Fertilization in Vitro

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

The objective was to develop recommendations for the diagnosis and treatment of endometriosis and its associated symptoms. A working group was convened comprised of practising gynaecologists and experts in evidence-based medicine from Europe, as well as an endometriosis self-help group representative. After reviewing existing evidencebased guidelines and systematic reviews, the expert panel met on three occasions for a day during which the guideline was developed and refined. Recommendations based solely on the clinical experience of the panel were avoided as much as possible. The entire ESHRE Special Interest Group for Endometriosis and Endometrium was given the opportunity to comment on the draft guideline, after which it was available for comment on the ESHRE website for 3 months. The working group then ratified the guideline by unanimous or near-unanimous voting; finally, it was approved by the ESHRE Executive Committee. The guideline will be updated regularly, and will be made available at http://www.endometriosis.org/guidelines.html with hyperlinks to the supporting evidence, and the relevant references and abstracts. For women presenting with symptoms suggestive of endometriosis, a definitive diagnosis of most forms of endometriosis requires visual inspection of the pelvis at laparoscopy as the 'gold standard' investigation. However, pain symptoms suggestive of the disease can be treated without a definitive diagnosis using a therapeutic trial of a hormonal drug to reduce menstrual flow. In women with laparoscopically confirmed disease, suppression of ovarian function for 6 months reduces endometriosis-associated pain; all hormonal drugs studied are equally effective although their side-effects and cost profiles differ. Ablation of endometriotic lesions reduces endometriosis-associated pain and the smallest effect is seen in patients with minimal disease; there is no evidence that also performing laparoscopic uterine nerve ablation (LUNA) is necessary. In minimal-mild endometriosis, suppression of ovarian function to improve fertility is not effective, but ablation of endometriotic lesions plus adhesiolysis is effective compared to diagnostic laparoscopy alone. There is insufficient evidence available to determine whether surgical excision of moderate-severe endometriosis enhances pregnancy rates. IVF is appropriate treatment especially if there are coexisting causes of infertility and/or other treatments have failed, but IVF pregnancy rates are lower in women with endometriosis than in those with tubal infertility. The management of severe/deeply infiltrating endometriosis is complex and referral to a centre with the necessary expertise is strongly recommended. Patient self-help groups can provide invaluable counselling, support and advice.

Objective: To summarize the role of reactive oxygen species (ROS) in the pathophysiology of human reproduction. Design: Review of literature. Setting: Fertility research center and obstetrics and gynecology department in a tertiary care facility. Result(s): ROS plays an essential role in the pathogenesis of many reproductive processes. In male-factor infertility, oxidative stress attacks the fluidity of the sperm plasma membrane and the integrity of DNA in the sperm nucleus. Reactive oxygen species induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility. ROS mediated female fertility disorders share many pathogenic similarities with the ones on the male side. These similarities include a potential role in the pathophysiology of endometriosis and unexplained infertility. High follicular fluid ROS levels are associated with negative IVF outcomes, particularly in smokers. Moreover, oxidative stress may be responsible in hydrosalpingeal fluid mediated embryotoxicity as well as poor in vitro embryonic development. Conclusion(s): High levels of ROS are detrimental to the fertility potential both in natural and assisted conception states. (Fertil Steril 2003;79:829 -43.

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Previously reported better fertilization rate after intracytoplasmic single sperm injection (ICSI) than after subzonal insemination of several spermatozoa was confirmed in a controlled comparison of the two procedures in 11 patients. Intracytoplasmic sperm injection was carried out in 150 consecutive treatment cycles of 150 infertile couples, who had failed to have fertilized oocytes after standard in-vitro fertilization (IVF) procedures or who were not accepted for IVF because not enough motile spermatozoa were present in the ejaculate. A single spermatozoon was injected into the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes (8.3%) were damaged by the procedure and 830 oocytes (64.2% of the successfully injected oocytes) had two distinct pronuclei the morning after the injection procedure. The fertilization rate was not influenced by semen characteristics. After 24 h of further in-vitro culture, 71.2% of these oocytes developed into embryos, which were transferred or cryopreserved. Only 15 patients did not have embryos replaced. Three-quarters of the transfers were triple-embryo transfers. High pregnancy rates were noticed since 67 pregnancies were achieved, of which 53 were clinical, i.e. a total and clinical pregnancy rate of 44.7% and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer. A total of 237 supernumerary embryos were cryopreserved in 71 treatment cycles.

