Reproductive BioMedicine Online - Articles in Press

Hellenic National Authority of Medically Assisted Reproduction - Accepted Manuscript

Haris E. Cazlaris, Xeni Skorini-Paparrigopoulou, Basil C. Tarlatzis — 2012-02-02

Ovarian biopsy has no role as a routine diagnostic test of ovarian reserve: a systematic review - Accepted Manuscript

Ricky Kwok, Neil P Johnson — 2012-02-02

Abstract: Ovarian reserve describes a woman’s reproductive potential and there are a variety of tests for this. This reflects the lack of a gold standard and the lack of a single test that provides sufficient accuracy. Ovarian biopsy has been proposed as a potential tool for assessing the ovarian reserve and, thus, the ability or inability for a woman to bear a child with or without treatment. The literature assessing the diagnostic accuracy of ovarian biopsy as a test of ovarian reserve for predicting fertility outcomes (live birth rate, ongoing pregnancy, clinical pregnancy, biochemical pregnancy, embryos available, oocytes retrieved or cancelled cycles) was systematically reviewed. There were no studies identified that assessed the diagnostic accuracy of ovarian biopsy for predicting fertility outcomes but a number of studies provided evidence that ovarian follicles are distributed unevenly and randomly throughout the ovarian cortex. This leads to sampling error when ovarian biopsy is used to sample the ovarian reserve. It is concluded that ovarian biopsy should not be used as a test of ovarian reserve.

Successful pregnancy outcome following gamete intra-Fallopian transfer in a patient with Müllerian dysgenesis - Accepted Manuscript

Colin S.S. Lee, Aldrin T.M. Lie — 2012-02-02

Abstract: A 29-year-old lady with Müllerian dysgenesis was keen to have a baby. Clinically, she was medium built with well-developed secondary female sexual characteristics. There was a short and blind vagina. She had undergone surgery for an imperforated hymen. Her FSH and LH concentrations were normal. Laparoscopy revealed a patent right Fallopian tube, a rudimentary right uterus and extensive pelvic endometriosis. She subsequently underwent gamete intra-Fallopian transfer (GIFT). Oocyte retrieval was carried out laparoscopically and a total of nine oocytes were retrieved. Four of the oocytes were transferred together with motile spermatozoa into the right Fallopian tube and the remaining five oocytes were inseminated with spermatozoa for IVF. Three embryos resulted and were frozen. She subsequently developed moderate ovarian hyperstimulation syndrome. Serum β-human chorionic gonadotrophin concentration 14 days after GIFT was 1612 IU/l. Her antenatal care was relatively uneventful until 31 weeks of gestation when she was diagnosed to have intrauterine growth retardation and oligohydramnios. She then underwent an emergency Caesarean section at 32 weeks of pregnancy delivering a normal baby.

The eutopic endometrium in endometriosis: are the changes of clinical significance? - Accepted Manuscript

Ivo Brosens, Jan J. Brosens, Giuseppe Benagiano — 2012-02-02

Abstract: The eutopic endometrium in women suffering from endometriosis is different in many ways from that of healthy controls. Both proliferative and secretory eutopic endometria exhibit changes in endometriosis with heterogeneous responses. In addition, nerve fibres appear in the endometrium and myometrium of these women. The endometrium is a rich source of pro-angiogenic factors and vascular events are often disrupted in endometriosis with an overall increase in angiogenesis. A number of investigations have shown that endometriosis is likely the most common cause of endometrial receptivity defects. Endometriosis is also associated with relative 17β-hydroxysteroid dehydrogenase type II deficiency and these molecular aberrations indicate that local oestrogen production sustains ectopic implants. Recently it has been shown that endometriosis, as a chronic inflammatory disorder, disrupts co-ordinated progesterone response throughout the reproductive tract, including the endometrium, leading to a condition of ‘progesterone resistance’. Investigators have searched for biomarkers of endometriosis, but these investigations are fraught with methodological difficulties. In conclusion, molecular phenotyping of the endometrium is changing the disease paradigm, from being foremost an oestrogen-dependent disease to a disorder characterized primarily by progesterone resistance.

Anti-Müllerian hormone is highly expressed and secreted from cumulus granulosa cells of stimulated preovulatory immature and atretic oocytes - Accepted Manuscript

2012-02-02

Abstract: This study investigated anti-Müllerian hormone (AMH) expression and secretion from cumulus granulosa cells (GC) and steroidogenesis in follicular fluids (FF) with relation to oocyte maturational stages and fertilization capacity in large preovulatory follicles. This prospective study included 53 ovulatory women undergoing intracytoplasmic sperm injection. FF and cumulus GC from 140 large preovulatory follicles were individually obtained during oocyte retrieval. Main outcome measures were oocyte maturation, fertilization and embryo quality. FF were assayed for AMH, progesterone, 17β-oestradiol and testosterone. Cumulus GC were assayed for AMH mRNA expression. AMH mRNA expression and secretion in cumulus GC in preovulatory follicles containing germinal-vesicle (GV) and metaphase-I (MI) oocytes were significantly higher than follicles containing MII oocytes (P < 0.01 and P < 0.0001, respectively). In addition, FF AMH concentrations from atretic oocytes were significantly higher than from MII oocytes. No correlation was found between AMH expression and secretion to fertilization or embryo quality. FF of MI and GV oocytes had higher concentrations of testosterone and lower progesterone/oestradiol ratios than MII oocytes, and FF of atretic oocytes contained higher testosterone concentrations than FF of MII oocytes. AMH is highly expressed in and secreted from cumulus GC of preovulatory follicles containing premature and atretic oocytes.

Response: Re: An exploration of the association between male body mass index and semen quality - Accepted Manuscript

Ahmad Ghiyath Shayeb, Kirsten Harrild, Eileen Mathers, Siladitya Bhattacharya — 2012-02-01

Perceptions of embryo status and embryo use in an Australian community - Accepted Manuscript

2012-01-30

Abstract: Increasingly, important areas of medical therapy and research rely on the donation and use of human embryos. Yet their use is commonly determined by community tolerance and ethico-legal regulation. The aim of this study was to explore the views of an Australian community about what an embryo is, how it should be used and who should make disposition decisions. The findings of a large representative population survey showed that most participants thought of an embryo as human or potentially human but that this did not affect a majority community view that embryos should be used rather than discarded. This study also found divergent views about what the community perceived to be acceptable uses of embryos. The majority perceived the couple as having the authority to make a disposition decision. Women held different views to men across all three questions. The way an embryo was perceived related significantly to how it should be used and who should decide its disposition. These differences and relationships should be considered when developing clinic practices and ethico-legal frameworks to regulate embryo use in science or treatment.

