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Category Archives: Human Reproduction

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 patient's 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.

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http://humrep.oxfordjournals.org/rss/current.xml

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Cyclosporin A promotes crosstalk between human cytotrophoblast and decidual stromal cell through up-regulating CXCL12/CXCR4 interaction

BACKGROUND

Our previous studies have demonstrated that cyclosporin A (CsA) can increase the cell number in and invasion by human first-trimester trophoblasts and induce maternal–fetal tolerance. C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine ligand 12 (CXCL12) are important mediators at the maternal–fetal interface during early pregnancy. In this study, we further investigate the molecular mechanisms underlying modulation by CsA of the crosstalk between human cytotrophoblast and decidual stromal cell (DSC).

METHODS

Human first-trimester cytotropoblast and DSC were treated with CsA in the absence or presence of U0126 pretreatment, and then the mRNA and protein levels of CXCL12 and CXCR4 were measured by RT–PCR, qPCR, in-cell western blots and enzyme-linked immunosorbent assay (ELISA), respectively. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and Matrigel invasion assays were used to determine the invasiveness of cytotrophoblast, respectively. The activity of matrix metalloproteinase (MMP)-9 and MMP-2 was detected by gelatin zymography. A co-culture with direct contact between cytotrophoblast and DSC was established and used to investigate the interaction between these two cells.

RESULTS

CsA up-regulated CXCL12 and CXCR4 expression in human first-trimester cytotrophoblast cells, but not in DSCs. Blocking the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK1/2) signaling by U0126 abrogated the CsA-induced increase in CXCL12 and CXCR4 expression and neutralizing antibodies to CXCL12 or CXCR4 completely inhibited the CsA-induced increase in cell number, invasion and MMP-9 and MMP-2 activity of cytotrophoblast. CsA also significantly promoted the activity of MMP-9 and MMP-2 in DSCs, but this was unaffected by CXCL12 or CXCR4 neutralizing antibody. Furthermore, the CsA-induced MMP-9 and MMP-2 activity and the invasiveness of cytotrophoblast in the cytotrophoblast and DSC co-culture were significantly increased compared with CsA-treated trophoblast cultured alone, and CXCR4 blocking antibody effectively abolished the increased MMP activity and invasion of cytotrophoblasts in the cytotrophoblast-DSC co-culture stimulated by CsA.

CONCLUSIONS

CsA can promote the crosstalk between cytotrophoblast and DSC through up-regulating CXCL12/CXCR4 interaction via MAPK signaling, resulting in the increased numbers of and invasion by human cytotrophoblast cells.

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http://humrep.oxfordjournals.org/rss/current.xml

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Expression of leukaemia inhibitory factor and interleukin 15 in endometrium of women with recurrent implantation failure after IVF; correlation with the number of endometrial natural killer cells

BACKGROUND

Several studies have suggested that endometrial interleukin 15 (IL-15) and the leukaemia inhibitory factor (LIF) may be important in embryo implantation. IL-15 is postulated to play a role in the control of uterine natural killer (uNK) cell proliferation and function, and uNK cells are also known to play a role in implantation. The aims of this study was to (1) compare endometrial levels of IL-15 and the LIF in women with recurrent implantation failure (RIF) after IVF with those in fertile women (controls) and (2) examine the relation of IL-15 and LIF levels to the uNK cell number.

METHODS

We investigated IL-15 and LIF in precisely timed endometrial biopsies (days LH + 7-LH + 9, where the day of the LH surge is LH + 0) obtained from control women (n = 15) and women with RIF (n = 45) by immunohistochemistry. A semi-quantitative analysis was performed by the H-score analysis of staining intensity in the stroma, glandular epithelium and luminal epithelium, separately. We also correlated expression of LIF and IL15 with uNK cell numbers (obtained in an earlier study of the same samples).

RESULTS

The quantity of the LIF protein in endometrial glandular epithelium in women with RIF [median and range; 179 (70–365)] was lower (P = 0.01) than in control women [median and range; 247 (120–287)]. In contrast, the level of the IL-15 protein in the stroma in women with RIF [median and range; 90 (0–175)] was higher (P = 0.009) than in control women [median and range; 60 (15–150)]. There was a significant correlation between the uNK cell number and stromal expression of IL-15 (r = 0.427, P = 0.001). No correlation between the LIF expression in any compartment and the uNK cell number was seen.

CONCLUSIONS

The results show an altered expression of LIF and IL-15 in the endometrium of women with RIF. Despite the limitation of not identifying uNK cells by phenotypic markers, the correlation between the uNK cell number and the stromal cell IL-15 suggests that IL-15 may play a role in the control of endometrial uNK cell function or proliferation.

