Idiopathic early ovarian aging: is there a relation with premenopausal accelerated biological aging in young women with diminished response to ART?

PURPOSE: To evaluate whether young women with idiopathic early ovarian aging, as defined by producing fewer oocytes than expected for a given age over multiple in vitro fertilization (IVF) cycles, have changes in telomere length and epigenetic age indicating accelerated biological aging (i.e., increased risk of morbidity and mortality). METHODS: A prospective cohort study was conducted at two Danish public fertility clinics. A total of 55 young women (≤ 37 years) with at least two IVF cycles with ≤ 5 harvested oocytes despite sufficient stimulation with follicle-stimulating hormone (FSH) were included in the early ovarian aging group. As controls, 52 young women (≤ 37 years) with normal ovarian function, defined by at least eight harvested oocytes, were included. Relative telomere length (rTL) and epigenetic age acceleration (AgeAccel) were measured in white blood cells as markers of premenopausal accelerated biological aging. RESULTS: rTL was comparable with a mean of 0.46 (± SD 0.12) in the early ovarian aging group and 0.47 (0.14) in the normal ovarian aging group. The AgeAccel of the early ovarian aging group was, insignificantly, 0.5 years older, but this difference disappeared when adjusting for chronological age. Sub-analysis using Anti-Müllerian hormone (AMH) as selection criterion for the two groups did not change the results. CONCLUSION: We did not find any indications of accelerated aging in whole blood from young women with idiopathic early ovarian aging. Further investigations in a similar cohort of premenopausal women or other tissues are needed to fully elucidate the potential relationship between premenopausal accelerated biological aging and early ovarian aging.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing.

PURPOSE: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M). METHOD: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS. RESULTS: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results. CONCLUSION: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS. TRIAL REGISTRATION NUMBER: N-20180001.

Preimplantation genetic testing legislation and accessibility in the Nordic countries.

INTRODUCTION: Assisted reproduction technologies are being rapidly developed and implementation of preimplantation genetic testing (PGT) has allowed patients with genetic disorders to initiate pregnancies while minimizing or eliminating the risk of transmitting these disorders to their offspring. Testing for numeric chromosomal anomalies has been proposed as a way to increase efficacy in assisted reproduction; however, this remains disputed. Legislation is lagging behind the rapid developments in this field. MATERIAL AND METHODS: We conducted a structured online survey of legislation and accessibility to preimplantation genetic testing in the Nordic countries to compare the regulation and uptake of this technique. The survey was designed and answered by the authors. RESULTS: Key elements in the regulation of preimplantation testing for monogenic disorders and structural rearrangements are similar in the Nordic countries, although accessibility varies since only Denmark, Finland, and Sweden have national clinics offering treatment. In addition, Denmark and Finland have private clinics offering PGT. Regulation is the most stringent in Norway where a national board evaluates all couples seeking treatment. Treatment volumes vary between the Nordic countries, with Norway and Finland having lowest treatment numbers. Preimplantation genetic testing for aneuploidy in the embryo varies between the Nordic countries: Finland and Iceland allow this form of treatment, Denmark and Sweden offer it only in the form of a research protocol, and Norway does not allow it at all. Therefore the number of treatment cycles involving testing for embryo aneuploidy are lower in the Nordic countries than in other countries where this treatment option is more common. CONCLUSIONS: Science needs to inform politics regarding the rapidly evolving field of reproductive medicine and we recommend harmonization of legislation and accessibility between the Nordic countries.

Preimplantation genetic testing practices in the Nordic countries.