Re: An exploration of the association between male body mass index and semen quality - Accepted Manuscript

Ahmet Gokce, Ege Can Serefoglu, Wayne J.G. Hellstrom — 2012-01-30

Taurine attenuates maternal and embryonic oxidative stress in a streptozotocin-diabetic rat model - Accepted Manuscript

Mahesh Mysore Shivananjappa, Muralidhara — 2012-01-30

Abstract: Oxidative stress mechanisms have been implicated in congenital anomalies and morbidity/mortality of fetus/newborn in diabetic pregnancy. Numerous antioxidant treatments have shown varied beneficial effects in improving both maternal and fetal outcomes. The present study examined the propensity of taurine to attenuate the degree of embryopathy and oxidative stress among pregnant diabetic rats. Adult rats (CFT-Wistar) were rendered diabetic with an acute dose of streptozotocin (STZ; 45 mg/kg bodyweight) on gestation day (GD) 4. Both Diabetic and non-diabetic dams were given oral supplements of taurine (0.5 and 1 g/kg bodyweight/day) from GD 5 to GD 12. Maternal diet intake, bodyweight gain and urine output were monitored and dams were killed on GD 13. Markers of oxidative stress were determined in embryos and maternal livers. STZ treatment induced marked embryopathy (32%) and taurine supplements markedly reduced the degree of embryopathy (54% protection). The STZ-induced higher oxidative stress was significantly attenuated in rats given taurine supplements (P < 0.05) and a similar effect was seen in embryos (P < 0.05). These data suggest that dietary taurine during pregnancy provides significant protection against diabetes-induced oxidative stress in both the mother and the embryos and thus may serve as a therapeutic supplement during diabetic pregnancy.

Disclosure patterns of embryo donation mothers compared with adoption and IVF - Accepted Manuscript

Fiona MacCallum, Sarah Keeley — 2012-01-30

Abstract: Attitudes towards information sharing in donor conception have changed in recent years in some parts of the world, with a move towards openness. This study follows up a sample of embryo donation mothers, examining their current disclosure patterns and comparing them with adoptive and IVF mothers to investigate any influence of the method of family creation. This study compared 17 mothers with an embryo donation child aged 5–9 years with 24 mothers with an adopted child and 28 mothers with a genetically related IVF child. Embryo donation mothers were far less likely to share information with the child; 43% were inclined towards disclosing, compared with all adoptive mothers and over 90% of IVF mothers. Furthermore, embryo donation and IVF mothers who had disclosed had often only given partial explanations of the child’s conception. Differences between embryo donation and adoption in particular should be taken into account when advising embryo recipients.

Effect of patient selection on estimate of reproductive success after surgery for rectovaginal endometriosis: literature review - Accepted Manuscript

2012-01-27

Abstract: The effect of rectovaginal endometriosis on fertility is unclear. Several authors favour radical surgery, including colorectal resection, as a fertility-enhancing procedure. However, interpretation of data is difficult, as the baseline fertility status is often undefined and it is not always possible to discriminate between spontaneous conceptions and those resulting from IVF. A systematic literature review was performed with the aim of defining the pregnancy rate specifically in patients who were infertile before surgery and who sought spontaneous pregnancy. A PubMed search was conducted to identify English language studies published between 2005 and 2011 evaluating reproductive performance after surgery for rectovaginal and rectosigmoid endometriosis. According to the results of the 11 selected studies, the mean post-operative conception rate in all women seeking pregnancy independently of preoperative fertility status and IVF performance was 39% (95% CI 35–43%; 223/571), but dropped to 24% (95% CI 20–28%; 123/510) in infertile patients who sought spontaneous conception (odds ratio 0.50, 95% CI 0.38–0.65%). Patient selection significantly influences the estimate of the effect of rectovaginal endometriosis excision on infertility (P < 0.0001). This should be carefully taken into consideration at preoperative counselling.

Characterization of a recurrent poor-quality embryo morphology phenotype and zygote transfer as a rescue strategy - Accepted Manuscript

2012-01-27

Abstract: Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate (>60%) of highly fragmented embryos (⩾40% of cytoplasmic fragments) leading to recurrent IVF failures. This study postulated that, for various maternal reasons, some embryos were unable to withstand the in-vitro environment and an early pronucleate-stage transfer was proposed to these couples. Fifty-three patients with recurrent IVF failures (a mean of 2.8 ± 1.0 previous attempts) characterized by low embryonic quality (a mean of 62.7% of the embryos with extended fragmentation) were included this transfer protocol. As in previous cycles, the mean number of oocytes retrieved and the fertilization rate were normal. The mean number of zygotes per transfer was 2.24. Fourteen clinical pregnancies were obtained, representing a pregnancy rate and a delivery rate per oocyte retrieval of 26.4% and 18.9%, respectively. Recurrent heavy and early embryo fragmentation in vitro characterizes around 3% of IVF couples and leads to lack of transfer or implantation failure. These data on fresh zygote transfers are encouraging and may provide a valid alternative solution for some of these patients.

Comparative incidence of ovarian hyperstimulation syndrome following ovarian stimulation with corifollitropin α or recombinant FSH - Accepted Manuscript

2012-01-27

Abstract: Corifollitropin α is a novel recombinant gonadotrophin with sustained follicle-stimulating activity. A single injection can replace seven daily injections of recombinant FSH (rFSH) during the first week of ovarian stimulation. All cases of ovarian hyperstimulation syndrome (OHSS) with corifollitropin α intervention in a gonadotrophin-releasing hormone antagonist protocol have been assessed in three large trials: Engage, Ensure and Trust. Overall, 1705 patients received corifollitropin α and 5.6% experienced mild, moderate or severe OHSS. In the randomized controlled trials, Engage and Ensure, the pooled incidence of OHSS with corifollitropin α was 6.9% (71/1023 patients) compared with 6.0% (53/880 patients) in the rFSH group. Adjusted for trial, the odds ratio for OHSS was 1.18 (95% CI 0.81–1.71) indicating that the risk of OHSS for corifollitropin α was similar to that for rFSH. The incidence of mild, moderate and severe OHSS was 3.0%, 2.2% and 1.8%, respectively, with corifollitropin α, with 1.9% requiring hospitalization, and 3.5%, 1.3% and 1.3%, respectively, in the rFSH arms, with 0.9% requiring hospitalization. Despite a higher ovarian response with corifollitropin α compared with rFSH for the first 7 days of ovarian stimulation, the incidence of OHSS was similar.

Genetic variants in microRNA biogenesis pathway genes are associated with semen quality in a Han-Chinese population - Accepted Manuscript

2012-01-27

Abstract: MicroRNA biogenesis genes have been confirmed involved in lots of diseases. This study evaluated the role of genetic variants in microRNA biogenesis genes in semen quality and idiopathic male infertility. Seven single-nucleotide polymorphisms (SNP) of DICER1 (rs13078, rs1057035 and rs12323635) and DROSHA (rs10719, rs2291109, rs17409893 and rs642321) were determined by TaqMan probes and SNPstream in 667 eligible infertile men and 419 fertile controls. Semen quality analysis was performed by computer-assisted sperm analysis. It was found that genetic variants of rs12323635 was associated with idiopathic male infertility. Additionally, in strategy analysis, the rs12323635 C allele might decrease the risk of oligozoospermia (OR 0.42, 95% CI 0.26–0.66; P = 0.0002). The rs642321 TT genotype may have a higher risk of oligozoospermia (OR 2.38, 95% CI 1.34–4.25; P = 0.003). These significant differences were retained after Bonferroni correction. The results showed that variants of DICER1 and DROSHA may modify the risk of abnormal semen parameters, which could result in male infertility.