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http://humrep.oxfordjournals.org/rss/current.xml

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Case report of apoptosis in testis of four AZFc-deleted patients: increased DNA fragmentation during meiosis, but decreased apoptotic markers in post-meiotic germ cells

AZFc deletions of the Y chromosome are the major known genetic cause of spermatogenetic failure. Meiotic studies have shown a prevalence of synaptonemal complex fragmentation and an excess of early-stage sperm cells, suggesting that the maturation block could involve apoptosis. We present a prospective and observational study of apoptotic markers in the sperm of four AZFc-deleted patients and two non-obstructive azoospermic controls without an AZFc deletion. Polycaspases assays and terminal deoxynucleotidyl transferase dUDP nick-end labelling (TUNEL) assays were combined to evaluate the incidence of apoptosis in pre-meiotic, meiotic and post-meiotic germs cells identified, respectively, using anti-melanoma-associated antigen A4 (MAGE-A4), anti-synaptonemal complex protein 3 (SCP3) and anti-sperm acrosome membrane-associated protein 1 (SPACA1) antibodies. We detected apoptosis at all stages of AZFc-deletion spermatogenesis. Using the caspase assay, the incidence of positive cells was found to be heterogeneous for pre-meiotic (from 4.8 to 84.5%) and meiotic stages (from 7.9 to 57.6%), while for post-meiotic cells, the mean incidence was 6% in AZFc-deleted patients compared with 26.5% in controls (P < 0.05). Using the TUNEL assay, the mean percentage with DNA fragmentation for meiotic cells was 54.0% in AZFc-deleted patients compared with 20.3% in controls (P < 0.05), while the percentage of TUNEL-positive post-meiotic cells ranged from 5.3 to 44.7%. Spermatocyte loss in AZFc-deleted patients occurs via the apoptotic pathway. In post-meiotic cells, the lower incidence of apoptosis in testis from three of the four AZFc-deleted patients, compared with controls, is consistent with AZFc deletions having little negative impact on sperm quality.

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http://humrep.oxfordjournals.org/rss/current.xml

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Evaluation of preparatory psychosocial counselling for medically assisted reproduction

BACKGROUND

This study evaluated couples' perceptions of preparatory psychosocial counselling prior to participation in medically assisted reproduction (MAR).

METHODS

Eighty-three couples about to undergo IUI treatment were asked about their expectations regarding a subsequent single psychosocial counselling session and assessed in terms of their levels of infertility-specific stress, anxiety and depressive symptoms. Afterwards, participants rated their satisfaction with different elements of the session.

RESULTS

Almost two-thirds of women and one-half of men expected counselling to be important, and the majority anticipated that the session would be helpful and informative. Views of preparatory counselling were significantly more positive afterwards, indicating that a focused session addressing issues of treatment concerns, goal setting and managing infertility stress was more beneficial than anticipated. Those experiencing higher levels of infertility-specific stress expected the counselling session to be more important, and elevated stress and greater utilization of social support were predictive of post-counselling satisfaction.

CONCLUSIONS

Preparatory psychosocial counselling provided with a specific and practical focus appears to be a potentially important and helpful service prior to MAR. Clinics should not assume that patients can accurately judge the benefits of counselling before actually engaging in the session. Identifying patients most likely to benefit and providing a clear rationale may further increase receptivity to this proactive counselling service. While patients characterize this intervention as beneficial, it is not yet known if these benefits translate into improved management of treatment procedures.

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http://humrep.oxfordjournals.org/rss/current.xml

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Guidelines for infertility counselling in different countries: Is there an emerging trend?

BACKGROUND

It is widely accepted that infertility and involuntary childlessness, and the decision to engage with assisted reproduction technology (ART) services as a patient, donor or surrogate can entail wide-ranging psychosocial issues. Psychosocial counselling has, therefore, become valued as an integral element of ART services. The objective of this study was to begin to map out what exists globally by the way of guidelines for infertility counselling.

Methods

Data were analysed from formal guidelines produced by seven national infertility counselling bodies, onetransnational infertility counselling organization, reports of the American Society for Reproductive Medicine Ethics Committee and Practice Committee and the ESHRE Task Force on Ethics and Law. Additional sources of data were the International Infertility Counseling Organization and counselling colleagues internationally.

Results

Four broad areas concerning contemporary practice in infertility counselling are identified: (i) the legal mandate for counselling; (ii) eligibility credentials for individuals carrying out professional counselling activities; (iii) different forms of counselling and (iv) counselling practice in relation to specific elements of assisted reproduction treatment.

CONCLUSIONS

Internationally, the development of infertility guidelines is best described as a ‘work in progress’, although key trends are evident.

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http://humrep.oxfordjournals.org/rss/current.xml

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