Since the number of ovarian follicles declines with increasing age, AMH levels might be used as a marker for ovarian ageing. Therefore, we studied the relationship between AMH levels and ovarian response during ovarian stimulation for IVF. METHODS: A total of 130 patients undergoing their first IVF treatment cycle using a long protocol with GnRH agonist was prospectively included. Blood withdrawal was performed and the number of antral follicles was assessed by ultrasound on day 3 of a spontaneous cycle. Poor response and the number of oocytes were used as primary outcome measures. In a random subset of 23 patients a GnRH agonist stimulation test was performed to investigate whether a rise in FSH and LH would affect AMH levels. RESULTS: The data of 119 patients were analysed. Serum AMH levels were highly correlated with the number of antral follicles (r ⍧ 0.77; P < 0.01) and the number of oocytes retrieved (r ⍧ 0.57, P < 0.01). A negative association was found between AMH levels and poor ovarian response (fewer than 4 oocytes or cycle cancellation; OR 0.82, 95% CI 0.75-0.90, P < 0.01). Inclusion of inhibin B and FSH concentrations to AMH in a multivariate model improved the prediction of ovarian response. The post GnRH agonist rise in FSH and LH levels did not influence AMH values. CONCLUSIONS: Poor response in IVF, indicative of a diminished ovarian reserve, is associated with reduced baseline serum AMH concentrations. In line with recent observations it appears that AMH can be used as a marker for ovarian ageing.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2± 6 mm follicles; (2) immature oocytes from b 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2±6 mm follicles. Results from b 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality. Mol. Reprod. Dev. 61: 234±248,

The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. on zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/ Dppa3/stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DnA methylation reprogramming processes in the mammalian zygote.

Interpretation We were able to identify by logistic regression the factors that significantly affect the outcome of IVF treatment, and to measure the magnitude of that effect. These factors should be taken into account in assessment of IVF results. After allowance for background clinic success rates, these factors can be used to predict outcome in individual cases.

We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a Ml99 granuloaa cell co-culture (Ml99 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined condltiis in SOFaaci with 5% cow serum (SOFaaci-CS) or Ml99 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n=30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocyats (n=lO) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investi ated. f! Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and Ml99 was 16 f 3 , 23 + 2*, 30 rt 8a and 36 + 7'?&, respectively (Pab< 0.05). Additional inclusion of myoinositol resulted in 42 It la% blastocysts in SOFaaci-PVA vs 19 + 3b% in SOFaac-PVA, 47 + 7a% in SOFaac-BSA, and 38 + 7a% in Ml99 co-culture, respectively (Pab< 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 + 4 and 49 + 5%, respectively. Five normal calves were produced after transfer of 10 blastccysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 + 3 and 55 + 3% vs 90 + 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 + 7 and 19 + 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVMIIVF for normal fertilization and subsequent high blastocyst development.

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co-culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.

background: Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. methods: Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted.

background: While live birth is the principal clinical outcome following in vitro fertilization (IVF) treatment, the number of eggs retrieved following ovarian stimulation is often used as a surrogate outcome in clinical practice and research. The aim of this study was to explore the association between egg number and live birth following IVF treatment and identify the number of eggs that would optimize the IVF outcome. methods: Anonymized data on all IVF cycles performed in the UK from April 1991 to June 2008 were obtained from the Human Fertilization and Embryology Authority (HFEA). We analysed data from 400 135 IVF cycles. A logistic model was fitted to predict live birth using fractional polynomials to handle the number of eggs as a continuous independent variable. The prediction model, which was validated on a separate HFEA data set, allowed the estimation of the probability of live birth for a given number of eggs, stratified by age group. We produced a nomogram to predict the live birth rate (LBR) following IVF based on the number of eggs and the age of the female. results: The median number of eggs retrieved per cycle was 9 [inter-quartile range (IQR) 6-13]. The overall LBR was 21.3% per fresh IVF cycle. There was a strong association between the number of eggs and LBR; LBR rose with an increasing number of eggs up to 15, plateaued between 15 and 20 eggs and steadily declined beyond 20 eggs. During 2006-2007, the predicted LBR for women with 15 eggs retrieved in age groups 18 -34, 35 -37, 38-39 and 40 years and over was 40, 36, 27 and 16%, respectively. There was a steady increase in the LBR per egg retrieved over time since 1991.