Gender incidence of intracytoplasmic morphologically selected sperm injection-derived embryos: a prospective randomized study - Accepted Manuscript

2012-01-27

Abstract: The aim of this prospective randomized study was to determine if the use of intracytoplasmic morphologically selected sperm injection (IMSI) is associated with gender incidence. Couples who underwent IVF-preimplantation genetic screening (PGS) cycles, as a result of advanced maternal age, were randomly allocated into two groups: intracytoplasmic sperm injection (ICSI; n = 80) or intracytoplasmic morphologically selected sperm injection (IMSI; n = 80). The incidences of genders were compared between ICSI- and IMSI-derived embryos. Considering all the biopsied embryos were characterized as normal for sex chromosome, the results showed that IMSI results in a significantly higher incidence of female embryos as compared with ICSI (65.1% versus 54.0%, respectively, P = 0.0277). After analysing only euploid embryos for the eight selected chromosomes, a significantly higher incidence of XX embryos derived from IMSI was also observed compared with ICSI cycles (66.9% versus 52.5%, respectively, P = 0.0322). This result was confirmed by logistic regression, which demonstrated a nearly 2-fold increase in euploid XX embryos derived from spermatozoa selected by high magnification (OR 1.83, 95% CI 1.05–3.35, P = 0.032). A higher proportion of morphologically normal spermatozoa analysed under high magnification seem to carry the X chromosome.

Matrix metalloproteinases 1, 2, 3 and 9 functional single-nucleotide polymorphisms in idiopathic recurrent spontaneous abortion - Accepted Manuscript

Nina Pereza, Saša Ostojić, Marija Volk, Miljenko Kapović, Borut Peterlin — 2012-01-27

Abstract: Idiopathic recurrent spontaneous abortion (IRSA) has been associated with abnormalities in the remodelling of endometrial extracellular matrix, as well as aberrant matrix metalloproteinase (MMP) gene expression in endometrium of IRSA women and chorionic villi of IRSA concepti. This study investigated the association of five functional MMP gene promoter polymorphisms (MMP1 –1607 1G/2G, MMP2 –735 C/T, MMP2 –1306 C/T, MMP3 –1612 5A/6A and MMP9 –1562 C/T) with IRSA. A total of 149 couples with at least three consecutive IRSA and 149 fertile couples were included in a case–control study. Genotype analysis was performed using PCR restriction fragment length polymorphism. Statistically significant differences were found in distributions of MMP2 –735 CT (chi-squared 10.21, P = 0.006; OR 2.15, 95% CI 1.34–3.45, P = 0.001), and MMP9 –1562 CC (chi-squared 9.06, P = 0.010; OR 2.21, 95% CI 1.30–3.80, P = 0.004) between IRSA women and controls. Combined analysis of MMP gene polymorphisms did not increase their predictive value. There were no statistically significant differences in genotype and allele frequencies of any polymorphism between IRSA men and controls. MMP2 –735 C/T and MMP9 –1562 C/T functional gene polymorphisms might be associated with an increased risk of IRSA in women.

Influence of Val16Ala SOD2 polymorphism on the in-vitro effect of clomiphene citrate in oxidative metabolism - Accepted Manuscript

2012-01-27

Abstract: This study investigated the in-vitro antioxidant properties of the ovulation induction drug, clomiphene citrate, and assessed whether its effects are influenced by the Val16Ala polymorphism in the SOD2 gene, which encodes manganese superoxide dismutase enzyme. The investigation involved an in-vitro experimental protocol testing the effect of different concentrations of clomiphene citrate on antioxidant capacity, reactive oxygen species (ROS) production and peripheral blood mononuclear cell (PBMC) culture viability. A total of 58 healthy adult women were genotyped for the Val16Ala SOD2 polymorphism, and blood samples were collected to perform in-vitro experiments. ROS production and cytotoxicity assays were performed on blood and PBMC from carriers of different Val16Ala SOD2 genotypes. Clomiphene citrate exhibited antioxidant capacity and effects and decreased ROS production. The AA genotype displayed a more responsive antioxidant effect with clomiphene citrate treatment than other genotypes. AA and AV PBMC showed an increase in viability following treatment with 10 μmol/l clomiphene citrate when compared with control groups. The results suggest that clomiphene citrate exhibits antioxidant activity siimilar to that observed with other selective oestrogen receptor modulators, and the intensity of the effect appears to be SOD2 polymorphism dependent.

Elevated progesterone during ovarian stimulation for IVF - Accepted Manuscript

2012-01-27

Abstract: There is an ongoing debate regarding the impact of premature progesterone rise on the IVF outcome. The objective of this review is to assess evidence of poorer ongoing pregnancy rate in IVF cycles with elevated serum progesterone at the end of follicular phase in ovarian stimulation. It also explores the origin of the progesterone rise, potential modifying factors and possible methods to prevent its rise during ovarian stimulation. This review draws on information already published from monitoring progesterone concentrations at the end of follicular phase in ovarian stimulation. The databases of Medline and PubMed were searched to identify relevant publications. Good-quality evidence supports the negative impact on endometrial receptivity of elevated progesterone concentrations at the end of the follicular phase in ovarian stimulation. Future trials should document the cause and origin of premature progesterone in stimulated IVF cycles.

Epigenetic features of the mouse trophoblast - Accepted Manuscript

Peter J. Rugg-Gunn — 2012-01-27

Abstract: Trophoblast cells are required for the growth and survival of the fetus during pregnancy, and failure to maintain appropriate trophoblast regulation is associated with placental insufficiencies and intrauterine growth restriction. Development of the trophoblast lineage is mediated by interactions between genetic and epigenetic factors. This review will focus on new insights that have been gained from analysis of mouse models into the epigenetic mechanisms that are required for the early establishment of the trophoblast lineage and for the development of specialized cell types of the fetal placenta. In particular, the importance of DNA methylation, 5-hydroxymethylcytosine and histone modifications in orchestrating trophoblast gene expression and functional outcome will be discussed. These insights are beginning to be extended towards human studies and initial results suggest that the causes and consequences of a variety of placental pathologies are related to epigenetic processes. Furthermore, the epigenetic landscape that regulates trophoblast cells seems to be particularly vulnerable to perturbation during development. This has major implications for diet and other environmental factors during pregnancy.

Forming a family with sperm donation: a survey of 244 non-biological parents - Accepted Manuscript

Lucy Frith, Neroli Sawyer, Wendy Kramer — 2012-01-27

Abstract: There has been little research on the views and experiences of non-biological parents of sperm donor children. This paper reports the results of a survey of non-biological mothers and fathers. An online survey was designed and conducted by the Donor Sibling Registry, a US-based non-profit organization that supports those who have used donor conception. A total of 244 people responded (199 non-biological mothers and 45 non-biological fathers). The survey aimed to understand the perspectives of the respondents who had used donor spermatozoa within heterosexual or same-sex relationships, by exploring their views on a number of key issues. Certain issues and concerns associated with not being genetically related to their offspring were experienced differently by men and women. However, there were many important areas of common ground: a concern for getting a healthy donor, the importance of matching the donor to the non-biological partner, and the amount of thought that went into selecting the donor. The implications of these results for policies concerning donor spermatozoa are discussed.

