Jan Gerris

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUNDnIn mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta.nnnMETHODS AND RESULTSnImmunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations.nnnCONCLUSIONSnOur findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection

Failed fertilization after intracytoplasmic sperm injection (ICSI) can occur due to an oocyte activation defect. In these cases assisted oocyte activation (AOA) may help but efficiency is still unknown. Prior to AOA, the mouse oocyte activation test (MOAT) can be carried out by injecting human spermatozoa into mouse oocytes to evaluate their activating capacity. According to the MOAT activation percentage achieved, patients were classified into three groups: 0-20% (16 patients); 20-85% (seven patients); 85-100% (seven patients). For AOA, CaCl(2) was injected together with spermatozoa followed by a double Ca(2+) ionophore treatment. The fertilization rates before application of AOA in 50 cycles were 6%, 22% and 14% in, respectively, groups 1, 2 and 3 without any pregnancy. Fertilization and pregnancy rates after AOA in 61 cycles were significantly increased to 75% and 34% for group 1, 73% and 43% for group 2, and 75% and 17% for group 3 (P < 0.0001 and P < 0.004, respectively). Application of AOA results in normal fertilization and pregnancy rates in patients whose spermatozoa show deficient activation. When MOAT reveals no activation deficiency in spermatozoa, AOA still allows for high fertilization and acceptable pregnancy rates. The obstetric and neonatal outcomes after AOA were normal as no malformations were observed.

Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency

BACKGROUNDnDespite the success of ICSI, total fertilization failure (TFF) still occurs in 1-3% of all ICSI cycles. ICSI followed by assisted oocyte activation (ICSI-AOA) can restore fertilization, most efficiently in cases of sperm-related fertilization deficiency. The indication for ICSI-AOA is less obvious when the capacity of the sperm to activate oocytes is considered normal, as proved by a heterologous ICSI model, such as the mouse oocyte activation test (MOAT). In this study, we verified whether ICSI-AOA is beneficial for patients in whom an oocyte-related activation deficiency is suspected.nnnMETHODSnA prospective study was conducted including patients presenting with a history of TFF or low fertilization (LF) following conventional ICSI in our centre (in-house cases, n= 2) or elsewhere (out-house cases, n= 12). In all cases a sperm deficiency was refuted by the MOAT. In a next treatment cycle, ICSI-AOA was performed on half of the sibling metaphase II oocytes and conventional ICSI on the rest (split ICSI-AOA cycle). The main outcome parameters were fertilization, pregnancy and live birth rates.nnnRESULTSnOverall, ICSI-AOA was able to improve fertilization rates in couples with a suspected oocyte-related fertilization problem, with a mean fertilization rate of 74.2% following ICSI-AOA compared with 43.5% following conventional ICSI (P< 0.001). Cumulative pregnancy rate and live birth rate per cycle were 35.7 and 14.3%, respectively. Considering the out-house patients only, fertilization rates with ICSI-AOA were higher in couples with previous TFF than with conventional ICSI (P< 0.001). Interestingly, for out-house patients who had experienced low, but not zero, fertilization elsewhere, ICSI-AOA could not enhance the fertilization rate. For the two in-house patients, both suffering from previous LF following conventional ICSI, the ICSI-AOA procedure enhanced the mean fertilization rate (25 versus 75%, respectively).nnnCONCLUSIONSnFor patients with a suspected oocyte-related activation deficiency, as diagnosed by a heterologuous ICSI model, the indication for ICSI-AOA still remains debatable. Our data show that ICSI-AOA is very efficient in patients with a suspected oocyte-related activation deficiency and previous TFF after conventional ICSI. In contrast, when there was a history of LF in another centre, one should be careful and test the efficiency of ICSI-AOA on half of the sibling oocytes, because ICSI-AOA is not always beneficial for patients with previous LF and a suspected oocyte-related activation deficiency. For these patients, a split ICSI-AOA cycle using sibling oocytes can help to distinguish between a molecular oocyte-related activation deficiency and a previous technical or other biological failure. Moreover, this split ICSI-AOA strategy enables us to set the appropriate strategy for future treatment cycles. Further research with larger groups of patients is now required.

Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes.

The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre‐maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3‐inhibitor (PDE3‐I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus–oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to10 µM) for 24 hr. Afterwards, oocytes were removed from PDE3‐I‐containing medium and underwent in vitro maturation (IVM) for 16–18 hr. A concentration of 1 µM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3‐I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3‐I is a safe and reliable method to improve oocyte quality after IVM. Mol. Reprod. Dev. 75: 1021–1030, 2007.

Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial

BACKGROUNDnIt has been suggested in the literature that low-dose aspirin leads to an increased number of oocytes in IVF/ICSI as well as a higher pregnancy rate. The aim of the present study was to investigate the effect of daily administration of low-dose aspirin, compared with placebo, on pregnancy rate in IVF and ICSI.nnnMETHODSnThis study was a prospective, randomized, double-blind placebo controlled trial, performed in the fertility centre of the University Hospital of Ghent. Concealed allocation by computerized randomization was done by the central pharmacy of the hospital. Daily oral administration of aspirin 100 mg or placebo started before stimulation and was continued until confirmation of pregnancy by detection of fetal heart activity on ultrasound. The primary outcome measure assessed in this trial was clinical pregnancy rate per cycle.nnnRESULTSnTwo hundred and one couples were included in this study, 193 women (aspirin group n = 97, placebo group n = 96) started treatment and 181 underwent an embryo transfer. There were 31 clinical pregnancies (31/97 or 32%) in the aspirin group versus 30 (30/96 or 31%; P = 0.916; OR 1.033; 95% CI 0.565-1.890) in the placebo group.nnnCONCLUSIONSnThis randomized controlled trial could not show a significant difference in clinical pregnancy rate between the aspirin and the placebo group in a first or second IVF/ICSI cycle. Given the lack of evidence for a beneficial effect of low-dose aspirin, it appears that low-dose aspirin should not be prescribed routinely in IVF/ICSI treatment. ClinicalTrials.gov Identifier: NCT00644085.

Placebo-controlled trial of high-dose Mesterolone treatment of idiopathic male infertility

The possible effect of Mesterolone (Schering N.V., Brussels, Belgium) (l α -methyl-5- α -androstane-17 β -ol-3-one) on semen quality and fertility of men with idiopathic oligoasthenospermia and/or teratozoospermia has been evaluated in a double-blind trial. The study included 52 patients who were treated during 12months with either 150mg/d of Mesterolone or placebo. The overall pregnancy rate was similar in the Mesterolone-treated cases (26%) and in the placebo control cases (48%), although a significant increase in motility and in the proportion of spermatozoa with normal morphology was recorded in the Mesterolone-treated cases. Because similar semen improvement also occurred in the placebo controls, our findings cast doubt on the possible usefulness of high-dose Mesterolone treatment of idiopathic male infertility.

Back muscle as a promising site for ovarian tissue transplantation, an animal model

BACKGROUNDnThe aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments in murine hosts. We compared the transplantation to the back muscle (B) versus the kidney capsule (K) in a mouse allograft model.nnnMETHODSnHemi-ovaries from 12-day-old mice were allografted into B and K of bilaterally ovariectomized same strain recipients which had undergone gonadotrophin stimulation (n = 15). Graft survival after 27 days, angiogenesis and follicle development were scored and compared to age-matched control ovaries (38-day old, n = 5). The ability of oocytes to be fertilized was studied after IVF, ICSI and embryos were transferred to recipient mothers. Anti-mouse CD 31+ antibody was used to evaluate neo-vascularization in grafts.nnnRESULTSnPrimordial follicle survival was higher (P < 0.01) and vascular support was better (P < 0.01) in B- than in K-grafts. From 34 oocytes retrieved from B-grafts (15 metaphase I, of which 14 matured in vitro, and 19 collected at metaphase II), 18 morulae were obtained. Transfer of 12 embryos obtained by ICSI led to three live offspring, and transfer of six IVF embryos to another recipient mother yielded four offspring, one of which was born dead and one showed placental anomalies.nnnCONCLUSIONSnThe back muscle is a promising site for ovarian allografts in mice. This is the first report of live offspring obtained after back muscle grafting using both IVF and ICSI.

Relation of androgen receptor sensitivity and mood to sexual desire in hormonal contraception users

BACKGROUNDnSince very little research in this field is available, this study aims to assess the role of psychosexual, relationship, hormonal and genetic measures in the sexual desire of users of three hormonal contraceptive products [low-dose combined oral contraceptive (20 mcg ethinylestradiol/150 mcg desogestrel), progestin-only pill (75 mcg desogestrel) and vaginal ring (daily dose of 15 mcg ethinylestradiol/120 mcg etonogestrel)].nnnSTUDY DESIGNnFifty-five couples were randomized over three groups in which the women consecutively used each product during 3 months. Both partners repeatedly filled out questionnaires on solitary and dyadic sexual desire (desire to behave sexually by oneself or towards a partner). Total and free testosterone, sex hormone binding globulin and a genetic marker of androgen receptor sensitivity [cytosine-adenine-guanine (CAG) repeat length] were assessed on blood samples of the female partners.nnnRESULTSnSexual desire was higher in women with either short or long CAG repeats (solitary, p=.004; dyadic, p=.008). Desire levels were higher during vaginal ring use (solitary, p=.018; dyadic, p=.007). The womans mood was found to impact her dyadic sexual desire (p<.001); this scale was also strongly associated with the male partners dyadic sexual desire (p<.001).nnnCONCLUSIONSnThe current study found evidence for a role of androgen receptor sensitivity and mood in the sexual desire of hormonal contraceptive users.

Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation

Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential.

Effect of ionomycin on oocyte activation and embryo development in mouse

Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 micromol/l. An improved effect of a second exposure to 5 micromol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.