The impact of paternal factors on cleavage stage and blastocyst development analyzed by time-lapse imaging-a retrospective observational study.

PURPOSE: Various time-lapse studies have postulated embryo selection criteria based on early morphokinetic markers. However, late paternal effects are mostly not visible before embryonic genome activation. The primary objective of this retrospective study was to investigate whether those early morphokinetic algorithms investigated by time-lapse imaging are reliable enough to allow for the accurate selection of those embryos that develop into blastocysts, while of course taking into account the correlation with the type of injected spermatozoa. METHODS: During a period of 18 months, a total of 461 MII oocytes from 43 couples with severe male factor infertility and previous "external" IVF failures after cleavage-stage embryo transfer (ET) were fertilized by intracytoplasmic morphologically selected sperm injection (IMSI). Thereof, 373 embryos were monitored in a time-lapse incubator until ET on day 5. Blastocyst outcome in combination with three previously postulated MKc (cc2: t3-t2, 5-12 h; t3, 35-40 h; t5, 48-56 h) and the morphology of the selected sperm were analyzed. RESULTS: A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P < 0.01) and top blastocysts (25.3 vs. 10.8 %; P < 0.001) was observed in the group of those meeting all three morphokinetic criteria (MKc3). However, MKc3 were only met in 23.3 % of all embryos. Moreover, TBR was influenced by the type of injected spermatozoa. In both groups, TBR decreased dramatically (MKc3, 35.0 vs. 17.0 %; MKc < 3, 14.2 vs. 8.4 %) when class II/III sperm instead of class I were injected. CONCLUSION: Early morphokinetic parameters might give some predictive information but fail to serve as a feasible selective tool for the prediction of blastocyst development given the influence of the type of spermatozoa injected.

Intrauterine administration of human chorionic gonadotropin does not improve pregnancy and life birth rates independently of blastocyst quality: a randomised prospective study.

BACKGROUND: Successful embryo implantation depends on a well-timed maternal-embryonic crosstalk. Human chorionic gonadotropin (hCG) secreted by the embryo is known to play a key role in this process and to trigger a complex signal transduction cascade allowing the apposition, attachment, and invasion of the embryo into the decidualized uterus. Production of hCG was reported to be dependent on blastocyst quality and several articles suggested that intrauterine hCG injection increases pregnancy and implantation rates in IVF patients. However, no study has as yet analysed birth rates as final outcome. Our objective was to determine whether clinical outcome after blastocyst transfer can be improved by intrauterine injection of hCG and whether this is dependent on blastocyst quality. METHODS: A prospective randomised study was conducted in two settings. In cohort A, hCG application was performed two days before blastocyst transfer. In cohort B, the administration of hCG occurred just prior to embryo transfer on day 5. For both cohorts, patients were randomised to either intrauterine hCG application or to the control group that received culture medium. Clinical outcome was analysed according to blastocyst quality of transferred embryos. RESULTS: The outcome of 182 IVF-cycles (cohort A) and 1004 IVF-cycles (cohort B) was analysed. All patients received a fresh autologous blastocyst transfer on day five. Primary outcomes were pregnancy rates (PR), clinical pregnancy rates (cPR), miscarriage rates (MR), and live birth rates (LBR). No improvement of clinical outcome after intrauterine hCG administration on day 3 (cohort A) or day 5 (cohort B) was found, independently of blastocyst quality transferred. The final outcome in cohort A: LBR after transfer of top blastocysts was 50.0 % with hCG and 53.3 % in the control group. With non-top blastocysts, LBR of 17.1 % (hCG) and 18.2 % (control) were observed (n.s.). In cohort B, LBR with top blastocysts was 53.3 % (hCG) and 48.4 % (control), with non-top blastocysts it came to 28.7 % (hCG) and 35.0 % (control). The differences between the groups were statistically not significant. Furthermore, we investigated a possible benefit of hCG administration in correlation with female age. In both age groups (<38 years and ≥ 38 years) we found similar LBR after treatment with hCG vs. medium. A LBR of 47.1 % vs. 48.7 % was obtained in the younger group and 26.6 % vs. 30.8 % in the older group. CONCLUSIONS: In contrast to previous studies indicating a substantial benefit from intrauterine hCG application in cleavage stage embryo transfers, in our study we could not find any evidence for improvement of clinical outcome in blastocyst transfer cycles, neither with top nor with non-top quality morphology.

Outpatient follicle monitoring: a plea for standardization in ultrasound based follicle monitoring and data transfer.

