A. De Vos

Preimplantation diagnosis for Huntington's disease (HD): clinical application and analysis of the HD expansion in affected embryos

Huntingtons disease (HD) is an autosomal dominant disease characterized by motor disturbance, cognitive loss and psychiatric manifestations, starting between the fourth and the fifth decade, followed by death within 10–20 years of onset of the disease. The disease‐causing mutation is an expansion of a CAG triplet repeat at the 5′ coding end of the Huntington gene. We have developed a single‐cell PCR assay for the HD gene in order to propose preimplantation genetic diagnosis (PGD) for the couples at risk. We present here our first results with our first nine PGD cycles and also discuss the behaviour of the disease‐causing expansion in pre‐implantation embryos. Copyright

Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers

BACKGROUNDnHuman embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated.nnnMETHODSnIn order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%).nnnCONCLUSIONSnThe clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.

Zona Hardening, Zona Drilling and Assisted Hatching: New Achievements in Assisted Reproduction

Prior to fertilization, the zona pellucida surrounding the mammalian oocyte acts as a species-specific sperm barrier and is involved in sperm binding. After fertilization, the zona plays a role in blocking polyspermic fertilization, it protects the integrity of the preimplantation embryo during early embryonic development, and also helps its oviductal transport. Zona hardening occurs naturally after fertilization in order to ensure this threefold function. A combination of lysins produced by the cleaving embryo or the uterus and physical expansion then reduces the zona thickness in preparation for hatching. Zona hardening, although not readily quantifiable, may also be induced by in vitro culture and by in vivo aging. Indeed, prolonged exposure of human oocytes and embryos to artificial culture conditions seems to impair their ability to implant. Implantation rates are also inversely correlated with advanced female age. Recently, failure of the embryonic zona pellucida to rupture following blastocyst expansion has been put forward as a possible contributing factor in implantation failure. In order to help embryos escape from their zonae during blastocyst expansion, different types of assisted hatching have been developed. Zona drilling involves the creation of an opening in the zona with acidified medium, whereas zona slitting is carried out in the same manner as partial zona dissection. In zona thinning, the zona is just made thinner over a certain area without a hole or a slit being created. More recently, laser-assisted hatching has been introduced. In vitro studies with both mouse and human embryos have indicated that an artificial gap in the zona pellucida significantly improves the hatching ability of blastocysts grown in vitro as compared to non-micromanipulated embryos. However, the clinical relevance of assisted hatching within an assisted reproduction program remains controversial and elusive. Very few randomized studies are available. Most reports are of retrospective analyses which report either no differences in implantation and pregnancy rates between assisted hatching and control embryos or better results after assisted hatching. Five randomized controlled studies suggest that assisted hatching - of no benefit to the overall patient population - might be of value in increasing embryo implantation rates only in selected cases. No further evidence exists for an age-related benefit from assisted hatching in patients with advanced maternal age.Prior to fertilization, the zona pellucida surrounding the mammalian oocyte acts as a species-specific sperm barrier and is involved in sperm binding. After fertilization, the zona plays a role in blo

Does intracytoplasmic morphologically selected sperm injection improve embryo development? A randomized sibling-oocyte study