INTRODUCTION: Preimplantation genetic testing (PGT) is growing in importance and volume internationally. International societies such as the European Society for Human Reproduction and Embryology compile international results and these data are published in scientific journals. We present the first compilation of practices, quality measuress and outcome data from Nordic clinics performing PGT. MATERIAL AND METHODS: We conducted a structured online survey of PGT practices in the Nordic countries to compare clinical and laboratory techniques, outcomes and quality measures applied in Nordic clinics. The survey was designed by the authors and answered by the authors and members of the study group. The outcome data represents results from 2018. Results and details were clarified through iteration with responding clinics while maintaining anonymity. Response rate in the study was 80%, with 8 of 10 clinics performing PGT responding. RESULTS: Most of the PGT cycles in the Nordic countries are funded through the public healthcare system with University Hospitals performing the majority of treatments, 716/848, or 84.4%, of oocyte retrievals in this dataset. The genetic analyses are in five cases performed by the affiliated local genetic laboratory, and the remaining three consult with large international private enterprise laboratories. Genetic counseling is widely used. Results in the Nordic clinics compare well with international data. Systematic quality control procedures are in place and the larger clinics and laboratories utilize ISO certification or accreditation in the quality management. Automatic witnessing with detailed electronic documentation of laboratory processes is not utilized in the responding clinics, although a majority uses manual witnessing procedures in the laboratory. The outcome after PGT in terms of clinical pregnancy per transfer is around 40% per embryo transfer and compares well with international data. CONCLUSIONS: Preimplantation genetic testing is organized in rather few clinics in the Nordic countries and most of them use local laboratories for genetic analyses of the biopsies. Laboratory procedures are largely in accordance with international guidelines and the outcome after PGT in terms of clinical pregnancy per transfer is comparable to results in international reports.

A systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: What is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening?

INTRODUCTION: In assisted reproductive technology, aneuploidy is considered a primary cause of failed embryo implantation. This has led to the implementation of preimplantation genetic testing for aneuploidy in some clinics. The prevalence of aneuploidy and the use of aneuploidy screening during preimplantation genetic testing for inherited disorders has not previously been reviewed. Here, we systematically review the literature to investigate the prevalence of aneuploidy in blastocysts derived from patients carrying or affected by an inherited disorder, and whether screening for aneuploidy improves clinical outcomes. MATERIAL AND METHODS: PubMed and Embase were searched for articles describing preimplantation genetic testing for monogenic disorders and/or structural rearrangements in combination with preimplantation genetic testing for aneuploidy. Original articles reporting aneuploidy rates at the blastocyst stage and/or clinical outcomes (positive human chorionic gonadotropin, gestational sacs/implantation rate, fetal heartbeat/clinical pregnancy, ongoing pregnancy, miscarriage, or live birth/delivery rate on a per transfer basis) were included. Case studies were excluded. RESULTS: Of the 26 identified studies, none were randomized controlled trials, three were historical cohort studies with a reference group not receiving aneuploidy screening, and the remaining were case series. In weighted analysis, 34.1% of 7749 blastocysts were aneuploid. Screening for aneuploidy reduced the proportion of embryos suitable for transfer, thereby increasing the risk of experiencing a cycle without transferable embryos. In pooled analysis the percentage of embryos suitable for transfer was reduced from 57.5% to 37.2% following screening for aneuploidy. Among historical cohort studies, one reported significantly improved pregnancy and birth rates but did not control for confounding, one did not report any statistically significant difference between groups, and one properly designed study concluded that preimplantation genetic testing for aneuploidy enhanced the chance of achieving a pregnancy while simultaneously reducing the chance of miscarriage following single embryo transfer. CONCLUSIONS: On average, aneuploidy is detected in 34% of embryos when performing a single blastocyst biopsy derived from patients carrying or affected by an inherited disorder. Accordingly, when screening for aneuploidy, the risk of experiencing a cycle with no transferable embryos increases. Current available data on the clinical effect of preimplantation genetic testing for aneuploidy performed concurrently with preimplantation genetic testing for inherited disorders are sparse, rendering the clinical effect from preimplantation genetic testing for aneuploidy difficult to access.

The effect of expressive writing intervention for infertile couples: a randomized controlled trial.

