Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females. On day three postinsemination, one or two blastomeres were removed by embryo biopsy, and used for dual fluorescent in situ hybridisation with X and Y chromosome-specific DNA probes. In two cases, two female embryos were transferred and one pregnancy, (sex confirmed), is ongoing at 19 weeks. All eight embryos from one couple were of such poor quality that diagnosis was possible in one only. In the remaining two cases no embryos were transferred due to the detection of an abnormal number of X chromosome signals. Investigation of the biopsied embryos that were not transferred revealed evidence of mitotic non-disjunction in one and of complete X monosomy in a second. A surviving fetus with this latter constitution would have developed Turner syndrome and would also have been at high risk of X-linked disease. The use of fluorescent in situ hybridisation rather than the polymerase chain reaction allowed the detection of abnormal copy numbers of X chromosomes thus preventing the transfer of potentially abnormal zygotes.

We undertook a prospective longitudinal study of thyroid function in 60 normal pregnant women and measured serum concentrations of T4, triiodothyronine (T3), T-uptake, thyroxine binding globulin (TBG), free thyroxine index (FTI), free T4, albumin and thyrotropin (TSH). From these data we established reference ranges for each of these analytes for each trimester and examined the inter-relationships between laboratory measurements of thyroid function tests. We observed significant increases in serum concentrations of thyrotropin and decreases in free T4, assays commonly used as first line investigations of thyroid activity during pregnancy. However, the 95th centile intervals for both analytes remained within the reference range for nonpregnant women.

Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.

The literature suggests that the results of in-vitro fertilization (IVF) for patients with endometriosis depend on the stage of the disease, and that patients with severe endometriosis have a higher failure rate. Miscarriage is said to be more prevalent in women treated for endometriosis. In the study reported here, 140 patients with endometriosis underwent 182 cycles of IVF using gonadotrophin-releasing hormone analogues (GnRHa). Patients with endometriosis only were allocated to one group (group 4). The results were compared with those of three other groups of patients undergoing the same treatment within the same period. Group 1 consisted of couples with male factor only (45 cycles), group 2, couples with unexplained infertility (196 cycles) and group 3, couples with a tubal factor only (1139 cycles). The mean age of the patients, mean number of human menopausal gonadotrophin (HMG) ampoules administered, oestradiol concentration on the day of human chorionic gonadotrophin administration, number of days of HMG, mean number of oocytes retrieved and retrieval rate were not significantly different. The fertilization rate was significantly lower in group 1; no difference was observed in the other three groups. The mean number of normally fertilized embryos was not significantly different. The number of transferred embryos in each cycle and the implantation rates were similar in the four groups. The overall pregnancy rate per transfer was 39% in group 1, 48% in group 2, 45% in group 3 and 40% in group 4.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of prenatal genetic diagnosis is to give parents the option of terminating affected pregnancies. For most X linked conditions specific diagnosis is not possible and the only option is induced abortion of all male fetuses, of which half would be unaffected. An altemative is offered by preimplantation diagnosis of sex in embryos obtained by in vitro fertilisation and selective transfer of female embryos. The sex of preimplantation embryos has been determined by amplification of a DNA fragment specific to the Y chromosome by the polymerase chain reaction after biopsy of the embryo at the eight cell stage.' Live births of female infants have followed this procedure, testifying to its safety.2 Misdiagnosis is, however, possible because of contamination, failure of amplification, or the sampling of anuclear cytoplasmic fragments.' We report the application of dual colour fluorescent in situ hybridisation for sexing interphase nuclei of three day old human preimplantation embryos. Methods and resultsSix couples at risk of transmitting an X linked disorder underwent seven cycles of routine in vitro fertilisation treatment, and biopsy specimens of one or two cells were taken from embryos at the 6-10 cell stage early on the third day after fertilisation.' The cells were pretreated with hypotonic solution, fixed, and spread on to glass slides as described previously.4 Dual fluorescent in situ hybridisation was performed by dehydrating the specimens, treating them with ribonuclease A and proteinase K, and hybridising them with biotinylated probe pBamX7 (specific for the X chromosome) and digoxigenin labelled probes pHY2. 1 and cY98 (specific for the Y chromosome). The two hour hybridisation was followed by post-hybridisation washes.4The biotin and digoxigenin labels were detected with fluorescein isothiocyanate (green fluorochrome) conjugated with avidin and tetramethyl rhodamine isothiocyanate (red fluorochrome) conjugated with anti-digoxigenin antibody. Both were applied simultaneously in a TRIS based buffer containing a fluorochrome blocking reagent. Slides were mounted in an anti-fade medium with the blue fluorescent DNA counterstain diamidinophenylindole and then viewed for blue, green, and red fluorescence separately. This was adapted from our earlier dual fluorescent in situ hybridisation technique4 to allow the whole procedure to be performed in one working day. Nuclei were located by means of the counterstain. Cells, and hence embryos, were diagnosed as female if two green (X chromosome) and no red (Y chromosome) signals could be seen and as male if one or more red signals was seen. Control lymphocyte material gave 95-100% ofthe expected number of signals from interphase nuclei, and no male nucleus was ever identified as female.A positive female diagnosis on a single embryonic cell was considered to be sufficient to allow transfer to proceed. Selective transfer of female embryos took place late on the third day after fertilisation. In six of the cycles of treatment two female embryos were transferred, while...