STUDY QUESTIONnDoes high-magnification sperm selection influence oocyte fertilization and further embryo development?nnnSUMMARY ANSWERnThe present study did not show a difference in oocyte fertilization rate, nor in embryo development between high-magnification intracytoplasmic morphologically selected sperm injection (IMSI) and conventional ICSI.nnnWHAT IS KNOWN ALREADYnThe presence of nuclear vacuoles in sperm seems to influence embryo development and more specifically blastocyst formation. The use of high magnification for morphological sperm selection prior to ICSI has been associated with higher pregnancy rates and lower miscarriage rates.nnnSTUDY DESIGN, SIZE, DURATIONnA prospective sibling-oocyte study was conducted, including 350 ICSI cycles to alleviate male infertility. Cycles were included from March 2010 to November 2011.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnOn the day of treatment, a high-magnification sperm morphology was assessed on at least 200 spermatozoa. Primary endpoints were oocyte fertilization rate and embryo development. Because embryo transfers were not randomized, the clinical outcome (clinical pregnancy rate per transfer cycle) was descriptive. However, the embryologist selecting the embryos for transfer was blinded for the sperm selection procedure.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIMSI morphology was assessed in 330 semen samples, resulting in the following distribution: 18.1 ± 14.8% Grade I, 15.2 ± 10.3% Grade II, 12.3 ± 9.1% Grade III and 54.4 ± 23.2% Grade IV. Oocyte fertilization rate was 79.1 and 77.3% after IMSI and ICSI, respectively (NS, paired t-test). Embryo development was similar in both treatment groups up to Day 5 of preimplantation development. Comparable numbers of IMSI-only (n = 125) and ICSI-only (n = 139) embryo transfers were performed. Clinical pregnancies with fetal heart beat were equally distributed over transfers with embryos from IMSI-only (34.4%) or ICSI-only treatment (36.7%).nnnLIMITATIONS, REASONS FOR CAUTIONnThe clinical outcome remains descriptive. No firm conclusions could be drawn on cycle rank as a possible indication for IMSI.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe prevalence of vacuoles in normal-shaped spermatozoa is as low as 27.5%. A routine application of IMSI in unselected artificial reproductive technology patients cannot be advocated.nnnSTUDY FUNDING/COMPETING INTEREST(S)nNone.

Preimplantation genetic diagnosis for sickle‐cell anemia and for β‐thalassemia

We developed single‐cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the β‐globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle‐cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two β‐thalassemia PGD protocols: one for the analysis of the 25‐26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non‐labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non‐labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR‐PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%. Copyright

Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos

STUDY QUESTIONnIs the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos?nnnSUMMARY ANSWERnVitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight.nnnWHAT IS KNOWN ALREADYnAfter slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage.nnnSTUDY DESIGN, SIZE, DURATIONnSurvival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnEmbryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed.nnnMAIN RESULTS AND ROLE OF CHANCEnSurvival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification.nnnLIMITATIONS, REASONS FOR CAUTIONnThis analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos.nnnWIDER IMPLICATIONS OF THE FINDINGSnAlthough it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed.nnnSTUDY FUNDING/COMPETING INTEREST(S)nnone.

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the ΔF508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non‐affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born. Copyright

The type of culture medium and the duration of in vitro culture do not influence birthweight of ART singletons

STUDY QUESTIONnDoes the type of in vitro culture medium or the duration of in vitro culture influence singleton birthweight after IVF/ICSI treatment?nnnSUMMARY ANSWERnIn a comparison of two culture media, neither the medium nor the duration of culture (Day 3 versus Day 5 blastocyst transfer) had any effect on mean singleton birthweight.nnnWHAT IS KNOWN ALREADYnPrevious studies indicated that in vitro culture of human embryos may affect birthweight of live born singletons. Both the type of culture medium and the duration of culture may be implicated. However, these studies are small and report conflicting results.nnnSTUDY DESIGN, SIZE, DURATIONnA large retrospective analysis was performed including all singleton live births after transferring fresh Day 3 or Day 5 embryos. IVF and ICSI cycles performed between April 2004 and December 2009 at a tertiary care centre were included for analysis.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnA total of 2098 singleton live births resulting from singleton pregnancies were included for analysis. Two different sequential embryo culture media were concurrently used in an alternating way: Medicult (n = 1388) and Vitrolife (n = 710). Maternal age, maternal and paternal BMI, maternal parity, maternal smoking, main cause of infertility, cycle rank, stimulation protocol, method of fertilization (IVF or ICSI), time in culture and number of embryos transferred were taken into account. Embryo transfers were performed either on Day 3 (n = 1234) or on Day 5 (n = 864). Singleton birthweight was the primary outcome parameter. Gestational age and gender of the newborn were accounted for in the multiple regression analysis.nnnMAIN RESULTS AND THE ROLE OF CHANCEnNo significant differences in mean singleton birthweight were observed between the two culture media: Medicult 3222 g (±15 SE) and Vitrolife 3251 g (±21 SE) (P = 0.264). The mean singleton birthweight was not different between Day 3 embryo transfers (3219 ± 16 g) and Day 5 blastocyst transfers (3250 ± 19 g; P = 0.209). Multiple regression analysis controlling for potential maternal, paternal, treatment and newborn confounders confirmed the non-significant differences in mean singleton birthweight between the two culture media. Likewise, the adjusted mean singleton birthweight was not different according to the duration of in vitro culture (P = 0.521).nnnLIMITATIONS, REASONS FOR CAUTIONnThe conclusions are limited by its retrospective design; however, the two different sequential culture systems were used in an alternating way during the same time period. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were not included in the analysis.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis large retrospective study does not support earlier concerns that both the type of culture medium and the duration of embryo culture influence singleton birthweight. However, a continuous surveillance of human embryo culture procedures (medium type, culture duration and other culture conditions) should remain a priority within assisted reproduction technology.nnnSTUDY FUNDING/COMPETING INTERESTSnNone.