STUDY QUESTION: Is expressive writing intervention (EWI) efficacious in reducing distress and improving pregnancy rates for couples going through ART treatment? SUMMARY ANSWER: Compared to controls, EWI statistically significantly reduced depressive symptoms but not anxiety and infertility-related distress. WHAT IS KNOWN ALREADY: ART treatment is considered stressful. So far, various psychological interventions have been tested for their potential in reducing infertility-related distress and the results are generally positive. It remains unclear whether EWI, a brief and potentially cost-effective intervention, could be advantageous. STUDY DESIGN SIZE, DURATION: Between November 2010 and July 2012, a total of 295 participants (163 women, 132 men) were randomly allocated to EWI or a neutral writing control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were couples undergoing IVF/ICSI treatment. Single women and couples with Preimplantation Genetic Diagnosis or acute change of procedure from insemination to IVF, were excluded. EWI participants participated in three 20-min home-based writing exercises focusing on emotional disclosure in relation to infertility/fertility treatment (two sessions) and benefit finding (one session). Controls wrote non-emotionally in three 20-min sessions about their daily activities. The participants completed questionnaires at the beginning of treatment (t1), prior to the pregnancy test (t2), and 3 months later (t3). In total, 26.8% (79/295) were lost to follow-up. Mixed linear models were chosen to compare the two groups over time for psychological outcomes (depression, anxiety and infertility-related distress), and a Chi2 test was employed in order to examine group differences in pregnancy rates MAIN RESULTS AND THE ROLE OF CHANCE: One hundred and fifty-three participants received EWI (women = 83; men = 70) and 142 participants were allocated to the neutral writing control group (women = 83; men = 62). Both women and partners in the EWI group exhibited greater reductions in depressive symptoms compared with controls (P = 0.049; [CI 95%: -0.04; -0.01] Cohen's d = 0.27). The effect of EWI on anxiety did not reach statistical significance. Overall infertility-related distress increased marginally for the partners in the EWI group compared to the partners in the control group (P = 0.06; Cohen's d = 0.17). However, in relation to the personal subdomain, the increase was statistically significant (P = 0.01; Cohen's d = 0.24). EWI had no statistically significant effect on pregnancy rates with 42/83 (50.6%) achieving pregnancy in the EWI group compared with 40/80 (49.4%) in the control group (RR = 0.99 [CI 95% = 0.725, 1.341]; P = 0.94). LIMITATIONS, REASONS FOR CAUTION: The results for depressive symptoms corresponded to a small effect size and the remaining results failed to reach statistical significance. This could be due to sample characteristics leading to a possible floor-effect, as we did not exclude participants with low levels of emotional distress at baseline. Furthermore, men showed increased infertility-related distress over time. WIDER IMPLICATIONS OF THE FINDINGS: EWI is a potentially cost-effective and easy to implement home-based intervention, and even small effects may be relevant. When faced with infertility, EWI could thus be a relevant tool for alleviating depressive symptoms by allowing the expression of feelings about infertility that may be perceived as socially unacceptable. However, the implications do not seem to be applicable for men, who presented with increased infertility-related distress over time. STUDY FUNDING/COMPETING INTERESTS: The present study was supported by research grants from Merck Sharpe and Dohme and The Danish Agency for Science Technology and Innovation as part of a publicly funded PhD. The funding bodies had no influence on the data collection, analysis or conclusions of the study. None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, trial no. NCT01187095. TRIAL REGISTRATION DATE: 7th September 2010 DATE OF FIRST PATIENT'S ENROLMENT: 23rd November 2010.

Comparison of two different methods for measuring anti-mullerian hormone in a clinical series.