The treatment of infertile women with polycystic ovary syndrome (PCOS) is surrounded by many controversies. This paper describes, on the basis of the currently available evidence, the consensus reached by a group of experts regarding the therapeutic challenges raised in these women. Before any intervention is initiated, preconceptional counselling should be provided emphasizing the importance of life style, especially weight reduction and exercise in overweight women, smoking and alcohol consumption. The recommended first-line treatment for ovulation induction remains the anti-estrogen clomiphene citrate (CC). Recommended second-line intervention, should CC fail to result in pregnancy, is either exogenous gonadotrophins or laparoscopic ovarian surgery (LOS). The use of exogenous gonadotrophins is associated with increased chances for multiple pregnancy and, therefore, intense monitoring of ovarian response is required. LOS alone is usually effective in <50% of women and additional ovulation induction medication is required under those circumstances. Overall, ovulation induction (representing the CC, gonadotrophin paradigm) is reported to be highly effective with a cumulative singleton live birth rate of 72%. Recommended third-line treatment is in vitro fertilization. More patient-tailored approaches should be developed for ovulation induction based on initial screening characteristics of women with PCOS. Such approaches may result in deviation from the above mentioned first-, second-or third-line ovulation strategies in well-defined subsets of patients. Metformin use in PCOS should be restricted to women with glucose intolerance. Based on recent data available in the literature, the routine use of this drug in ovulation induction is not recommended. Insufficient evidence is currently available to recommend the clinical use of aromatase inhibitors for routine ovulation induction. Even singleton pregnancies in PCOS are associated with increased health risk for both the mother and the fetus.

Even when conditions are optimal, the maximum chance of a clinically recognized pregnancy occurring in a given menstrual cycle is 30±40%. Increasing evidence points to preclinical pregnancy loss rather than failure of conception as the principal cause for the relatively low fecundity observed in humans. While sensitive assays for hCG have provided a glimpse of the events occurring between implantation and the missed menstrual period, new cytogenetic techniques have further opened this`black box', providing novel insights into the causes of early pregnancy wastage. In this article, the evidence and causes of preclinical or`occult' pregnancy are reviewed, and the implications for the infertile patient are addressed.

BACKGROUND: European results of assisted reproductive techniques (ARTs) from treatments initiated during 2003 are presented in this seventh report. METHODS: Data were mainly collected from already existing national registers. From 28 countries, 725 clinics reported 365 103 treatment cycles with: IVF 132 932, ICSI 162 149, frozen embryo replacement (FER) 60 412, oocyte donation (OD) 7548, PGD/PGS 1956 and IVM 109. Overall, this represents a 13% increase since 2002. For the third time, results on European data on intrauterine inseminations (IUIs) were reported from 19 countries. A total of 99 577 cycles (IUI-H, 82 834; IUI-D, 16 743) were included. RESULTS: In those 15 countries

To determine the relationship between sperm chromatin structure assay (SCSA) parameters (DNA fragmentation index [DFI] and high DNA stainability [HDS]), and conventional IVF and IVF/intracytoplasmic sperm injection (ICSI) outcomes. Design: Retrospective review and prospective study. Setting: Private IVF clinic. Patient(s): Two hundred forty-nine couples undergoing first IVF and/or ICSI cycle. Intervention(s): IVF, ICSI, blastocyst culture. Main Outcome Measure(s): DFI, HDS, conventional semen parameters, IVF, ICSI. Result(s): IVF and ICSI fertilization rates were not statistically different between high-and low-DFI groups. More men with Ն15% HDS had lower (Ͻ25% and Ͻ50%) IVF fertilization rates. High DNA stainability was not related to ICSI fertilization rates. High DNA stainability did not affect blastocyst rates or pregnancy outcomes. Men with Ն30% DFI were at risk for low blastocyst rates (Ͻ30%) and no ongoing pregnancies. Men with Ն30% DFI had more male factors. World Health Organization thresholds were not predictive of ongoing pregnancy.