China’s model of egg donation is a policy lesson for Britain - Corrected Proof

Kamal K. Ahuja — 2012-01-13

Abstract: In a move designed to increase the UK’s supply of donor eggs, the Human Fertilisation and Embryology Authority has recently agreed to a fixed-sum compensation policy for donors “which better reflects their expenses” and inconvenience. Such a policy, however, which is reliant on non-patient donors for its success, is in contrast to what appears the system in China, where egg donors can only be recruited from those having IVF (i.e. patient donors) and on a conditional egg-sharing basis. Commitment to an egg-sharing policy in the UK would provide a more equitable system of egg donation than a compensation policy.

Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos - Uncorrected Proof

2012-01-12

Abstract: Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB.Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.

Temperature gradients in female reproductive tissues - Accepted Manuscript

R.H.F. Hunter — 2012-01-09

Abstract: Deep body temperature in mammals is generally but incorrectly regarded as uniform. Alterations of temperature in oviducts and preovulatory Graafian follicles may play a vital role in gamete maturation, fertilization and early embryonic development. At a molecular level, the conformation of regulatory proteins is susceptible to changes in temperature. Deviation from physiological temperature during IVF procedures could thereby exert a profound influence on patterns of gene expression as the embryonic genome unfolds during early cleavage stages and act to generate specific anomalies. Systematic studies are urgently required.

Polymorphisms in DLGH1 and LAMC1 in Mayer–Rokitansky–Kuster–Hauser syndrome - Accepted Manuscript

2012-01-09

Abstract: Müllerian agenesis, also termed the Mayer–Rokitansky–Kuster–Hauser syndrome (MRKHS) is a disorder with an incidence of approximately 1 in 4500 newborn girls. This study screened 12 patients with MRKHS for mutations in two genes, LAMC1 and DLGH1, involved in the development of Müllerian structures and found 10 previously described variants and no novel variants in the coding sequence. It is highly unlikely that these variants are pathological since these are common in the general population. It is the first time that an extensive study of LAMC1 and DLGH1 has been undertaken in patients with MRKHS. The data support the notion that mutations in the coding sequence of LAMC1 and DLGH1 may not be associated with MRKHS.

PGD for inherited cardiac diseases - Uncorrected Proof

2012-01-09

Abstract: Preimplantation genetic diagnosis (PGD) has been applied for more than 200 different inherited conditions, with expanding application to common disorders with genetic predisposition. One of the recent indications for PGD has been inherited cardiac disease, for which no preclinical diagnosis and preventive management may exist and which may lead to premature or sudden death. This paper presents the first, as far as is known, cumulative experience of PGD for inherited cardiac diseases, including familial hypertrophic and dilated cardiomyopathy, cardioencephalomyopathy and Emery–Dreifuss muscular dystrophy. A total of 18 PGD cycles were performed, resulting in transfer in 15 of them, which yielded nine unaffected pregnancies and the births of seven disease- or disease predisposition-free children. The data open the prospect of PGD for inherited cardiac diseases, allowing couples carrying cardiac disease predisposing genes to reproduce without much fear of having offspring with these genes, which are at risk for premature or sudden death.Preimplantation genetic diagnosis (PGD) is currently an established clinical procedure in assisted reproduction and genetic practices. Its application has been expanding beyond traditional indications of prenatal diagnosis and currently includes common disorders with genetic predisposition, such as inherited forms of cancer. This applies also to the diseases with no current prospect of treatment, which may manifest despite presymptomatic diagnosis and follow up, when PGD may provide the only relief for the at-risk couples to reproduce. One of the recent indications for PGD has been inherited cardiac disease, for which no preclinical diagnosis and preventive management may exist and which may lead to premature or sudden death. We present here our first cumulative experience of PGD for inherited cardiac diseases, including familial hypertrophic and dilated cardiomyopathy, cardioencephalomyopathy and Emery–Dreifuss muscular dystrophy. A total of 18 PGD cycles for these disorders was performed, resulting in transfer in 15 of them, which yielded nine unaffected pregnancies and birth of six disease- or disease predisposition-free children. The data open the prospect of PGD for inherited cardiac diseases, allowing couples carrying cardiac disease predisposing genes to reproduce without much fear of having offspring with these genes at risk for premature or sudden death.

Peritoneal fluid modifies the response of human spermatozoa to follicular fluid - Accepted Manuscript

Adriana M. Caille, Cesar L. Berta, Patricia S. Cuasnicú, Maria Jose Munuce — 2012-01-09

Abstract: The aim of this study was to elucidate the mechanism involved in the acrosome reaction (AR) induced by follicular fluid (FF) in spermatozoa previously exposed to peritoneal fluid (PF). The influence of progesterone was also investigated. Semen samples were from 18 normozoospermic donors. PF samples were from 13 women with unexplained infertility and from a woman treated with synthetic progestagen. FF samples were collected from six women undergoing IVF/embryo transfer and pooled. Motile spermatozoa were capacitated overnight and a kinetic and inhibition study on the FF-induced AR was performed. Spermatozoa pretreated with PF were challenged with either FF or progesterone. The ability of progesterone- and progestagen-supplemented PF to induce AR was analysed. Enzyme-digested PF was also tested. Pre-incubation with PF for 60 min completely prevented the FF-induced AR; spermatozoa treated with PF were unable to respond to FF or progesterone and this effect was not reversible. Progesterone- and progestagen-supplemented PF stimulated the AR relative to controls. Enzyme-digested PF did not have an inhibitory capacity. These data strongly suggest that there are one or more inhibitory proteins in PF that interact with spermatozoa so as to prevent access of progesterone to its receptor and thus inhibit the occurrence of the AR.

A case–control pilot study of low-intensity IVF in good-prognosis patients - Accepted Manuscript

Norbert Gleicher, Andrea Weghofer, David H. Barad — 2012-01-09

Abstract: Low-intensity IVF (LI-IVF) is rapidly gaining in popularity. Yet studies comparing LI-IVF to standard IVF are lacking. This is a case–control pilot study, reporting on 14 first LI-IVF and 14 standard IVF cycles in women with normal age-specific ovarian reserve under age 38, matched for age, laboratory environment, staff and time of cycle. LI-IVF cycles underwent mild ovarian stimulation, utilizing clomiphene citrate, augmented by low-dose gonadotrophin stimulation. Control patients underwent routine ovarian stimulation. LI-IVF and regular IVF patients were similar in age, body mass index, FSH and anti-Müllerian hormone. Standard IVF utilized more gonadotrophins (P < 0.001), yielded more oocytes (P < 0.001) and cryopreserved more embryos (P < 0.001). With similar embryo numbers transferred, after ethnicity adjustments, standard IVF demonstrated better odds for pregnancy (OR 7.07; P = 0.046) and higher cumulative pregnancy rates (63.3% versus 21.4%; OR 6.6; P = 0.02). Adjustments for age, ethnicity and diagnosis maintained significance but oocyte adjustment did not. Cost assessments failed to reveal differences between LI-IVF and standard IVF. In this small study, LI-IVF reduced pregnancy chances without demonstrating cost advantages, raising questions about its utility. In the absence of established clinical and/or economic foundations, LI-IVF should be considered an experimental procedure.