BACKGROUND: Anti Mullerian hormone (AMH) has previously been measured using a manual method, but a fully automated assay from Roche Diagnostics was recently introduced. The aim of this study was to compare the results from the AMH gen II ELISA and Elecsys Cobas AMH methods in a clinical setting to evaluate whether the assays achieve the goals of analytical performance. A prospective observational study with 23 women seeking laparoscopic sterilization was conducted. Blood samples were collected preoperatively as well as 1 week and 1, 3 and 6 months postoperatively; they were evaluated with the AMH gen II ELISA and Elecsys Cobas AMH methods. The assays were validated according to the optimal performance of biochemical assays: CV Analytical < 0.25* CV Within Biological Variation. FINDINGS: We found a good correlation between the two methods; there was a bias of approximately 32 %. The total within-person biological variability ranged from approximately 21 to 32 %. The analytical variability of the AMH gen II ELISA and Elecsys Cobas methods ranged from 5.5 to 10.3 % and 2.8 to 3.3 %, respectively. Applying the goals for optimal assay performance, the Elecsys Cobas method achieved optimal performance throughout the measuring range, whereas the AMH Gen II only achieved optimal performance in the high end of the measuring range. Furthermore, the Elecsys Cobas assay had a low limit of quantitation of 0.5 pmol/l compared to 3.0 pmol/l for the AMH gen II ELISA. CONCLUSIONS: In the clinical setting, the Elecsys Cobas AMH assay performs well according to the optimal standard for biochemical assays.

Unaltered timing of embryo development in women with polycystic ovarian syndrome (PCOS): a time-lapse study.

PURPOSE: Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Factors other than anovulation, such as low embryo quality have been suggested to contribute to the infertility in these women. This 2-year retrospective study used timelapse technology to investigate the PCOS-influence on timing of development in the pre-implantation embryo (primary endpoint). The secondary outcome measure was live birth rates after elective single-embryo transfer. METHODS: In total, 313 embryos from 43 PCOS women, and 1075 embryos from 174 non-PCOS women undergoing assisted reproduction were included. All embryos were monitored until day 6. Differences in embryo kinetics were tested in a covariance regression model to account for potential confounding variables: female age, BMI, fertilization method and male infertility. RESULTS: Time to initiate compaction and reach the morula stage as well as the duration of the 4th cleavage division was significantly shorter in PCOS embryos compared with non-PCOS embryos. No other kinetic differences were found at any time-points annotated. The proportion of multi-nucleated cells at the 2-cell stage was significantly higher in PCOS embryos compared with non-PCOS embryos. The live birth rates were comparable between the two groups. CONCLUSION: The findings suggest that the causative factor for subfertility in PCOS is not related to timing of development in the pre-implantation embryo.

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.

STUDY QUESTION: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? SUMMARY ANSWER: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. WHAT IS KNOWN ALREADY: There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. STUDY DESIGN, SIZE, DURATION: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. LIMITATIONS, REASONS FOR CAUTION: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. STUDY FUNDING/COMPETING INTERESTS: The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest.

Limitations of a time-lapse blastocyst prediction model: a large multicentre outcome analysis.

The goal of embryo selection models is to select embryos with the highest reproductive potential, whilst minimizing the rejection of viable embryos. Ultimately, any embryo selection model must be tested on clinical outcome. We therefore retrospectively tested a published blastocyst prediction model on a large combined set of transferred embryos with known clinical outcome. The model was somewhat effective in that we found a relative increase of 30% for implantation in the model-selected group of embryos. There was, however, a concomitant large rejection of embryos from our test cohort, which actually resulted in pregnancy. This hypothetical experiment highlights the limitations of predicting blastulation only. Crucially, it illustrates that both sensitivity and specificity are important parameters when developing embryo selection models for prospective clinical use.

Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method: a time-lapse study.

PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p < 0.001), displayed longer duration of the 3-cell stage (p < 0.001), and arrested development from the compaction stage and onwards (p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells (p < 0.001). CONCLUSIONS: This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos.

Inter- and intra-observer variability of time-lapse annotations.

