Johannes L.H. Evers

Effect of in vitro culture of human embryos on birthweight of newborns

BACKGROUND In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it is not known whether culture conditions play a role in this. METHODS We compared pregnancy rates and perinatal outcomes from singleton pregnancies resulting from a total of 826 first IVF treatment cycles in which oocytes and embryos were randomly allocated to culture in either of two commercially available sequential media systems. RESULTS When the 110 live born singletons in the Vitrolife group were compared with the 78 singletons in the Cook group, birthweight +/- SEM (3453 +/- 53 versus 3208 +/- 61 g, P = 0.003), and birthweight adjusted for gestational age and gender (mean z-score +/- SEM: 0.13 +/- 0.09 versus -0.31 +/- 0.10, P = 0.001) were both significantly higher in the Vitrolife group. When analyzed by multiple linear regression together with several other variables that could possibly affect birthweight as covariates, the type of culture medium was significantly (P = 0.01) associated with birthweight. CONCLUSIONS In vitro culture of human embryos can affect birthweight of live born singletons.

HIGH-DOSE HUMAN MENOPAUSAL GONADOTROPIN STIMULATION IN POOR RESPONDERS DOES NOT IMPROVE IN VITRO FERTILIZATION OUTCOME

OBJECTIVE To study the outcome in poor responders to three ampules (225 IU) of hMG per day in subsequent IVF treatment cycles in which six ampules (450 IU)of hMG per day were administered. DESIGN Retrospective chart review. SETTING Academic tertiary center. PATIENTS Between January 1988 and May 1995, 126 poor response patients had a first treatment cycle on three ampules and a second cycle on six ampules of hMG per day. MAIN OUTCOME MEASURES Numbers of follicles, oocytes, and embryos, and pregnancy rates. RESULTS On six ampules, patients had significantly more follicles and oocytes. The number of embryos did not differ significantly. The pregnancy rate on six ampules were low (3.2% pregnancies per cycle started). CONCLUSION Poor responders do not benefit from high-dose hMG stimulation; their reproduction outcome is poor.

Assessment of efficacy of varicocele repair for male subfertility: a systematic review

BACKGROUND Varicocele repair is a widely used treatment for subfertility. Our aim was to identify and combine the results from randomised controlled trials published to ascertain whether the pregnancy rates after varicocele repair are higher than those with no treatment. METHODS We did a systematic review of seven studies identified by searching Medline and a register of controlled trials. We also searched the contents of specialist journals and the annual meeting programmes of relevant societies by hand. Inclusion criteria were treatment of varicocele in subfertile couples, random allocation to treatment and control groups, and pregnancy or livebirth rates as an outcome measure. We pooled data by use of fixed and random effects models. FINDINGS None of seven eligible studies published between 1979 and 2002 described a strategy for concealment of the allocation sequence. There were 61 pregnancies among 281 treated couples and 50 pregnancies among 259 controls. The overall relative benefit of treatment was 1.01 (95% CI 0.73-1.40) by the fixed effects model and 1.04 (0.62-1.75) by the random effects model. The overall risk difference was 0.2% (-7 to 7) and 3% (-7 to 14), respectively. In subgroup analyses, varicocele treatment was not effective in trials restricted to male subfertility with clinical varicocele, or in those that included men with subclinical varicocele or normal semen analysis. However, this systematic review, done with a meta-analytical method, might have had insufficient power to detect small effects because of the small number of patients in some subgroups. INTERPRETATION Varicocele repair does not seem to be an effective treatment for male or unexplained subfertility.