Accumulation of oocytes: a new strategy for managing low-responder patients - Accepted Manuscript

A. Cobo, Nicolás Garrido, Juana Crespo, Remohí José, Antonio Pellicer — 2012-01-09

Abstract: Accumulation of oocytes from several ovarian stimulation cycles is currently possible using novel vitrification technologies. This strategy could increase the inseminated cohort, creating a similar situation to normoresponders. This study included 242 low-responder (LR) patients (594 cycles) whose mature oocytes were accumulated by vitrification and inseminated simultaneously (LR-Accu-Vit) and 482 patients (588 cycles) undergoing IVF/embryo transfer with fresh oocytes in each stimulation cycle (LR-fresh). Drop-out rate in the LR-fresh group was >75%. The embryo-transfer cancellation per patient was significantly lower in the LR-Accu-Vit group (9.1%) than the LR-fresh group (34.0%). Live-birth rate (LBR)/patient was higher in the LR-Accu-Vit group (30.2%) than the LR-fresh group (22.4%). Cumulative LBR/patient was statistically higher in the LR-Accu-Vit group (36.4%) than the LR-fresh group (23.7%) and a similar outcome was observed among patients aged ⩾40 years (LR-Accu-Vit 15.8% versus LR-fresh 7.1%). The LR-Accu-Vit group had more cycles with embryo cryopreservation (LR-Accu-Vit 28.9% versus LR-fresh 8.7%). Accumulation of oocytes by vitrification and simultaneous insemination represents a successful alternative for LR patients, yielding comparable success rates to those in normoresponders and avoiding adverse effects of a low response.

Head birefringence properties are associated with acrosome reaction, sperm motility and morphology - Corrected Proof

2012-01-09

Abstract: Birefringence in sperm heads reflects an organized and very compacted texture, indicating nuclear and acrosomal structural normality. This study performed a direct analysis of the acrosome integrity in single spermatozoa to verify whether a pattern of total or partial head birefringence reflected the acrosome status. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, a reacted acrosome was present in 96% of spermatozoa with partial birefringence and only in 35% of those with totally birefringent heads. A great proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P<0.01), while in morphologically abnormal spermatozoa the frequency of total birefringence was comparable to that of partial birefringence irrespective of motility (26% and 27%, respectively, in motile spermatozoa; 22% and 19%, respectively, in immotile spermatozoa). These data support a strong association between partial birefringence and reacted acrosome and show that the patterns of birefringence vary depending on sperm motility and morphology.Human sperm cells are naturally birefringent when observed in a polarizing light due to the presence of longitudinally oriented protein filaments. The detection of birefringence in sperm heads reflects an organized and very compacted texture that indicates nuclear and acrosomal structural normality. Basically, three types of head birefringence have been observed: total, partial (localized in the post acrosomal region) and abnormal (absent or irregular). Data from electron microscopy has suggested a correlation between acrosome integrity and birefringence patterns. The aim of this study was to verify this hypothesis by direct analysis of the acrosome status in single spermatozoa. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, 96% of spermatozoa with partial birefringence had a reacted acrosome, while 65% of totally birefringent heads had an intact acrosome. A large proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P<0.01), while in morphologically abnormal spermatozoa the frequency of total birefringence was comparable to that of partial birefringence irrespective of motility (26% and 27%, respectively, in motile spermatozoa; 22% and 19%, respectively, in immotile spermatozoa). These data support a strong association between partial birefringence and reacted acrosome and show that the patterns of birefringence vary depending on sperm motility and morphology.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus - Corrected Proof

2011-12-22

Abstract: Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin βA subunit (Inhba) and the inhibin α subunit (Inha) in uterine tissues during early pregnancy (days 1–4, preimplantation) and in response to exogenous 17β-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17β-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus.Follistatin and activin are proteins with important functions in female reproduction. There are two forms of follistatin (FST288 and FST315), which interact with activin in different ways and subsequently may have different functions. We are interested in the function of these two forms of follistatin in the uterus. In this study, we examined how the expression of the two follistatin variants and activin changed in the uterus during early pregnancy in mice, as well as in response to the female hormones oestrogen and progesterone. We saw an increase in the expression of the genes for FST288, FST315 and activin in the uterus during early pregnancy. We also saw an increase in these proteins in the mouse uterus in response to progesterone treatment. We concluded that the hormone progesterone regulates expression of both follistatin variants, as well as activin, during early pregnancy in the mouse uterus.

Does exogenous LH in ovarian stimulation improve assisted reproduction success? an appraisal of the literature - Corrected Proof

Micah J. Hill, Gary Levy, Eric D. Levens — 2011-12-22

Abstract: A review of the scientific literature on the use of exogenous LH in assisted reproductive technology was performed by searching the MEDLINE, PubMed and Cochrane online databases. Scientific evidence was reviewed comparing recombinant FSH-only protocols to protocols supplemented with exogenous LH activity: human menopausal gonadotrophin (HMG), recombinant LH and mid-follicular human chorionic gonadotrophin (HCG). Studies were further compared based on pituitary suppression with gonadotrophin-releasing hormone (GnRH) antagonist and agonist protocols. Primary focus was given to randomized controlled trials and meta-analyses. Data from hypogonadotrophic hypogonadal patients demonstrated the importance of LH activity for success of assisted reproduction treatment. However, the majority of normogonadotrophic patients had adequate endogenous LH to successfully drive ovarian steroidogenesis and oocyte maturation. Exogenous LH supplementation was consistently associated with higher peak oestradiol concentrations. The use of HMG in long GnRH agonist cycles was associated with a 3–4% increase in live birth rate. There was insufficient evidence to make definitive conclusions on the need for exogenous LH activity in GnRH antagonist cycles or the benefit of recombinant LH and HCG protocols. Poor responders and patients 35years of age and older may benefit from exogenous LH.LH is essential in natural human reproduction. In many infertile patients treated with assisted reproductive technology, LH concentrations are naturally sufficient to allow for successful conception. The need for the physician to supplement the patient with additional LH as a medication has been a matter of medical controversy. This article reviews the scientific evidence available on the importance of LH in assisted reproduction treatment. Using a form of LH combined with FSH derived from humans (human menopausal gonadotrophin) results in a 3–4% higher live birth rate. Insufficient scientific evidence exists to recommend other forms of LH and more research is needed to clarify this question. Patients who are over the age of 35 or who are poor responders to ovarian stimulation may also benefit from the addition of LH to their ovarian stimulation protocol.

Contraceptive counselling and factors affecting women’s contraceptive choices: results of the CHOICE study in Austria - Corrected Proof

2011-12-19

Abstract: Empirical evidence of the impact of contraceptive counselling and factors affecting women’s contraceptive choices are limited. CHOICE (Contraceptive Health Research Of Informed Choice Experience) was a large-scale study in 11 European countries. Women in Austria aged 15–40 years considering a short-acting, reversible form of combined hormonal contraceptive were eligible to participate. The choices included the combined daily pill, weekly transdermal patch, and monthly vaginal ring. This study assessed and compared 2478 women’s original preferences with their post-counselling choices and evaluated their perceptions and criteria for their choice. Women who were ‘undecided’ decreased from 18.1% pre-counselling to 3.2% post-counselling; significantly more women post-counselling chose the monthly ring (8.7% to 23.8%; difference 15.1%, 95% CI 13.3–16.8%; P<0.0001) or the weekly patch (6.2% to 7.8%; difference 1.7%, 95% CI 0.5–2.9%; P=0.0014). Women’s primary reasons for choosing a method included ‘easy to use’ (daily pill, weekly patch and monthly ring) and ‘still effective if I experience vomiting, diarrhoea’ (weekly patch and monthly ring). Structured and balanced counselling led to changes in the method chosen.The impact of contraceptive counselling and factors affecting women’s contraceptive choices are not well investigated. In the present study, women in Austria aged 15–40 years considering a short-acting, reversible form of combined hormonal contraceptive were eligible to participate. We assessed and compared women’s original preferences with their choices after extensive counselling and evaluated their perceptions and criteria for their choice. Women who were ‘undecided’ decreased from 18.1% before counselling to 3.2% after counselling; significantly more women chose the monthly ring after counselling or the weekly patch. Women’s primary reasons for choosing a method included ‘easy to use’ (pill, patch and ring) and ‘still effective if I experience vomiting, diarrhoea’ (patch and ring). A balanced counselling led to changes in the method chosen, even in women possessing profound knowledge about their preferred method.