STUDY QUESTION: How consistent is the time-lapse annotation of dynamic and static morphologic parameters of embryo development, within and between observers? SUMMARY ANSWER: The assessment of dynamic parameters is characterized by almost perfect agreement within and between observers. WHAT IS KNOWN ALREADY: The commonly employed method used to assess embryos in IVF treatments is based on static evaluation of morphology in a microscope, but this is limited by substantial intra- and inter-observer variation. Time-lapse imaging has been proposed as a method to refine embryo selection by adding new dynamic predictors of viability to the assessment. Yet, there are no data regarding the consistency of estimates of the time-lapse parameters. STUDY DESIGN, SIZE, DURATION: Infertile patients were recruited at the Fertility Clinic, Arhus University Hospital from February 2011 to June 2012. All embryos were cultured for 6 days in a time-lapse incubator (EmbryoScope(™)). Automated image recording was performed every 20 min. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 158 fertilized embryos from 20 different patients were annotated. Three observers made independent annotations on time-lapse recordings. One observer performed the assessment twice. Twenty-five parameters were annotated and the inter- and intra-observer agreement was assessed by calculating intra-class correlation coefficients (ICCs). MAIN RESULTS AND THE ROLE OF CHANCE: Extremely close agreement (ICC 0.99) was found for dynamic parameters including the timing of the following: pronuclei breakdown, completion of blastocyst hatching and the appearance and disappearance of the first nucleus after the first division. Observations of cleavage divisions were strongly correlated (ICC > 0.8), indicating close agreement. Measurements of the static morphologic parameters, i.e. multi-nucleation and evenness of blastomeres at 2-cell stage showed fair-to-moderate agreement (ICC ≤ 0.5). LIMITATIONS, REASON FOR CAUTION: The study was conducted at a single clinic. Only embryos with a good prognosis were included. The influence of training sessions was not measured. WIDER IMPLICATIONS OF THE FINDINGS: Consistency is crucial to the validity of embryo scoring and selection. All of the time-lapse parameters suggested by the literature showed in our study high intra- and inter-observer correlation, thus validating the precision of time-lapse annotations. This provides the basis for further investigation of embryo assessment and selection by time-lapse imaging in prospective trials. STUDY FUNDING/COMPETING INTEREST(S): Research at the Fertility Clinic was funded by an unrestricted grant from Ferring and MSD. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: NCT01139268.

Time-lapse monitoring as a tool for clinical embryo assessment.

As elective transfer of a single embryo (eSET) becomes increasingly accepted, the need to improve implantation rates becomes crucial. Selecting the most competent embryo therefore constitutes a major challenge in assisted reproductive technology. Embryo morphology and developmental stage at given time points are closely correlated with developmental competence and assessment of morphological parameters at discrete inspection points thus remains the preferred way of evaluating embryonic potential. Lately, more attention has been given to the assessment of dynamic embryo development as a tool for evaluating embryonic potential. The introduction of time-lapse equipment approved for use on human embryos offers novel clinical opportunities for continuous monitoring of embryos, enabling flexible evaluation of known morphological parameters and potentially introducing new dynamic markers of viability. Due to lack of larger, randomized clinical studies it remains to be elucidated whether embryo selection using dynamic parameters improves clinical outcome and which parameters are of significance. Before such randomized controlled studies are organized, the most promising parameters to evaluate must be identified. This mini-review summarizes the current knowledge about dynamic markers of viability and discusses the potential clinical role of time-lapse analysis in embryo assessment and selection.

Human embryonic development after blastomere removal: a time-lapse analysis.

