Marc Dhont

Perinatal outcome of pregnancies after assisted reproduction: A case-control study

PURPOSEnA matched case-control study of all pregnancies obtained after either IVF or ICSI was conducted to investigate the perinatal outcome.nnnMETHODSnThree hundred eleven singleton and 115 twin pregnancies obtained after assisted reproduction were studied. Controls were selected from a regional register and were matched for maternal age, parity, singleton or twin pregnancy, and date of delivery.nnnRESULTSnNo significant difference was observed for gestational age at delivery, birth weight, incidence of congenital anomalies, and incidence of perinatal mortality between ART (singleton and twin) pregnancies and spontaneous controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies (52 vs 42%; P < 0.05) and needed more neonatal intensive care (47 vs 26%; P < 0.05).nnnCONCLUSIONSnFrom this case-control study it is concluded that the perinatal outcome of ART singleton pregnancies is not different from that in matched controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies and needed more neonatal intensive care.

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.

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity

OBJECTIVEnTo investigate a method of assisted activation of human oocytes for the treatment of infertility resulting from globozoospermia associated with deficient oocyte activation capacity.nnnDESIGNnThe mouse oocyte activation test was used to analyze the oocyte activation capacity of the sperm cells of a patient with globozoospermia. Fresh donor human oocytes were used for determining the most appropriate procedure for oocyte activation.nnnSETTINGnInfertility Center, University Hospital of Ghent.nnnPATIENT(S)nA couple with infertility resulting from globozoospermia.nnnINTERVENTION(S)nIntracytoplasmic sperm injection, assisted oocyte activation, and embryo transfer.nnnMAIN OUTCOME MEASURE(S)nOocyte activation and fertilization rates, implantation, and pregnancy.nnnRESULT(S)nDeficiency in oocyte activation capacity was found in the sperm of a patient with globozoospermia. This deficiency was proven by the mouse oocyte activation test and was confirmed further by lack of activation of human oocytes after simple sperm injection. Only human oocytes that were injected with sperm and subjected to calcium chloride and ionophore treatment underwent activation. Transfer of embryos obtained by this procedure of assisted oocyte activation resulted in an ongoing pregnancy.nnnCONCLUSION(S)nAssisted oocyte activation of human oocytes is useful when globozoospermia is associated with absence of oocyte activation capacity in the sperm. These cases can be identified by the mouse oocyte activation test.

Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5-year appraisal

One of the most problematic issues of assisted reproduction is the high incidence of multiple pregnancies, resulting from the transfer of more than one embryo. Particularly at risk are young women who have good quality embryos. The only strategy to reduce the incidence of multiple pregnancies, including twin pregnancies, after assisted reproduction is single embryo transfer (SET). In 1997, the present authors therefore introduced elective SET (eSET) in this particular target group. The proportion of eSET increased from 1.5 (1997-1998) to 17.5% (1999-2002) of all transfers. In 2002, 20% of all transfers were SET. Comparing these two periods, an overall pregnancy rate of 35 and 34% per transfer, respectively, was obtained, while the overall twinning rate dropped from 30 to 21%. The twinning rate dropped to 14% in 2002, and in the eSET group there was only one monozygotic twin. These results demonstrate that a decline in the twinning rate is feasible without a drop in overall pregnancy rates. Comparing eSET with elective double embryo transfer (eDET), it was found that ongoing pregnancy and implantation rates were the same in both groups, but the proportion of twins was clearly different. It was further observed that the mean birthweight of singleton children born after eSET was significantly higher than that after DET. This could reflect a better developmental or implantation potential of these embryos, but this finding remains to be confirmed.

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.

Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice

OBJECTIVEnTo investigate follicle growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice.nnnSTUDY DESIGNnFresh or frozen-thawed human ovarian cortex was grafted subcutaneously or under the kidney capsule of 43 mice (35 nude mice and eight SCID mice), 14 of which were non-stimulated controls, 21 injected intra-peritoneally with gonadotrophins during 2 weeks and eight injected during 3 months. Follicle count was compared by Chi-square.nnnRESULTSnProportions of primordial follicles were significantly lower in grafts than in the tissue before transplantation in gonadotrophin-stimulated mice (37% versus 79%), but not in non-stimulated mice (51% versus 74%). Proportions of primary and secondary follicles were increased after transplantation indicating early follicular growth. One antral follicle was observed in a graft in a mouse stimulated for 3 months.nnnCONCLUSIONnPrimordial follicles in fresh or frozen-thawed human ovarian cortex transplanted under the kidney capsule or subcutaneously can grow and are responsive to hormonal stimulation.nnnCONDENSATIONnPrimordial follicles in fresh and cryopreserved human ovarian cortical grafts can initiate growth after transplantation to immunodeficient mice

Perinatal outcome of pregnancies after assisted reproduction: A case-control study.

AbstractPurpose: A matched case–control study of all pregnancies obtained after either IVF or ICSI was conducted to investigate the perinatal outcome.Methods: Three hundred eleven singleton and 115 twin pregnancies obtained after assisted reproduction were studied. Controls were selected from a regional register and were matched for maternal age, parity, singleton or twin pregnancy, and date of delivery.Results: No significant difference was observed for gestational age at delivery, birth weight, incidence of congenital anomalies, and incidence of perinatal mortality between ART (singleton and twin) pregnancies and spontaneous controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies (52 vs 42%; P < 0.05) and needed more neonatal intensive care (47 vs 26%; P < 0.05).n Conclusions: From this case–control study it is concluded that the perinatal outcome of ART singleton pregnancies is not different from that in matched controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies and needed more neonatal intensive care.

Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions

OBJECTIVEnTo study the influence of low-sodium cryopreservation media (CPM) on the survival and development of frozen-thawed germinal vesicle (GV) stage and in vitro matured human oocytes.nnnDESIGNnProspective experimental study.nnnSETTINGnAcademic hospital-based fertility center.nnnPATIENT(S)nExperimental groups: Oocytes cryopreserved at the GV (group A, n = 63 and group B, n = 64) or M II stage (group C, n = 62) with use of conventional (group A) or low-sodium CPM (groups B and C). Control groups: Sibling GV stage oocytes subjected to in vitro maturation (IVM; control group A, n = 64; control group B, n = 64).nnnINTERVENTION(S)nIVM, intracytoplasmic sperm injection and subsequent culture.nnnMAIN OUTCOME MEASURE(S)nRates of survival, maturation, fertilization, and cleavage.nnnRESULT(S)nThe postthaw survival was significantly lower in groups A (57.1%) and B (48.4%) compared to C (84.4%). In group A, maturation and cleavage rates were significantly lower, and fertilization rate was similar to controls (GVBD: 72.2% vs. 90.6%; progression to M II: 33.3% vs. 76.6%; cleavage: 42.9% vs. 88.2%; and fertilization: 58.3% vs. 69.4% in group A vs. control group A, respectively). There was no such difference in group B. In group C, despite a slight but significant lowering of the rate of 2 PN and an increase in that of 3 PN (2 PN: 47.4% vs. 70.2% and 3 PN: 15.8% vs. 3.2% in group C vs. total controls, respectively), embryonic cleavage per GV oocyte was significantly higher (25.8%) compared to group A (4.8%) but not to group B (15.6%). The rate of maturation and cleavage per surviving GV oocyte was significantly higher in group B than group A.nnnCONCLUSION(S)nLow-sodium-based CPM is beneficial for in vitro matured M II stage oocytes and is significantly better than the conventional sodium-based media for the GV stage oocytes.

Maturation of Mouse Primordial Follicles by Combination of Graftingand In Vitro Culture

Abstract Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j × CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-μl droplets of α-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO2 in air at 37°C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14–16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 ± 4.45 μm) was similar to that of oocytes from the control group (70.5 ± 2.35 μm). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.

Effect of culture media on in vitro development of cloned mouse embryos.

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.