BACKGROUND: The complexity of assisted reproductive technology (ART) increased during the last decades. New scientific and medical findings as well as the statutory requirements for improving the safety and the outcome of ART were the main impetus for its development. While therapy planning is done and ART is used by the IVF centers, the medical support and monitoring of patients is conducted by referring gynecologists. Reported follicle measurements by the gynecologist allow the adoption of the therapy plan. Most notably, the crucial aspect is processing and interpretation of ultrasound scan (US). The results of the received US, the transfer of data between IVF center(s) and referred physician(s) as well as the subjective interpretation often culminate in interpretation and logistical problems. This might increase the error probability with considerable detriments for the patients and ART outcome. METHODS: The follicle monitoring was performed using Voluson I ultrasound system combined with SonoAVC(®) software. Results were communicated via DICOM language to DynaMed(®) software, a medical program for managing an IVF center with seamless integration of all processes needed for an accurate and precise workflow. RESULTS: In this study, no loss of data was detected. All data were integrated by DynaMed(®) software and were recallable in a fast and easy manner. CONCLUSION: The broad usage of Voluson I ultrasound SonoAVC(®) software and communication of the results via Picture Archiving and Communication System (PACS) server between the IVF center and local gynecologist would provide more assistance for the patients and consequently the ART outcomes can be improved.

Transfer of blastocysts with deviant morphological and morphokinetic parameters at early stages of in-vitro development: a case series.

Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage.

The combination matters--distinct impact of lifestyle factors on sperm quality: a study on semen analysis of 1683 patients according to MSOME criteria.

BACKGROUND: Poor sperm quality can negatively affect embryonic development and IVF outcome. This study is aimed at investigating the influence of various lifestyle factors on semen quality according to MSOME (motile sperm organelle morphology examination) criteria. METHODS: 1683 male patients undergoing assisted reproductive technologies (ART) in our clinic were surveyed about their age, BMI (body mass index), ejaculation frequency, nutrition, sports, sleeping habits and social behavior. Semen samples were collected and evaluation of semen parameters according to MSOME and WHO criteria was performed. Results were grouped and statistically analyzed. RESULTS: Although single parameters had minor effects on sperm parameter, the combination of age, BMI, coffee intake, ejaculatory frequency and duration of sexual abstinence were identified as factors having a negative effect on sperm motility. Additionally, we could demonstrate that MSOME quality was reduced. The negative impact of age, BMI and coffee intake on sperm quality could be compensated if patients had a high ejaculation frequency and shorter periods of sexual abstinence. CONCLUSIONS: Combinations of adverse lifestyle factors could have a detrimental impact on sperm, not only in terms of motility and sperm count but also in terms of sperm head vacuolization. This negative impact was shown to be compensated by higher ejaculation frequency and a shorter period of sexual abstinence. The compensation is most likely due to a shorter storage time in the male gonads, thus reducing the duration of sperms' exposure to reactive oxygen species (ROS).

Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment.

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle. In stimulated IVF cycles, high doses of hormones are given to stimulate multifollicular growth. One drawback of the hormonal substitution is that the uterine environment is not at the same time optimally prepared for embryo implantation. A solution, which is increasingly under discussion, is to cryopreserve the embryos obtained in the stimulated cycle and to transfer them back into the optimal uterine environment in a subsequent cryo-cycle. This procedure requires highly secure and safe cryopreservation protocols in order to ensure benefits for both pregnancy and birth rates. We have established a protocol for the vitrification of zygote-stage embryos in aseptic devices, which minimize the potential risk of contamination during cooling and storage. The vitrified zygotes showed the same blastocyst development as compared with sibling zygotes in fresh culture. A clinical study comprising 173 cryo-cycles with transfer of blastocysts originating from vitrified zygotes shows an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification conditions contributes to a change in transfer strategy and encourages us to increase the cryo-embryo transfer rate for an optimal uterine environment.

Pregnancy after ovarian superovulation by transdermal delivery of follicle-stimulating hormone.

Because of its size of 32 kDa and physicochemical properties, urinary FSH cannot permeate intact skin. We report the first pregnancy after laser microporation and transdermal delivery of FSH for ovarian superovulation as a substitute for SC or IM injections.

Dopamine agonist bromocriptine for the prevention of ovarian hyperstimulation syndrome.

The aim of this retrospective study is to investigate the frequency and severity of ovarian hyperstimulation syndrome and the pregnancy rate in a patient collective at risk who received bromocriptine treatment.