Epigenetics and the placenta

BACKGROUND The placenta is of utmost importance for intrauterine fetal development and growth. Deregulation of placentation can lead to adverse outcomes for both mother and fetus, e.g. gestational trophoblastic disease (GTD), pre-eclampsia and fetal growth retardation. A significant factor in placental development and function is epigenetic regulation. METHODS This review summarizes the current knowledge in the field of epigenetics in relation to placental development and function. Relevant studies were identified by searching PubMed, Medline and reference sections of all relevant studies and reviews. RESULTS Epigenetic regulation of the placenta evolves during preimplantation development and further gestation. Epigenetic marks, like DNA methylation, histone modifications and non-coding RNAs, affect gene expression patterns. These expression patterns, including the important parent-of-origin-dependent gene expression resulting from genomic imprinting, play a pivotal role in proper fetal and placental development. Disturbed placental epigenetics has been demonstrated in cases of intrauterine growth retardation and small for gestational age, and also appears to be involved in the pathogenesis of pre-eclampsia and GTD. Several environmental effects have been investigated so far, e.g. ethanol, oxygen tension as well as the effect of several aspects of assisted reproduction technologies on placental epigenetics. CONCLUSIONS Studies in both animals and humans have made it increasingly clear that proper epigenetic regulation of both imprinted and non-imprinted genes is important in placental development. Its disturbance, which can be caused by various environmental factors, can lead to abnormal placental development and function with possible consequences for maternal morbidity, fetal development and disease susceptibility in later life.

Early endometriosis invades the extracellular matrix.

OBJECTIVES To investigate whether the aminoterminal propeptides of type III procollagen are increased in patients with early endometriosis and to demonstrate that the subtle lesion of endometriosis is an active stage of the disease. DESIGN Aminoterminal propeptide of type III procollagen was determined in serum and peritoneal fluid (PF) of 100 consecutive patients undergoing laparoscopy. SETTING Academisch Ziekenhuis Maastricht, The Netherlands, a tertiary care center. RESULTS Aminoterminal propeptide PF levels were significantly higher in women with early lesions of endometriosis compared with levels in two groups of controls, i.e., fertile, cycling, women without the disease (P = 0.019) and women on oral contraceptives without the disease (P = 0.036). No difference was found in aminoterminal propeptide PF levels when comparing patients with early lesions of endometriosis and patients with unexplained infertility, the third control group. Aminoterminal propeptide PF levels of patients with endometriosis without early lesions were not different from PF levels in controls. CONCLUSION The early lesion is an active stage of endometriosis, invading the extracellular matrix. In women with unexplained infertility active, microscopic endometriosis may be present.

Expression of integrins E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, endometriosis

Objective To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle. Design An immunohistochemical study. Setting Tertiary care university medical center. Patients Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles. Interventions A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead. Main Outcome Measures The expression of cell adhesion molecules, including the integrin α 2 β 1, α 3 β 1, α 4 β 1, α 5 β 1, and α 6 β 1 and E-cadherin, as determined by immunohistochemistry on frozen sections. Results All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins α 2 β 1 and α 3 β 1 was found in epithelial cells in 13 of 16 cases. Integrin α 5 β 1 was detected in 11 of 16 samples, and integrins α 4 β 1 and α 6 β 1 were detected in 5 of 16 samples. In PF, integrin α 3 β 1 was found in epithelial cells in 12 of 16 samples, integrin α 5 β 1 in 5 of 16, and integrins α 4 β 1 and α 6 β 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin. Conclusion Integrins α 2 β 1, α 3 β 1, α 4 β 1, α 5 β 1, and α 6 β 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis

Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of IVF and more particular of elective single-embryo transfer. Genome-wide gene expression in cumulus cells was studied, since these cells surround the oocyte inside the follicle and therefore possibly reflect oocyte developmental potential. Early cleavage (EC) was chosen as a parameter for embryo viability. Gene expression in cumulus cells from eight oocytes resulting in an EC embryo (EC-CC; n = 8) and from eight oocytes resulting in a non-EC (NEC) embryo (NEC-CC; n = 8) was analysed using microarrays (n = 16). A total of 611 genes were differentially expressed (P < 0.01), mainly involved in cell cycle, angiogenesis, apoptosis, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor signalling, general vesicle transport and chemokine and cytokine signalling. Of the 25 selected differentially expressed genes analysed by quantitative real-time PCR 15 (60%) genes could be validated in the original samples. Of these 8 (53%) could also be validated in 24 (12-EC-CC and 12 NEC-CC) extra independent samples. The most differentially expressed genes among these were CCND2, CXCR4, GPX3, CTNND1 DHCR7, DVL3, HSPB1 and TRIM28, which probably point to hypoxic conditions or a delayed oocyte maturation in NEC-CC samples. This opens up perspectives for new molecular embryo or oocyte selection parameters which might also be useful in countries where the selection has to be made at the oocyte stage before fertilization instead of at the embryonic stage.