Thrombophilia and impaired placental vasculature are a major cause of adverse pregnancy outcome. In 2007, a new hereditary factor for obstetric complications and recurrent pregnancy loss (RPL) was identified as a sequence variation in the core promoter of the annexin A5 gene, ANXA5, called the M2 haplotype. M2 carriership has been demonstrated in couples with recurrent miscarriage and its origin is embryonic rather than specifically maternal, confirmed by subsequent papers. The M2 haplotype is the first report of a hereditary factor related to pregnancy pathology caused by embryonic-induced anticoagulation. It has been demonstrated that couples with RPL had equal and significantly increased M2 carriership and that maternal and paternal carriership confers equal risk. Given its importance for patients with RPL, and potentially implantation failure, this study assessed the incidence of carrier status for the M2 ANXA5 haplotype in both the male and female of couples attending five CARE IVF centres. In 314 patients (157 couples), 44% of couples (one or both partners), 24% of females, 26% of males and 37% of couples with unexplained infertility were M2 carriers. This high incidence has provoked further urgent studies on specific patient populations and on the value of post embryo-transfer therapy.

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BackgroundPregnancy failure and placenta mediated pregnancy complications affect > 25% of pregnancies. Although there is biological plausibility for a procoagulant mechanism underlying some of these events, antithrombotic intervention trials demonstrate limited benefit, possibly through lack of stratification in heterogeneous patient groups. The ANXA5 M2 haplotype is a possible procoagulant biomarker and was tested pragmatically to determine whether this screening and LMWH treatment normalized the outcome for ANXA5 M2 positive couples.This was a pragmatic study that aimed to measure the effectiveness of a testing (for the M2 haplotype) and treatment (LMWH) pathway in routine clinical practice where there is variation between patients. Such a study in couples with fertility problems can inform choices between treatments; it is then the management protocol which is the subject of the investigation, not the individual treatments.MethodsCouples (N = 77) with one or both partners ANXA5 M2 positive demonstrated association of this haplotype with adverse IVF outcome. A pragmatic, multicenter, prospective cohort study of ANXA5 M2 haplotype screening, and LWMH treatment following embryo transfer (ET) in 103 IVF couples positive for ANXA5 M2 was performed. They were compared with a group of 1000 contemporaneous randomly selected unscreened and untreated couples undergoing assisted conception, from which 103 matched control couples were derived. The primary outcome measure was live birth incidence. Secondary outcomes were results following embryo transfer (ET) and live birth outcome by gender and M2 carriage, and allelic dose influence.FindingsThe tested and treated cohort of ANXA5 M2 carriers achieved a similar live birth rate (37.9%) per ET cycle compared to both the more fertile comparison group (38.5%), and to the 103 matched controls (33.0%). Significantly more treated male carrier only couples had a live birth versus female M2 only (47.7% vs. 25.0% p = 0.045).InterpretationPragmatic ANXA5 M5 screening and treatment with LMWH in couples undergoing IVF is associated with similar outcome to couples with more favorable prognostic factors. The difference in live birth outcome for treated male only carrier couples may be consistent with an additional maternal thrombophilic factor that may adversely affect pregnancy, although other mechanisms are possible. This study suggests that LMWH treatment should be started prior to clinical pregnancy.