Short-term, low-dose, non-steroidal anti-inflammatory drug application diminishes premature ovulation in natural-cycle IVF - Corrected Proof

2011-12-16

Abstract: A retrospective cohort study was conducted in a private infertility centre to evaluate the use of non-steroidal anti-inflammatory drugs (NSAID) in natural-cycle IVF (nIVF) treatment. A total of 1865 first-rank nIVF cycles performed during 2009–2010 were evaluated. Low-dose, post-trigger NSAID was administered in a non-randomized way in cycles at higher ovulation risk where an imminent LH surge was detected on triggering day. Main outcome measures were premature ovulation rate, embryo transfer rate per scheduled cycle and clinical pregnancy and live birth rates per embryo transfer. NSAID use was associated with a significantly lower risk of premature ovulation (3.6% versus 6.8%, adjusted OR 0.24, 95% CI 0.15–0.39, P<0.0001) and higher embryo transfer rate (46.8% versus 39.5%, adjusted OR 1.38, 95% CI 1.06–1.61, P=0.012) per scheduled cycle. Clinical pregnancy (39.1% versus 35.9%) and live birth rates per embryo transfer (31.3% versus 31.4%) were comparable. In this retrospective series, short-term low-dose NSAID application positively influenced nIVF cycles by diminishing the rate of unwanted premature ovulations and increasing the proportion of cycles reaching embryo transfer.In recent years, increasing interest has emerged in milder approaches to IVF treatment such as natural-cycle IVF (nIVF), which could decrease the physical burden and psychological distress for patients, increase patient convenience and reduce treatment costs. The main drawback of nIVF is its low per cycle efficiency, which is partly related to high cancellation rates due to premature LH surge and premature ovulation detected at the time of oocyte retrieval. Non-steroidal anti-inflammatory drugs (NSAID) could efficiently delay or even prevent follicular rupture and they were recently proposed as a way to control spontaneous ovulations during nIVF. A retrospective cohort study was conducted in a Japanese private infertility centre to evaluate the use of NSAID in a total of 1865 nIVF cycles during 2009–2010. The NSAID use was associated with a significantly lower risk of premature ovulation (3.6% versus 6.8%, adjusted odds ratio 0.24) and higher embryo transfer (46.8% versus 39.5%, adjusted odds ratio 1.38). In our retrospective series, short-term, low-dose NSAID application positively influenced nIVF outcome by diminishing the rate of unwanted premature ovulation and increasing the proportion of cycles reaching embryo transfer. A large randomized clinical trial is still warranted to evaluate more precisely the effect of low-dose NSAID administration in nIVF cycles.

Interleukin-1 gene cluster variants and recurrent pregnancy loss among North Indian women: retrospective study and meta-analysis - Corrected Proof

S. Agrawal, F. Parveen, R.M. Faridi, S. Prakash — 2011-12-12

Abstract: The aim of this study was to determine whether or not interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β) and IL-1 receptor antagonist (IL-1RA) polymorphisms are associated with risk of unexplained recurrent pregnancy loss (RPL) among North Indian women. This retrospective case–control study examine 200 well-characterized RPL cases for IL-1 gene cluster variants, determined by restriction fragment length polymorphism-PCR. The observed allele, genotype and haplotype distributions were compared with those obtained from 300 ethnically matched negative controls. Invariant distribution of IL-1 gene cluster single-nucleotide polymorphisms was observed among RPL cases and controls. Meta-analysis of IL-1β −511, +3953 and IL-1RN 86-bp variable number tandem repeat from the reported literature and this study did not reveal any significant association with the risk of RPL. In conclusion, no significant difference between RPL and control groups was observed at the allele, genotype or haplotype levels when tested for association using the dominant, recessive and additive models of inheritance for IL-1 gene cluster variants. As far as is known, this is the first report from India pertaining to IL-1 gene cluster variants’ association with the risk of RPL from North India.One of the most unfortunate complications of incomplete motherhood is recurrent pregnancy loss (RPL) particularly of unknown aetiology. RPL is defined as the loss of three or more consecutive pregnancies before the 24th week of gestation. Both genetic and environmental factors may play a role in the maintenance of pregnancy. From a traditional immunological perspective, survival of the semi-allogenic fetus is dependent on suppression of the maternal immune response. However, the function of immune cells changes during pregnancy, no generalized suppression of the maternal immune response has been recorded. Cytokines in the interleukin-1 system (IL-1α, IL-1β and receptor antagonist (IL-1RA)) are produced at the fetal–maternal interface during early pregnancy and are believed to influence the immune responsiveness of Th1/Th2 cells and has been implicated in the establishment of successful pregnancy. This study examines IL-1 gene cluster variants among well-characterized RPL patients and compares them with those of healthy controls. IL-1 gene cluster single-nucleotide polymorphisms were found to be invariably distributed among RPL patients and controls. Meta-analysis of results of IL-1β −511, IL-1β +3953 and IL-1RN 86-bp variable number tandem repeat studies in RPL, as reported previously, also did not show any effect of these variations on the risk of occurrence of the disease.

Patient evaluation of infertility management in an ISO 9001:2008-certified centre for reproductive medicine - Corrected Proof

2011-12-08

Abstract: Quality management according to ISO 9001:2008 guidelines includes infertility care quality assessment and improvement. This study aimed to describe the development process of a questionnaire for infertility management. A literature review, qualitative interviews with experts and patients resulted in a content-valid and face-valid questionnaire. Three cross-sectional surveys were performed in 2004, 2007 and 2008 in a tertiary university infertility centre. First (2004), the questionnaire – measuring eight a-priori dimensions of infertility management – was tested. Second (2007), improvement projects for infertility management were evaluated. Third (2008), factor analysis was performed and internal consistency was documented. The developed patient questionnaire to evaluate infertility management and pre-set desired levels of agreement served to set targets for and assess quality improvement projects. The final patient questionnaire to evaluate infertility management within an ISO framework was valid and reliable and contained 14 items covering four dimensions of infertility management: Telephone Access, Reception, Information and Patient-centeredness. ISO 9001:2008-certified infertility centres can evaluate infertility management with this 14-item questionnaire. This top-down approach to evaluate the patients’ perspective on quality aspects selected by health professionals can be complementary to the bottom-up approach evaluating the patients’ complete experiences of quality of care.Quality assessment and improvement in infertility care are central concepts in quality management according to ISO 9001:2008 guidelines. This paper describes the development process of a questionnaire for infertility management. The questionnaire was constructed by conducting a literature review, qualitative interviews with experts and patients in order to obtain a content-valid and face-valid questionnaire. Three cross-sectional surveys were conducted at a tertiary university infertility centre. First (2004), the questionnaire – measuring eight a-priori dimensions of infertility management – was tested. Second (2007), improvement projects for infertility management were evaluated. Third (2008), psychometric properties of the questionnaire were evaluated. The final patient questionnaire to evaluate infertility management within an ISO framework contained 14 items covering four dimensions of infertility management: Telephone Access, Reception, Information and Patient-centeredness. The patient questionnaire has been shown to be valid and reliable and can be used to set targets for and assess quality improvement projects. This top-down approach to evaluate the patients’ perspective on quality aspects selected by health professionals can be complementary to the bottom-up approach to evaluate the patients’ complete experiences of quality of care.