The rationale behind collecting umbilical cord blood.

Umbilical cord blood (UCB) is an increasingly important and rich source of stem cells. These cells can be used for the treatment of many diseases, including cancers and immune and genetic disorders. For patients for whom no suitable related donor is available, this source of hematopoietic stem cells offers substantial advantages, notably the relative ease of procurement, the absence of risk to the donor, the small likelihood of transmitting clinically important infections, the low risk of severe graft-versus-host disease (GVHD) and the rapid availability of placental blood for transplantation centers. Even though almost 80 diseases are treatable with cord blood stem cells, 97 percent of cord blood is still disposed of after birth and lost for patients in need! To improve availability of stem cells to a broader community, efforts should be undertaken to collect cord blood and expectant parents should be properly informed of their options with regard to cord blood banking.

Improved monitoring of ovarian stimulation using 3D transvaginal ultrasound plus automated volume count.

Two-dimensional transvaginal ultrasound (2D) is typically performed to monitor follicle growth in IVF and to determine the optimal time for administering human chorionic gonadotrophin. However, 2D only provides an approximation of the real volume of follicles and therefore cannot be used to guarantee standards for follicular measurement. The automated measurement of follicular size in three dimensions (3D) using a software programme that identifies and quantifies hypoechoic regions within a 3D dataset might provide an objective, fast, valid and reliable standard for such measurements. A prospective controlled study (group I: 20 patients, 2D; group II: 20 patients, 3D) investigated how the criteria for triggering oocyte maturation that are normally used in 2D compare to the new and more accurate method of measuring follicles using 3D-based automated volume count. Significantly more oocytes were fertilized (group 1: 7.1 +/- 4.5, group 2: 11.5 +/- 6.4; P < 0.03) when using 3D technology and automated volume count. The study assumes that the automated volume count more closely mirrors the biological reality, which means that it can also be used to guarantee the quality standards established by the European Union directive on tissues and cells (2004/23/EC). This new technology therefore holds great promise of becoming the new standard for monitoring follicular growth in IVF.

Non-informative results and monosomies in PGD: the importance of a third round of re-hybridization.

The incidence of non-informative results after fluorescence in-situ hybridization (FISH) was analysed in preimplantation genetic diagnosis (PGD). FISH was performed on seven chromosomes (13, 16, 18, 21, 22, X, and Y) in two rounds of hybridization (one biopsied blastomere per day 3 embryo). A third round with telomeric probes was performed in order to analyse the chromosome(s) in question. A total of 702 embryos out of a total of 719 embryos from 95 cycles were analysed. The remaining 17 embryos were anucleated and/or had poor quality and could not be diagnosed. After FISH analysis, 52.7% of blastomeres were found to be abnormal, 27.1% euploid, and 20.2% had non-informative results. Abnormalities considered as non-informative included 'monosomy in question' (46.5%), 'trisomy in question' (40.2%), compound aneuploidy (8.5%), and 'no result' (4.9%) for a tested chromosome. Following re-hybridization with telomeric probes, euploidy was found in 42.4% of 'monosomies in question,' in 82.4% of 'trisomies in question,' in 16.7% of compound aneuploidies, and in 71.4% of 'no results' for a tested chromosome. Only 4.2% of non-informative results could not be rescued. This study clearly demonstrates the importance of re-hybridizing non-informative results and monosomies using a third round of hybridization with telomeric probes for chromosome(s) in question.

Protocols for hematopoietic stem cell expansion from umbilical cord blood.

The reconstitution of adult stem cells may be a promising source for the regeneration of damaged tissues and for the reconstitution of organ dysfunction. However, there are two major limitations to the use of such cells: they are rare, and only a few types exist that can easily be isolated without harming the patient. The best studied and most widely used stem cells are of the hematopoietic lineage. Pioneering work on hematopoietic stem cell (HSC) transplantation was done in the early 1970s by ED. Thomas and colleagues. Since then HSCs have been used in allogenic and autologous transplantation settings to reconstitute blood formation after high-dose chemotherapy for various blood disorders. The cells can be easily harvested from donors, but the cell number is limited, especially when the HSCs originate from umbilical cord blood (UCB). It would be desirable to set up an ex vivo strategy to expand HSCs in order to overcome the cell dose limit, whereby the expansion would favor cell proliferation over cell differentiation. This review provides an overview of the various existing HSC expansion strategies-focusing particularly on stem cells derived from UCB-of the parameters that might affect the outcome, and of the difficulties that may occur when trying to expand such cells.