Endometrial wavelike movements during the menstrual cycle

OBJECTIVE To qualify and quantify endometrial waves in regularly cycling women. DESIGN A prospective transvaginal ultrasound study was performed throughout 23 cycles of 16 healthy women. Wave type and wave frequency were evaluated. SETTING Normal human volunteers in an academic research environment. PATIENTS Sixteen healthy regularly cycling women. RESULTS Of 23 cycles, 19 proved to be ovulatory. Five types of endometrial waves could be distinguished. Waves from cervix to fundus and opposing waves were seen predominantly periovulatorily. Waves from fundus to cervix were absent in the postovulatory phase. Endometrial wavelike activity was most pronounced in the periovulatory phase. CONCLUSIONS Standardized qualification and quantification of endometrial waves, as described in this study, might shed new light on the underlying mechanisms in selected cases of hitherto unexplained subfertility.

Patient dropout in an assisted reproductive technology program: implications for pregnancy rates

OBJECTIVE To study patient dropout and its impact on crude and cumulative pregnancy rates (PRs) after assisted reproductive technology (ART). DESIGN Retrospective cohort study. SETTING University hospital-based tertiary care fertility clinic. PATIENT(S) Two hundred two couples applying for their first ART treatment cycle. MAIN OUTCOME MEASURE(S) Drop-out rate and PR per cycle, cumulative drop-out rate and cumulative PR. RESULT(S) After three treatment cycles, the cumulative drop-out rate was 126 of 202 (62.4%); 13.9% (n = 28) was due to active censoring. Active censoring was shown to flatter cumulative PRs by life-table analysis. CONCLUSION Dropout from ART is high, even when ART costs are covered by health cost insurance. Life-table analysis overestimates cumulative PRs in ART.

Further evidence that culture media affect perinatal outcome: findings after transfer of fresh and cryopreserved embryos

BACKGROUND We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer of fresh embryos. In the present study, we hypothesize that in vitro culture of embryos during the first few days of preimplantation development affects perinatal outcome, not only in singletons conceived in all rank order cycles but also in twins and in children born after transfer of frozen embryos. Furthermore, we investigated the effect of culture medium on gestational age (GA) at birth. METHODS Oocytes and embryos from consecutive treatment cycles were alternately assigned to culture in either medium from Vitrolife or from Cook. Data on a cohort of 294 live born singletons conceived after fresh transfer during any of a patients IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. RESULTS In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P= 0.03), and in more singletons with low birthweight (LBW) <2500 g (P= 0.006) and LBW for GA ≥ 37 weeks (P= 0.015), when compared with singletons born after culture in medium from Vitrolife AB. GA at birth was not related to the medium used (adjusted difference, 0.05 weeks, P = 0.83). Among twins in the Cook group, higher inter-twin mean birthweight disparity and birthweight discordance were found. Z-scores after FET were -0.04 (± 0.14) in the Cook group compared with 0.18 (± 0.21) in the Vitrolife group (P> 0.05). CONCLUSIONS Our findings support our hypothesis that culture medium influences perinatal outcome of IVF singletons and twins. A similar trend is seen in case of singletons born after FET. GA was not affected by culture medium. These results indicate that in vitro culture might be an important factor explaining the poorer perinatal outcome after assisted reproduction technology (ART). Further research is needed to confirm this culture medium-induced effect in humans and to provide more insight into whether it is caused by epigenetic disturbance of imprinted genes in fetal or placental tissues. Moreover, embryo culture media and their effects need to be investigated thoroughly to select the best embryo culture medium in order to minimize or prevent short-term risks and maybe even long-term disease susceptibility.