BACKGROUND: Blastomere biopsy of human embryos is performed for preimplantation genetic diagnosis (PGD). The impact on further development is largely unexplored, though studies on mice suggest an influence on the hatching process. The objective of this study was to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. METHODS: Embryos from couples undergoing PGD treatment or IVF/ICSI were included. In the PGD group, 56 human embryos had one blastomere biopsied. As controls, 53 non-biopsied IVF/ICSI embryos were selected. All embryos were cultured until 5 days after fertilization in a time-lapse incubator (EmbryoScope™). Images of embryos were acquired every 20 min. Time-points of key embryonic events were registered, and development in the two groups was compared. RESULTS: Duration of the biopsied cell-stage in the PGD group was longer than in the control group (P < 0.001), causing biopsied embryos to reach subsequent embryonic stages until hatching at significantly later time-points (P(compaction) < 0.001; P(morula) < 0.001; P(earlyblast) < 0.001; P(fullblast) = 0.01), but with unchanged intervals. Embryos in the PGD group started hatching at the same time-point as the control group, but had a smaller diameter (P < 0.001), and a thicker zona pellucida (P < 0.001) when hatching. Time-lapse videos revealed that in the control group, expansion of the blastocyst caused continuous thinning of zona pellucida until the blastocyst hatched, whereas in the PGD group the blastocyst hatched through the opening in zona pellucida artificially introduced prior to the biopsy. CONCLUSIONS: We find that blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.

Preimplantation genetic diagnosis with HLA matching - a way to save a child.

Preimplantation genetic diagnosis can be used to establish a pregnancy with an embryo that is human leukocyte antigen (HLA)-matched to a sibling having a hematological or immunological disease and needing a life-saving bone marrow transplantation. The ethical aspects of this procedure have been discussed intensively. The procedure applies where no unrelated HLA-matching donor is available or when transplantation from an HLA-matching sibling is considered a better solution. It is only offered in a limited number of centers in Europe as this is a challenging procedure. Where both HLA matching and diagnosis of a dominant disease are necessary, only a small proportion of the embryos can be used, and the procedure is not always technically feasible. The clinical pregnancy rate per cycle started is much lower than following normal in vitro fertilization (IVF) due to a high cycle cancellation rate, but the success rate is only somewhat lower when measured per transfer.

Preimplantation genetic diagnosis for HLA typing in a case of X-linked chronic granulomatous disease.

Bone marrow transplantation may be life saving in cases of hematopoietic disease, severe congenital immunodeficiency or malignancy. An HLA-matching sibling often gives the best success, but this may not be an option, nor may an HLA-matching unrelated donor be found. Preimplantation genetic diagnosis with HLA-matching embryos may then be a solution. We report the first Danish child born after such diagnosis with identification of healthy embryos and HLA matching for a sibling with chronic granulomatous disease. Our patient had 13 in vitro fertilization (IVF) cycles; 286 oocytes were collected and 74 embryos analysed. Sixteen of these (22%) were either healthy or carriers. Five embryos were transferred in four stimulated fresh IVF cycles. Four embryos were frozen, and two were later transferred. Two successive clinical pregnancies ensued. In the first, prenatal diagnosis revealed trisomy 21, and the fetus was aborted. In the second pregnancy, chorionic villus sampling revealed a normal karyotype, the diagnosis was confirmed, and the pregnancy was normal. At delivery, 82 mL of cord blood was collected for later transplantation to the diseased sibling.

A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator.

PURPOSE: Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study was to evaluate the safety of a commercially available time-lapse incubator. METHODS: In a two center, randomized, controlled, clinical trial 676 oocytes from 59 patients in their 2nd or third treatment cycle, age <38 years and ≥ 8 oocytes retrieved were cultured in the time-lapse incubator or in a conventional incubator. The primary outcome was proportion of 4-cell embryos on day 2. Secondary outcomes were proportion of 7-8 cell embryos on day 3 and proportion of blastocysts on day 5. Implantation pregnancy rates were registered based on presence of fetal heart activity visualized by ultrasound 8 weeks after embryo transfer. RESULTS: No significant difference was found between the time-lapse incubator (TLI) and conventional incubator (COI) in proportion of 4-cell embryos on day 2 irrespective of whether data was analyzed according to ITT (RR(TLI/COI): 0.81 (0.65; 1.02)) or PP (RR(TLI/COI): 0.80 (0.63; 1.01)). Nor were any significant differences detected in the secondary endpoints; i.e. proportion of 7-8-cell embryos on day three ITT (RR(TLI/COI): 0.96 (0.73; 1.26)); PP (RR(TLI/COI): 0.95 (0.72; 1.26)) and proportion of blastocysts on day five ITT (RR(TLI/COI): 1.09 (0.84; 1.41)); PP (RR(TLI/COI): 1.09 (0.83: 1.41)). We found no differences in clinical pregnancy rate or implantation rate. CONCLUSION: Culture in the time-lapse incubator supports embryonic development equally to a conventional incubator.