Closed blastocyst vitrification of biopsied embryos: evaluation of 100 consecutive warming cycles

BACKGROUNDnThe aim of this study was to analyse the outcome of closed blastocyst vitrification of embryos biopsied at the cleavage stage.nnnMETHODSnVitrification of supernumerary blastocysts was performed using the closed CBS-VIT High Security straws. Warming cycles (n = 100) for patients with preimplantation genetic diagnosis (PGD) and/or aneuploidy screening in the fresh cycle were analysed. The outcome parameters were morphological survival and transfer rates after warming, clinical pregnancy rate and implantation rate (with fetal heart beat). Clinical outcome was compared with two control groups of (i) vitrified/warming transfer cycles without embryo biopsy and (ii) fresh Day 5 transfer of biopsied embryos.nnnRESULTSnIn total, 131 blastocysts were warmed with a morphological survival of 83.2% (109/131) and a transfer rate of 79.4% (104/131). Day 5 blastocysts survived significantly better (90.4%) than Day 6 blastocysts (70.8%, P < 0.01). No difference in survival rate was observed between early cavitating (89.2%) and full/expanded blastocysts (93.3%). In nine cycles, no blastocyst was available for transfer. The clinical pregnancy rate was 19.2% (15/78) after single-embryo transfer (SET) and 38.5% (5/13) after double-embryo transfer (DET). In SET, the implantation rate for blastocysts frozen on Day 5 was 13.7% (7/51), which was not different from the implantation rate of Day 6 blastocysts (18.5%, 5/27). The overall implantation rate of vitrified PGD biopsied blastocysts (14.4%) was comparable with that of vitrified blastocysts without biopsy (20.4%), but lower than the implantation rate obtained in the fresh PGD cycles (24.4%).nnnCONCLUSIONnBlastocysts on Day 5 and Day 6 of development derived from biopsied embryos can be successfully vitrified using a closed system.

Integrated Molecular Diagnosis of Theileria and Babesia Species of Cattle in Italy

Abstract: A reverse line blot hybridization (RLB) test was developed to specifically identify six Theileria spp. (T. annulata, T. parva, T. mutans, T. velifera, T. taurotragi, and T. buffeli/orientalis) and three Babesia spp. (B. bovis, B. bigemina, and B. divergens). No cross reaction was observed with other livestock pathogens (such as Anaplasma marginale, A. centrale, A. ovis, Cowdria ruminantium, Trypanosoma brucei, T. congolense, and T. vivax). This method was used to test bovine blood samples collected in Sicily in April and November, 1998. Preliminary results indicated that T. annulata and T. buffeli/orientalis were the main species observed in cattle blood. Babesia species represented 1.8% and 23.5% in April and November, respectively.