and integrins, the biomolecular markers of endometrial receptivity. We studied using scanning electron microscopy, the expression of pinopodes on tubal samples and their corresponding endometria, from 21 women of reproductive age (7 from proliferative phase, 7 from day LH +5 and 7 from day LH +7). In addition, we examined the immunohistochemical staining of integrins alpha v beta 3, alpha v beta 5 and their ligands, fibronectin (FN) and osteopontin (OPN) in the same tubal epithelium samples. Pinopodes were detected on the tubal epithelium exclusively during day LH +7, coincident with their formation in the endometrium and synchronous to alpha v beta 3 sharp increase in the oviduct epithelium, suggesting a regulation similar to the endometrium. In contrast, alpha v beta 5, FN and OPN remained unchanged during the cycle. These results show for the first time the formation of pinopodes in the tubal epithelium at the time of endometrial receptivity and correlate it with the upregulation of the intact dimmer alpha v beta 3 in the tubes.

Management of severe OHSS using GnRH antagonist and blastocyst cryopreservation in PCOS patients treated with long protocol.

Despite the fact that many methods have been proposed for the management of severe ovarian hyperstimulation syndrome (OHSS), its prevention is mainly achieved by withholding human chorionic gonadotrophin (HCG) administration and cycle cancellation. Currently no curative therapy is available. Three women diagnosed with polycystic ovarian syndrome underwent ovarian stimulation for IVF using a long gonadotrophin-releasing hormone (GnRH) agonist protocol. Six days after oocyte retrieval, severe early OHSS was diagnosed by analysis of haematocrit, white blood cell (WBC) count, serum urea, and ultrasonographic assessment of ovarian size and ascitic fluid. On the same day, antagonist administration was administrated and continued daily for 1 week, while resulting blastocysts were cryopreserved. Progression of severe early OHSS was inhibited in all three patients. A marked decrease of haematocrit, WBC, ascitic fluid, oestradiol, progesterone and ovarian volume was observed, during 1 week of follow-up suggesting a luteolytic effect of GnRH antagonist. None of the patients required hospitalization. In conclusion, GnRH antagonist administration combined with blastocyst cryopreservation 6 days post retrieval might represent a new approach for the effective management of patients with established severe OHSS. The flexibility of the approach allows the elongation of the monitoring period up to 8 days following HCG administration.

Management of severe early ovarian hyperstimulation syndrome by re-initiation of GnRH antagonist.

Several approaches have been proposed for the management of OHSS that reduce, but do not completely eliminate the incidence of human chorionic gonadotrophin (HCG)-induced early severe OHSS. Three women diagnosed with polycystic ovarian syndrome underwent ovarian stimulation for IVF using a gonadotrophin-releasing hormone (GnRH) antagonist protocol. Three days after oocyte retrieval, severe early OHSS was diagnosed by analysis of haematocrit (Ht), white blood cell (WBC) count, serum urea, and ultrasonographic assessment of ovarian size and ascitic fluid. On the same day, antagonist administration was re-initiated and continued daily for a week, while all embryos were cryopreserved. No progression of severe early OHSS was observed in any of the patients. A marked decrease of Ht, WBC count, ovarian volume and ascitic fluid was observed during 1 week of follow-up, and none of the patients required hospitalization. GnRH antagonist re-initiation might represent a new strategy for flexible management of patients with established severe early OHSS. Based on the flexibility of the approach, if severe OHSS does not occur, patients may proceed to embryo transfer, while if severe early OHSS ensues, antagonist administration combined with embryo cryopreservation appear to be associated with prevention of life-threatening OHSS, facilitation of regression of severe OHSS to a moderate form and avoidance of patient hospitalization.

Intratumoral CRH modulates immuno-escape of ovarian cancer cells through FasL regulation.

Although corticotropin-releasing hormone (CRH) and Fas ligand (FasL) have been documented in ovarian carcinoma, a clear association with tumour progression and immuno-escape has not been established. FasL plays an important role in promoting tumour cells' ability to counterattack immune cells. Here, we examined immunohistochemically the expression of CRH, CRHR1, CRHR2 and FasL in 47 human ovarian cancer cases. The ovarian cancer cell lines OvCa3 and A2780 were further used to test the hypothesis that CRH might contribute to the immune privilege of ovarian tumours, by modulating FasL expression on the cancer cells. We found that CRH, CRHR1, CRHR2 and FasL were expressed in 68.1, 70.2, 63.8 and 63.8% of the cases respectively. Positivity for CRH or FasL expression was associated with higher tumour stage. Finally, CRH increased the expression of FasL in OvCa3 and A2780 cells through CRHR1 thereby potentiated their ability to induce apoptosis of activated peripheral blood lymphocytes. Corticotropin-releasing hormone produced by human ovarian cancer might favour survival and progression of the tumour by promoting its immune privilege. These findings support the hypothesis that CRHR1 antagonists could potentially be used against ovarian cancer.

Solid cerebral echinococcosis mimicking a primary brain tumor.

Solitary brain affection is rare in echinococcosis. We report the case of a 35-year-old woman presenting with symptomatic grand-mal epilepsy due to a right frontal, partially cystic space-occupying lesion. Pre-operative computed tomography and magnetic resonance imaging (MRI) suggested a cystic astrocytoma. However, histological examination yielded the diagnosis of a 'chitinoma', a rare subtype of solid cerebral hydatid disease (echinococcosis). It mimicked a primary brain tumor and, therefore, posed a diagnostic problem. We present the--to our knowledge--first MRI scans in a case of a histologically proven chitinoma.

Microsurgical Study of the Dorello's Canal.

The microsurgical anatomy of Dorello's canal was examined in 20 cadaver specimens. The study included dissections of this anatomic structure, under operative microscope and using microsurgical instruments. The detailed microanatomy of the canal was defined, as well as its relation to the sixth nerve, the clivus, the petrous apex, and the adjacent neurovascular elements.