BACKGROUND: To evaluate the safety of ICSI, this study compared data of IVF and ICSI children by collecting data on neonatal outcome and congenital malformations during pregnancy and at birth. METHODS: The followup study included agreement to genetic counselling and eventual prenatal diagnosis, followed by a physical examination of the children after 2 months, after 1 year and after 2 years. 2840 ICSI children (1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999) and 2955 IVF children (1983)(1984)(1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999) were liveborn after replacement of fresh embryos. ICSI was carried out using ejaculated, epididymal or testicular sperm. RESULTS: In the two cohorts, similar rates of multiple pregnancies were observed. ICSI and IVF maternal characteristics were comparable for medication taken during pregnancy, pregnancy duration and maternal educational level, whereas maternal age was higher in ICSI and a higher percentage of first pregnancies and first children born was observed in the ICSI mothers. Birthweight, number of neonatal complications, low birthweight, stillbirth rate and perinatal death rate were compared between the ICSI and the IVF groups and were similar for ICSI and IVF. Prematurity was slightly higher in the ICSI children (31.8%) than in the IVF children (29.3%). Very low birthweight was higher in the IVF pregnancies (5.7%) compared with ICSI pregnancies (4.4%). Major malformations (defined as those causing functional impairment or requiring surgical correction), were observed at birth in 3.4% of the ICSI liveborn children and in 3.8% of the IVF children (P ⍧ 0.538). Malformation rate in ICSI was not related to sperm origin or sperm quality. The number of stillbirths (born ≥20 weeks of pregnancy) was 1.69% in the ICSI group and 1.31% in the IVF group. Total malformation rate taking into account major malformations in stillborns, in terminations and in liveborns was 4.2% in ICSI and 4.6% in IVF (P ⍧ 0.482). CONCLUSIONS: The comparison of ICSI and IVF children taking part in an identical follow-up study did not show any increased risk of major malformations and neonatal complications in the ICSI group.

The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c-myc under control of the zebrafish Rag2 promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c-myc- induced carcinogenesis.

To compare success rates between fresh ETs after ovarian stimulation and frozen-thawed ETs (FET) after artificial endometrial preparation, to compare endometrial receptivity. Randomized, controlled trial. Private fertility center. There were 53 patients completing fresh blastocyst transfer (fresh group) and 50 patients completing FET (cryopreservation group). All were first-time IVF patients aged &amp;amp;amp;amp;amp;lt;41 years, with cycle day 3 FSH &amp;amp;amp;amp;amp;lt;10 mIU/mL and 8-15 antral follicles. Randomized to fresh or thawed ET. Clinical pregnancy rate per transfer. The clinical pregnancy rate per transfer was 84.0% in the cryopreservation group and 54.7% in the fresh group. The implantation rates were 70.8% and 38.9%, respectively. The ongoing pregnancy rates per transfer (at 10 weeks&amp;amp;amp;amp;amp;#39; gestation) were 78.0% and 50.9%, respectively. The attributable risk percentage of implantation failure due to reduced endometrial receptivity in the fresh group was 64.7%. The clinical pregnancy rate per transfer was significantly greater in the cryopreservation group than in the fresh group. These results strongly suggest impaired endometrial receptivity in fresh ET cycles after ovarian stimulation, when compared with FET cycles with artificial endometrial preparation. Impaired endometrial receptivity apparently accounted for most implantation failures in the fresh group. ClinicalTrials.gov Identifier: NCT00963625.

Pregnancy rates in women of advanced maternal age undergoing in vitro fertilization (IVF) are disappointingly low. It has been suggested that the use of preimplantation genetic screening of cleavage-stage embryos for aneuploidies may improve the effectiveness of IVF in these women.

Objective: To assess the value of antimullerian hormone (AMH) as a test to predict poor ovarian response and pregnancy occurrence after IVF and to compare it with the performance of the antral follicle count (AFC). Design: A systematic review of existing literature and a meta-analysis were carried out. After a comprehensive search, studies were included if 2 Â 2 tables for outcomes poor response and pregnancy in IVF patients in relation to AMH or AFC could be constructed. Setting: Academic referral center for tertiary care. Patient(s): Cases indicated for IVF. Intervention(s): None. Main Outcome Measure(s): Poor response and nonpregnancy after IVF. Result(s): A total of 13 studies were found reporting on AMH and 17 on AFC. Because of heterogeneity among studies, calculation of a summary point estimate for sensitivity and specificity was not possible. However, for both tests summary receiver operating characteristic curves for the outcome measures poor response and nonpregnancy could be estimated and compared. The curves for the prediction of poor response indicated no significant difference between the performances of AMH and AFC. For the prediction of nonpregnancy, poor performance for both AMH and AFC was found. Conclusion(s): In this meta-analysis it was shown that AMH has at least the same level of accuracy and clinical value for the prediction of poor response and nonpregnancy as AFC. (Fertil Steril Ò 2009;91:705-14. Ó2009 by American Society for Reproductive Medicine.