Sleep, psychological distress, and clinical pregnancy outcome in women and their partners undergoing in vitro or intracytoplasmic sperm injection fertility treatment.

OBJECTIVES: To explore the prevalence of poor sleep quality in couples undergoing fertility treatment and study possible associations. PARTICIPANTS: 163 women and 132 partners receiving in vitro (IVF) or intracytoplasmic sperm injection (ICSI) fertility treatment. SETTING: Three public Danish fertility clinics. DESIGN AND MEASUREMENTS: Participants completed the Pittsburgh Sleep Quality Index (PSQI) at three time-points as part of a larger RCT. Additional data from patient records and questionnaires were included to evaluate possible associations with treatment protocol type, psychological distress, and pregnancy outcome. RESULTS: Mean PSQI global scores before treatment were 8.1 (standard deviation = 2.3), with 91% of participants having PSQI scores > 5, indicating poor sleep quality. Scores did not differ between women and their partners and did not change during treatment. Statistically significant associations were found between sleep quality and depressive symptoms and state anxiety (p < .001). No difference in PSQI scores was found between protocol types. While there was a trend towards higher clinical pregnancy rates among women with good sleep quality (PSQI ≤ 5 = 72.7%, PSQI 6-10 = 52.6% and PSQI ≥ 11 = 42.3%), the differences did not reach statistical significance (p = .10-.21). CONCLUSIONS: Poor sleep quality is a prevalent problem among couples undergoing fertility treatment and is associated with psychological distress and possibly with pregnancy outcomes. Success rates after fertility treatment remain moderate, and poor sleep quality, a potentially modifiable factor, could be relevant to screen for and treat among couples undergoing fertility treatment. The high prevalence of poor sleep quality calls for further investigation.

Tubal factor infertility is associated with antibodies against Chlamydia trachomatis heat shock protein 60 (HSP60) but not human HSP60.

BACKGROUND: Serum antibodies against major outer membrane protein (MOMP) and heat shock protein 60 (HSP60) from Chlamydia trachomatis are correlated with sequelae following infection. Since bacterial and human HSP60 share considerable sequence homology, cross-reactivity to human HSP60 is suggested as being involved in tubal factor infertility (TFI). The aim was to investigate whether antibodies to human HSP60 are associated with TFI, and to evaluate antibody testing in TFI diagnosis. METHODS: Serum levels of antibodies against chlamydial MOMP and HSP60 from C. trachomatis, Salmonella enterica Enteritidis, Campylobacter jejuni and human HSP60 were analysed by enzyme-linked immunosorbent assay in three groups of infertile women: women with TFI (n = 70), controls with normal fallopian tubes (control group 1, n = 92) and a subgroup of women with normal fallopian tubes and sero-positive for either chlamydial MOMP or chlamydial HSP60 (control group 2, n = 28). RESULTS: Serum levels of immunoglobulin (Ig)G1 and IgG3 antibodies against MOMP and HSP60 from C. trachomatis were elevated in patients with TFI compared with non-TFI individuals (group 1; P < 0.001), while levels of IgG3 against MOMP and IgG1 against HSP60 were higher in the TFI group compared with control group 2 (P = 0.04 and P = 0.03, respectively). Levels of antibodies against human HSP60 did not differ between groups. CONCLUSIONS: Our findings confirm an association between TFI and antibodies to MOMP and HSP60 from C. trachomatis, suggesting antibody testing as a supplement in TFI diagnosis. No connection was observed between TFI and antibodies to human HSP60, pointing to an infectious rather than an autoimmune inflammation as the cause of TFI.