Five chromosome segregation in polar bodies and the corresponding oocyte - Corrected Proof

2011-12-06

Abstract: For a comprehensive picture of the meiotic process and to follow up its products, five chromosomes were tested by fluorescent in-situ hybridization in both polar bodies (PB) and corresponding 145 oocytes. Results were obtained in 143 sets and the prediction of euploidy or aneuploidy based on PB analysis was confirmed by direct analysis in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes, while in the remaining eight, at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in PB: 58% in PB1 and 42% in PB2. Premature separation of chromatids occurred in 89% of aneuploid PB1, whereas only 11% of errors derived from bivalent non-disjunction. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division. In conclusion, the testing of PB predicted reliably the oocyte’s chromosome condition. Although limited to five chromosomes, the follow up of meiosis by fluorescent in-situ hybridization provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte.One essential requirement for normal embryo development is the presence of 23 pairs of chromosomes, a condition defined as euploidy. An incorrect number of chromosomes, or aneuploidy, is relatively frequent in human oocytes and is rarely compatible with implantation. The aim of this study was to verify the efficiency of polar body (PB) analysis in predicting the oocyte chromosomal status and to estimate meiotic errors for five chromosomes by fluorescent in-situ hybridization (FISH) in the complete set of female meiosis products, PB1, PB2 and oocyte. In all, 145 oocyte sets were analysed with results obtained in 143; the prediction of euploidy or aneuploidy made by PB testing was confirmed in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes; while in eight at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in the PB: 58% in PB1 and 42% in PB2. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division and, accordingly, the corresponding oocytes resulted to be normal for the chromosomes involved in the sequential errors. In conclusion, the testing of PB reliably predicted the oocyte chromosome condition. Although limited to five chromosomes, the follow up of meiosis by FISH provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte.

Sperm cryopreservation before cancer treatment: a 15-year monocentric experience - Corrected Proof

2011-12-05

Abstract: Sperm banking is an important procedure to preserve fertility before cancer therapy. The aim of this study was to comprehensively analyse cryopreservation activity retrospectively for 1080 patients referred to the sperm bank for sperm cryopreservation before cancer treatment. This study included 1007 patients diagnosed with testicular cancer (TC) (41.7%), lymphoma (26%), other haematological cancers (9.4%) or other types of cancer (22.8%); of these, 29 patients did not produce any semen sample and cryopreservation was impossible for 67 patients. Semen characteristics before treatment were within normal ranges, except moderate asthenospermia. Sperm concentration was significantly lower in TC than in non-TC. Straws from 57 patients (6.3%) were used in assisted reproductive technologies, which led to a 46.8% cumulative birth rate. Straws were destroyed for 170 patients (18.7%) and 140 patients performed semen analyses after cancer therapy. After an average delay of 22.5 months after the end of therapy, 43 patients (30.7%) exhibited azoospermia. This study of a large population of cancer patients revealed a high level of successful sperm storage. Utilization of cryopreserved spermatozoa led to good chances of fatherhood. Nevertheless, sperm banks should be aware of the low rates of straw use and straw destruction by cancer patients.Sperm banking is an important procedure to preserve fertility before cancer therapy. The aim of this study was to analyse comprehensive cryopreservation activity. We retrospectively studied 1080 patients referred to the sperm bank for sperm cryopreservation before cancer treatment. We included 1007 patients diagnosed with testicular cancer (TC) (41.7%), lymphoma (26.0%), other haematological cancers (9.4%) or other types of cancer (22.8%). Of these, 29 patients did not produce any semen sample and cryopreservation was impossible for 67 patients. Semen characteristics before treatment were within normal ranges, except moderate decrease in sperm motility. Sperm concentration was significantly lower in TC than in non-TC. Frozen spermatozoa from 57 patients (6.3%) were used in assisted reproductive treatment, which led to a 46.8% cumulative birth rate. Frozen spermatozoa were destroyed for 170 patients (18.7%). A total of 140 patients performed semen analyses after cancer therapy. After an average delay after the end of therapy of 22.5 months, 43 patients (30.7%) exhibited an absence of spermatozoa in the semen. This study of a large population of cancer patients revealed a high level of successful sperm storage. Utilization of cryopreserved spermatozoa in assisted reproduction led to good chances of fatherhood. Nevertheless, sperm banks should be aware of the low rates of frozen sperm use and frozen sperm destruction by cancer patients.

Simple vitrification for small numbers of human spermatozoa - Corrected Proof

2011-12-05

Abstract: Conventional freezing procedures and containers are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility, and various types of container have been utilized to freeze small numbers of spermatozoa. This study tried to develop a vitrification method for small numbers of spermatozoa using the Cell Sleeper, which is a closed type of cell-cryopreservation container. The container with spermatozoa were cooled in liquid nitrogen vapour and then stored in a cryotank. Sperm motility parameters improved significantly (P<0.05) by vitrification in oil-free droplets rather than in droplets covered with oil. After vitrification of five spermatozoa per container, all spermatozoa were recovered and the viable sperm rate was significantly higher when spermatozoa were vitrified in a 3.5-μl droplet rather than in 0.5μl (72.0% versus 38.0%; P<0.01). Recovery, motility and viability rates of vitrified–warmed spermatozoa were similar between the Cell Sleeper and the CryoTop groups. In conclusion, the Cell Sleeper is a highly effective tool for the cryopreservation of small numbers of spermatozoa and limited cells can be vitrified quickly and simply without significant loss.Conventional freezing procedures are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility. Techniques for the cryopreservation of small numbers of spermatozoa have attempted to use various types of containers, but these are the only currently available options and the lack of an easily implemented technology has remained a major bottleneck. In the current study, we successfully established a simple cryopreservation method for small numbers of human spermatozoa using the Cell Sleeper, which is a vial type of cell-cryopreservation container and is equipped with an inner tray. Cell Sleeper is commercially available and easy to prepare for use. On single-sperm cryopreservation, the best result was obtained when spermatozoa were cryopreserved in an oil-free 3.5-μl droplet and it was the most convenient volume for handling small numbers of spermatozoa using an intracytoplasmic sperm injection pipette equipped with a micromanipulator. Furthermore, we could recover spermatozoa efficiently and quickly without significant loss. It may be concluded that the Cell Sleeper is a useful container for the cryopreservation of small numbers of spermatozoa.