Objective: To evaluate the outcome of oocyte vitrification using the Cryotop method, observed in an egg donation program by simultaneously evaluating embryos derived from vitrified and fresh oocytes coming from the same stimulated cycle. Design: Cohort prospective randomized study. Setting: Instituto Valenciano de Infertilidad (IVI) Valencia, Spain. Patient(s): Thirty oocyte donors and 30 recipients with informed consents. Intervention(s): Vitrification by the Cryotop method. Warming 1 hour after vitrification. Microinjection of surviving MII and fresh oocytes, evaluation of fertilization, embryo development, and clinical results. Main Outcome Measure(s): Survival, fertilization, and cleavage rate. Embryo quality, pregnancy rate (PR), and implantation rate. Result(s): Survival rate observed was 96.7%. There was no difference in fertilization rates (76.3% and 82.2%), day 2 cleavage (94.2% and 97.8%), day 3 cleavage (80.8% and 80.5%), and blastocyst formation (48.7% and 47.5%) for vitrified and fresh oocytes, respectively. Embryo quality on day 3 and on day 5-6 were similar for vitrification and fresh oocyte group (80.8% vs. 80.5% and 81.1% vs. 70%, respectively). A total of 23 embryo transfers were carried out in the vitrification group. Pregnancy rates, implantation rates, miscarriage rates, and ongoing PR were 65.2%, 40.8%, 20%, and 47.8%, respectively. Conclusion(s): The Cryotop method preserves the potential of vitrified oocytes to fertilize and further develop, which is similar, when evaluated simultaneously, to fresh counterparts. Excellent clinical outcome indicates the possible use of this technology for egg donation programs, as well as a high potential for establishing oocyte banking. (Fertil Steril Ò 2007;-:---.

There is an absence of population-based long-term studies on the risk of neurological sequelae in children born after in-vitro fertilisation (IVF). Our aim was to compare the frequency of such problems between IVF-born children and controls. We did a population-based retrospective cohort study in which we compared development of neurological problems in 5680 children born after IVF, with 11360 matched controls. For 2060 twins born after IVF, a second set of controls (n=4120), all twins, were selected. We obtained data on neurological problems from the records of the Swedish habilitation centres. Children born after IVF are more likely to need habilitation services than controls (odds ratio 1.7, 95% CI 1.3-2.2). For singletons, the risk was 1.4 (1.0-2.1). The most common neurological diagnosis was cerebral palsy, for which children born after IVF had an increased risk of 3.7(2.0-6.6), and IVF singletons of 2.8 (1.3-5.8). Suspected developmental delay was increased four-fold (1.9-8.3) in children born after IVF. Twins born after IVF did not differ from control twins with respect to risk of neurological sequelae. Low-birthweight and premature infants were more likely to need habilitation than fullterm babies. Maternal age did not affect risk. Our study suggests that children born after IVF have an increased risk of developing neurological problems, especially cerebral palsy. These risks are largely due to the high frequency of twin pregnancies, low birthweight, and prematurity among babies born after IVF. To limit these risks, we recommend that only one embryo should be transferred during IVF.

Major depressive disorder (MDD) is a psychiatric illness that presents as a deficit of serotonergic neurotransmission in the central nervous system. MDD patients also experience alterations in cortisol and cytokines levels. Treatment with selective serotonin reuptake inhibitors (SSRIs) is the first-line antidepressant regimen for MDD. The aim of this study was to determine the effect of a combination of SSRIs and an immunomodulator-human dialyzable leukocyte extract (hDLE)-on cortisol and cytokines levels. Patients received SSRIs or SSRIs plus hDLE. The proinflammatory cytokines IL-1 , IL-2, and IFN-; anti-inflammatory cytokines IL-13 and IL-10; and 24-h urine cortisol were measured at weeks (W) 0, 5, 20, 36, and 52 of treatment. The reduction in cortisol levels in the SSRI-treated group was 30% until W52, in contrast, the combined treatment induced a 54% decrease at W36. The decline in cortisol in patients who were treated with SSRI plus hDLE correlated with reduction of anti-inflammatory cytokines and increases levels of proinflammatory cytokines at the study conclusion. These results suggest that the immune-stimulating activity of hDLE, in combination with SSRIs, restored the pro-and anti-inflammatory cytokine balance and cortisol levels in depressed patients versus those who were given SSRIs alone.