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines.

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.

Oocyte-like structures arising from cells of follicular fluid are not captured in aspirates.

Recently, cells from ovarian surface epithelium (OSE) of post-menopausal women and women with premature ovarian failure were investigated and oocyte-like cells with diameters up to 95 microm were found to arise after a certain time in culture. In addition, it seems that a mixed population of germ cells and germline stem cells exists in non-follicle ovarian structures. Relating to an earlier publication, where it was shown that pre-antral follicles with immature oocytes could be captured in follicular fluid (FF) aspirates due to the incorporated tissue in the puncture needle, it was reasoned that OSE or otherwise germline stem cells, possibly captured equally through ovarian puncture, might give rise to oocyte-like cells. The aim of this study was therefore to try to derive such oocyte-like cells from FF aspirates of patients undergoing IVF after culture. Additionally, FF-derived cells were aggregated with human embryonic stem cells to see if an embryonic environment had the ability to enable cells from the FF aspirate to acquire an oocyte-like morphology. Investigations could not confirm the development of oocyte-like cells from cells of FF aspirates.

Impact of meiotic and mitotic non-disjunction on generation of human embryonic stem cell lines.

At least 50-60% of oocytes derived from IVF procedures are chromosomally abnormal due to meiotic I or II errors. Through the use of polar body and blastomere diagnosis, euploid embryos suitable for transfer can be identified. Those embryos that are aneuploid are usually discarded, or otherwise can be used to generate chromosomally abnormal human embryonic stem cell (hESC) lines. The authors' centre has one of the largest repositories of hESC lines with genetic and chromosomal disorders generated from preimplantation genetic diagnosis (PGD) abnormal embryos. The results, studying hESC lines derived from PGD abnormal zygotes, imply that aneuploidies resulting from meiotic non-disjunction have a greater impact on viability of cells of the human embryos than those originating from post-zygotic mitotic non-disjunction.

Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.

Spermatozoa selection at high magnification before intracytoplasmic sperm injection seems to be positively associated with pregnancy rates after day 3 embryo transfers. The aim was to demonstrate an association between the presence of vacuoles in sperm nuclei and the competence of embryos to develop to day 5. Grading of spermatozoa at x 6000-x 12,500 magnification: grade I, no vacuoles; grade II, or=1 large vacuole; grade IV, large vacuoles with other abnormalities. The outcome of embryo development in a group of 25 patients after sibling oocyte injection with the four different grades of spermatozoa showed no significant difference in embryo quality up to day 3. However, the occurrence of blastocyst formation was 56.3 and 61.4% with grade I and II spermatozoa respectively, compared with 5.1% with grade III and 0% with grade IV respectively (P < 0.001). Spermatozoa selection at high magnification using Nomarski interference contrast is useful to identify more precisely the size and the number of nuclear vacuoles that greatly exert a negative effect on embryo development to the blastocyst stage. These observations confirm previous studies pointing to possible 'early and late paternal effects', both of which may have an impact on early embryonic development.

Monochorionic-diamniotic twins discordant in gender from a naturally conceived pregnancy through postzygotic sex chromosome loss in a 47,XXY zygote.

OBJECTIVE: It is generally believed that monochorionic-diamniotic twin pregnancies result from one fertilized oocyte with both siblings having the same genotype and phenotype. In rare instances, due to somatic mutations or chromosome aberrations, the karyotypes and phenotypes of the two twins can differ. METHOD: We report cytogenetic, molecular genetic and clinical examinations in monochorionic-diamniotic twins discordant in gender. RESULTS: The monochorionic-diamniotic status of the twins was diagnosed by ultrasound and histologic examination of the placenta. Prenatal chromosome examination performed on amniocytes revealed a normal female karyotype in one and a 46,XX(26)/46,XY(3) karyotype in the other twin. Molecular examinations confirmed monozygosity despite discordant sex. Based on the cytogenetic and molecular results of lymphocytes and placental cells, the only explanation for gender discordance was that the conceptus originally had a 47,XXY chromosome complement. CONCLUSION: A 47,XXY zygote appears to have undergone a twinning process. A postzygotic loss of the X chromosome in some cells and the Y chromosome in other cells, either before or after twinning, resulted in 46,XX/46,XY mosaicism in both monozygotic (MZ) twins. The sex discordance of the MZ twins can be explained by different proportions of the 46,XX and 46,XY cell lines in the gonads and other tissues.