BACKGROUND: Sperm DNA damage is common amongst infertile men and may adversely impact natural reproduction, IUI-assisted reproduction and to a lesser degree IVF pregnancy. The aim of this study was to examine the influence of sperm DNA damage on the risk of spontaneous pregnancy loss after IVF and ICSI. METHODS: We conducted a systematic review and meta-analysis of studies on sperm DNA damage and pregnancy loss after an IVF and/ or ICSI pregnancy. RESULTS: Two by two tables were constructed and odds ratios (ORs) were derived from 11 estimates of pregnancy loss (five IVF and six ICSI studies from seven reports). These 11 studies involved 1549 cycles of treatment (808 IVF and 741 ICSI cycles) with 640 pregnancies (345 IVF and 295 ICSI) and 122 pregnancy losses. The combined OR of 2.48 (95% CI 1.52, 4.04, P < 0.0001) indicates that sperm DNA damage is predictive of pregnancy loss after IVF and ICSI. CONCLUSIONS: In conclusion, sperm DNA damage is associated with a significantly increased risk of pregnancy loss after IVF and ICSI. These data provide a clinical indication for the evaluation of sperm DNA damage prior to IVF or ICSI and a rationale for further investigating the association between sperm DNA damage and pregnancy loss.

BACKGROUND: Breast cancer chemotherapy commonly causes premature ovarian failure and infertility. Because increased estrogen levels are thought to be potentially risky in breast cancer patients, natural cycle IVF (NCIVF) has been used to preserve fertility and treat infertility in these women. METHODS: Twelve women with breast cancer received 40±60 mg tamoxifen for 6.9 T T 0.6 days beginning on days 2±3 of their menstrual cycle (15 cycles), and had IVF (TamIVF) with either fresh embryo transfer (six cycles) or cryopreservation (nine cycles). They were compared to a retrospective control group (n = 5) who had natural cycle IVF (NCIVF, nine cycles). RESULTS: Cycle cancellation was signi®cantly less frequent in TamIVF, compared with NCIVF (1/15 versus 4/9, P < 0.05). Compared with NCIVF, TamIVF patients had a greater number of mature oocytes (1.6 T T 0.3 versus 0.7 T T 0.2, P = 0.03) and embryos (1.6 T T 0.3 versus 0.6 T T 0.2, P = 0.02) per initiated cycle. TamIVF resulted in the generation of embryo(s) in every patient (12/12) while only three out of ®ve patients had an embryo following NCIVF. Two out of six patients in TamIVF, and 2/5 in NCIVF conceived. One patient in the TamIVF group delivered a set of twins. After a mean follow up of 15 T T 3.6 months (range 3±54), none of the patients had a recurrence of cancer. CONCLUSIONS: Tamoxifen stimulation appears to result in a higher number of embryos and may provide a safe method of IVF and fertility preservation in breast cancer patients.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P Ͻ 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P Ͻ 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P Ͻ 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P Ͻ 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Case report gestation to a 47 year old recipient, after vitrification of Four patients of mean age 32.5 years (range 27-35) attending mature oocytes from four in-vitro fertilization (IVF) the SISMER in-vitro fertilization (IVF) clinic (Bologna, Italy) patients. A total of 17 oocytes was vitrified in 1-2 µl of agreed to have their surplus oocytes vitrified with the intention ethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in of reducing the number of surplus zygotes and embryos that open pulled straws. Eleven oocytes survived after vitrificaneeded to be frozen in the particular cycle of treatment. A tion and five pronuclear zygotes were obtained after intratotal of 17 oocytes from the four patients was vitrified, 13 cytoplasmic sperm injection (ICSI). Three embryos were were intended to be used by the patients to form embryos for transferred to three patients, two of whom were the original their own use and four were donated to recipients seeking oocyte donors and pregnancy was not established. The donor oocytes.

To determine the efficiency of oocyte cryopreservation relative to IVF with unfrozen oocytes. Design: Meta-analysis. Setting: Academic assisted reproduction center. Patient(s): Results of all reports from January 1997 to June 2005 with the patients undergoing IVF-intracytoplasmic sperm injection (ICSI) with cryopreserved cycles between 1996 and 2004 were compared with those of patients who underwent IVF-ICSI with unfrozen oocytes in 2002 and 2003 in our program. Intervention(s): Mean age and number of ET cycles originating from unfrozen oocytes was matched with those for thaw cycles originating from oocytes cryopreserved with a slow-freezing (SF) protocol. Vitrification (VF) reports were not included in the comparative analysis because of a small number of pregnancies (10) before June 2005. Main Outcome Measure(s): The comparison of fertilization rate, clinical pregnancy, and live-birth rates per injected oocyte, clinical pregnancy and live-birth rates per transfer, and implantation rate between IVF-ICSI cycles with frozen and unfrozen oocytes. Result(s): Live-birth rates per oocyte thawed were 1.9% and 2.0% for SF and VF, respectively, before June 2005. Live-birth rates per injected oocyte and ET, respectively, were 3.4% and 21.6% for SF and were 6.6% and 60.4% for IVF with unfrozen oocytes. Compared to women who underwent IVF after SF, IVF with unfrozen oocytes resulted in significantly better rates of fertilization (odds ratio [95% confidence interval]); 2.22 (1.80, 2.74), of live birth per injected oocyte; 1.5 (1.26, 1.79), and of implantation; 4.66 (3.93, 5.52). These odds ratios were lower when oocyte cryopreservation success rates from 2002-2004 were compared with those for IVF with unfrozen oocytes. When the reports after June 2005 were considered, this trend did not appear to continue. With the consideration of VF reports after June 2005, however, higher pregnancy rates were achieved.

BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and <0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology.

To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. Design: A randomized, controlled study. Setting: Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. Patient(s): In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of Ն36 years (n ϭ 157), Ն3 previous IVF failures (n ϭ 54), and an altered karyotype (n ϭ 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. Intervention(s): Analysis of chromosomes XY, was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. Main Outcome Measure(s): Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions.

The assessment of oocyte quality in human in vitro fertilization (IVF) is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF) is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics

To identify and quantify predictors of poor ovarian response in in vitro fertilization (IVF). Design: Prospective study. Setting: Tertiary fertility center. Patient(s): One hundred twenty women undergoing their first IVF cycle. Intervention(s): Measurement of the number of antral follicles and the total ovarian volume by ultrasound, and of basal levels of FSH, E 2 , and inhibin B on cycle day 3. Main Outcome Measure(s): Ovarian response, and clinical and ongoing pregnancy rates.

Objective: To examine the available evidence to assess if cryopreservation of all embryos and subsequent frozen embryo transfer (FET) results in better outcomes compared with fresh transfer. Design: Systematic review and meta-analysis. Setting: Centers for reproductive care. Patient(s): Infertility patient(s). Intervention(s): An exhaustive electronic literature search in MEDLINE, EMBASE, and the Cochrane Library was performed through December 2011. We included randomized clinical trials comparing outcomes of IVF cycles between fresh and frozen embryo transfers. Main Outcome Measure(s): The outcomes of interest were ongoing pregnancy rate, clinical pregnancy rate, and miscarriage. Result(s): We included three trials accounting for 633 cycles in women aged 27-33 years. Data analysis showed that FET resulted in significantly higher ongoing pregnancy rates and clinical pregnancy rates. Conclusion(s): Our results suggest that there is evidence that IVF outcomes may be improved by performing FET compared with fresh embryo transfer. This could be explained by a better embryo-endometrium synchrony achieved with endometrium preparation cycles. (Fertil Steril Ò 2012;-:---. Ó2012 by American Society for Reproductive Medicine.) Key Words: Fresh embryo transfer, frozen embryo transfer, endometrial receptivity, pregnancy outcome Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/roquem-fresh-versus-frozen-embryo-transfer/

Is there an association between high levels of sperm DNA damage and miscarriage? summary answer: Miscarriage rates are positively correlated with sperm DNA damage levels. what is known already: Most ejaculates contain a subpopulation of sperm with DNA damage, also referred to as DNA fragmentation, in the form of double or single-strand breaks which have been induced in the DNA prior to or following ejaculation. This DNA damage may be particularly elevated in some subfertile men, hence several studies have examined the link between sperm DNA damage levels and conception and miscarriage rates. study design, size, duration: A systematic review and meta-analysis of studies which examined the effect of sperm DNA damage on miscarriage rates was performed. Searches were conducted on MEDLINE, EMBASE and the Cochrane Library without any language restrictions from database inception to January 2012. participants/materials, setting, methods: We used the terms 'DNA damage' or 'DNA fragmentation' combined with 'miscarriage', 'abortion' or 'pregnancy' to generate a set of relevant citations. Data extraction was performed by two reviewers. Study quality was assessed using the Newcastle-Ottawa Scale. Meta-analysis of relative risks of miscarriage was performed with a random effects model. Subgroup analyses were performed by the type of DNA damage test, whether the sperm examined were prepared or from raw semen and for pregnancies resulting from IVF or ICSI treatment. main results and the role of chance: We identified 16 cohort studies (2969 couples), 14 of which were prospective. Eight studies used acridine orange-based assays, six the TUNEL assay and two the COMET assay. Meta-analysis showed a significant increase in miscarriage in patients with high DNA damage compared with those with low DNA damage [risk ratio (RR) ¼ 2.16 (1.54, 3.03), P , 0.00001)]. A subgroup analysis showed that the miscarriage association is strongest for the TUNEL assay (RR ¼ 3.94 (2.45, 6.32), P , 0.00001). limitations, reasons for caution: There is some variation in study characteristics, including the use of different assays and different thresholds for DNA damage and the definition of pregnancy loss. wider implications of the findings: The use of methods which select sperm without DNA damage for use in assisted conception treatment may reduce the risk of miscarriage. This finding indicates that assays detecting DNA damage could be considered in those suffering from recurrent pregnancy loss. Further research is necessary to study the mechanisms of DNA damage and the potential therapeutic effects of antioxidant therapy. study funding/competing interest(s): None.

BACKGROUND: The relationship between early embryo post-implantation development in couples undergoing assisted reproductive techniques (ARTs) and sperm chromatin alterations has not been satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters (concentration, motility and morphology) and ART outcome, especially with regard to clinical pregnancy and pregnancy loss (spontaneous miscarriage or biochemical pregnancy). METHODS: DNA fragmentation was evaluated by TUNEL assay, performed on sperm suspensions after density gradient separation, in 132 men undergoing an ART cycle (82 IVF and 50 ICSI) and correlated with sperm parameters and ART outcome. RESULTS: A highly significant negative correlation was found between DNA fragmentation and sperm parameters. There was a close relationship between DNA fragmentation and post-implantation development in ICSI patients: the clinical pregnancy and pregnancy loss rates significantly differed between patients with high and low sperm DNA fragmentation (P = 0.007 and P = 0.009, respectively). CONCLUSIONS: Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise 'embryo viability', resulting in pregnancy loss.

In women, anti-Müllerian hormone (AMH) is exclusively produced by granulosa cells of ovarian follicles during the early stages of follicle development. After an initial increase until early adulthood, AMH concentrations slowly decrease with increasing age until becoming undetectable ∼5 years before menopause when the stock of primordial follicles is exhausted. However, major individual variability exists in the pace of follicle pool depletion and the initial size of the follicle pool, reflected by a wide range of age at menopause. Individual AMH serum concentration does accurately reflect the size of the pool of antral follicles, representing the quantity of the remaining primordial follicles. Accordingly, AMH levels may vary significantly in women of the same chronological age, allowing AMH to predict the remaining length of a woman&#39;s reproductive lifespan. Following 10 years of intense clinical research in this area (with over 300 papers published in core clinical journals eve...

Simple media were developed to study the metabolic requirements of bovine embryos up to Day 7 (Day 0 = day of oocyte aspiration) in vitro. Embryos were derived from oocytes matured and fertilized in vitro. At 45 ± 2 h post insemination, embryos (-2 cells) were randomly allotted to treatments. Examined in experiments 1 and 3 was the effect of pyruvate concentration in the presence of lactate. In the presence of lactate, pyruvate (0.2-5.0 mM) had no effect (p > 0.05) on the percentage of morulae or blastocysts. However, increasing the concentration of hemicalcium L-lactate from 5 mM to 10 mM decreased (p < 0.001 ) the percentage of embryos reaching the morula or blastocyst stage (experiment 3). Neither magnesium sulfate (0.5 mM) nor EDTA (10 mM) improved embryo development when added to the medium CRI (experiment 2). Increasing the calcium level to 5 mM or the lactate level to 10 mM had no effect (p > 0.05) on embryo development (experiment 4). However, the interaction of adding calcium and lactate resulted in a decreased (p < 0.05) percentage of morulae. Determined in experiment 6 were the independent effects of pyruvate, lactate, and glucose on embryo development in vitro. As pyruvate or lactate level was increased from 1 to 10 mM, the percentage of blastocysts was decreased (p < 0.05). These experiments indicate that adding pyruvate to a medium containing lactate is not necessary for development of bovine embryos in vitro.

background: The influence of elevated serum progesterone levels during in vitro fertilization/intracytoplasmic sperm injection (IVF/ ICSI) cycles on pregnancy rates is a matter of continued debate among fertility clinicians. Efforts to resolve this question have been impeded by the various assays used to measure progesterone and the different, arbitrary threshold values for defining 'high' progesterone levels.

Objective: Exploration of the possibility that local injury of the endometrium increases the incidence of implantation. Design: Prospective study. Setting: Clinical IVF unit. Patient(s): A group of 134 patients, defined as good responders to hormonal stimulation, who failed to conceive during one or more cycles of IVF and embryo transfer (ET).