Screening for mutations of the FOXO4 gene in premature ovarian failure patients - Corrected Proof

2011-12-05

Abstract: FOXO4 constitutes a coherent candidate gene associated with premature ovarian failure (POF) pathogenesis. This study sequenced the coding and exon-flanking regions of this gene in a panel of 116 POF patients and 143 controls of Tunisian origin. In both groups, the IVS2+41T>G sequence variant was identified. It is concluded that coding mutations of FOXO4 should not be a common cause of the disease in women from the Tunisian population. However, this study cannot exclude that FOXO4 dysfunctions, originated from open reading frame or promoter sequence variations, might be associated with the pathogenesis of the disease in other ethnical groups.

Testosterone concentrations in early pregnancy: relation to method of conception in an infertile population - Corrected Proof

2011-12-05

Abstract: This prospective cohort study of infertility patients compared testosterone concentrations in early pregnancy in infertility patients who conceived naturally or after treatment. Although all groups demonstrated some increase in pregnancy testosterone from baseline concentrations, subjects who conceived following ovulation induction showed a significantly increased rise in testosterone as compared with controls (P<0.01).

Oestradiol valerate pretreatment in GnRH-antagonist cycles: a randomized controlled trial - Corrected Proof

2011-12-02

Abstract: This randomized controlled trial analyses the ability to control the oocyte retrieval schedule of gonadotrophin-releasing hormone antagonist cycles through the administration of oestradiol valerate during the luteo-follicular transition period prior to the initiation of ovarian stimulation. Eighty-six women undergoing ovarian stimulation for IVF/intracytoplasmic sperm injection were enrolled in the study. The control group (n=42) received a standard ovarian stimulation protocol. In the pretreatment group (n=44), patients were administered oestradiol valerate at a daily dose of 2×2mg from day 25 of the preceding cycle onwards, during 6–10 consecutive days, depending on the day of the week. The primary endpoint was the proportion of patients undergoing oocyte retrieval during a weekend day (i.e. Saturday or Sunday), which was significantly lower in the pretreatment group (1/37, 2.7%) compared with the control group (8/39, 20.5%; P value=0.029). The clinical pregnancy rates per started cycle were similar in the pretreatment group (38.6%) compared with the control group (38.1%). Pretreatment with oestradiol valerate results in a significantly lower proportion of patients undergoing oocyte retrieval during a weekend day and can be a valuable tool for the organization of an assisted reproduction centre.The past decade has seen growing interest in gonadotrophin-releasing hormone (GnRH)-antagonist protocols in an effort to reduce the incidence of potential complications. Nevertheless, GnRH agonists remain the GnRH analogue of choice in many assisted reproduction clinics, since they allow a more flexible and better controlled schedule of oocyte retrievals. The present study prospectively evaluates the efficacy of scheduling assisted reproduction cycles in a gonadotrophin-releasing hormone (GnRH)-antagonist protocol through the flexible pretreatment with oestradiol valerate during the luteo-follicular transition period of the menstrual cycle. Eighty-six women were enrolled in the study. The control group (n=42) received a standard ovarian stimulation protocol, whereas in the pretreatment group (n=44), patients were administered oestradiol valerate (Progynova) at a daily dose of 2×2mg from day 25 of the preceding cycle onwards, during 6–10 consecutive days, depending on the day of the week. After discontinuing the oestradiol valerate pretreatment, ovarian stimulation was initiated using the same protocol as the control group. The primary endpoint was the proportion of patients undergoing oocyte retrieval during weekend days, which was significantly lower in the pretreatment group (1/37, 2.7%) compared with the control group (8/39, 20.5%; P-value=0.029). The clinical pregnancy rates per started cycle were similar in the pretreatment group (38.6%) compared with the control group (38.1%). It can be concluded that pretreatment with oestradiol valerate results in a significant lower proportion of patients undergoing oocyte retrieval during a weekend day and can be a valuable tool for the organization of an assisted reproduction centre.

Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates - Corrected Proof

James Stanger, Jesmine Wong, Jason Conceicao, John Yovich — 2011-12-02

Abstract: Occasionally, clinical scenarios arise where embryos, previously cryostored and warmed, need to be recryopreserved. The outcome of 30 such transfer cycles from 25 women where embryos were recryopreserved is detailed. In 16 cases, embryos were initially cryopreserved by slow freezing and in 14 cases by vitrification. The cryopreservation stages were the pronuclear stage (n=16), day-3 cleavage stage (n=12), blastocyst (n=1) and oocytes (n=1). All recryopreservation was by Cryotop-based vitrification. From this mixed source, 30/31 twice-cryopreserved embryos survived warming and were transferred, resulting in 13 pregnancies, 11 deliveries with normal gestational age and birthweight, one pre-term birth at 33 weeks and two miscarriages. There were no malformations reported for the live births. Recryopreservation using vitrification by CryoTop has been used in a variety of clinical scenarios to preserve surplus cryopreserved embryos. The current study, although limited in numbers, resulted in high survival rates, clinical pregnancy rates similar to once-cryopreserved embryos and healthy live births independently of the initial stage and cryopreservation method. The technique may increasingly be applicable to elective single-embryo transfer and blastocyst transfer to maximize the pregnancy rate while minimizing the number of cryopreserved embryo transfers.Freezing of embryos has significantly influenced the likelihood of pregnancy after an IVF stimulation cycle. Since it is often unclear which embryos have the potential to continue developing, more embryos may be thawed than transferred. If, in such cases, the surplus embryos remain viable, the ability to refreeze them is an important laboratory tool. In this study, we report on 30 cycles from 25 women where, for a variety of clinical scenarios, surplus embryos previously cryopreserved by either slow freezing or more recently by vitrification were recryopreserved using the Cryotop vitrification protocol. The report details 13 pregnancies, including 11 live births. The study group included one case where oocytes rather than embryos were initially vitrified. When these recryopreserved embryos were subsequently warmed, they exhibited a high survival rate and, after embryo transfer, a pregnancy rate similar to once-cryopreserved embryos. While recryopreservation has to date demonstrated that the embryos retain viability, leading to healthy children, and is, as such, an additional tool for embryologists, its application should proceed with caution.

Targeted gonadotrophin stimulation using the PIVET algorithm markedly reduces the risk of OHSS - Corrected Proof

John Yovich, James Stanger, Peter Hinchliffe — 2011-12-02

Abstract: PIVET Medical Centre has developed an empirical algorithm for the dose of FSH administration based upon day-2 FSH, antral follicle count, anti-Müllerian hormone, body mass index, age and smoking parameters in an attempt to reduce the incidence of ovarian hyperstimulation syndrome particularly in at-risk women with elevated antral follicle count and anti-Müllerian hormone. The algorithm utilized the incremental dosage capabilities of the recombinant FSH pens to fine-tune the daily concentration of FSH. Application of the algorithm aimed to minimize any form of excessive follicle recruitment that necessitated increased clinical awareness. The measure used to assess the impact of the algorithm was the number of women who, after oocyte retrieval, were considered to be potentially at risk of any degree of OHSS and were allocated to increased monitoring. Compared with the previous 20-month period, introduction of the algorithm significantly reduced both the incidence of referral for increased monitoring, treatment for OHSS and the incidence of freeze-all cycles (all P<0.05). This was particularly focused on those considered to be at risk without reducing the fresh cycle pregnancy rate.Women with an elevated antral follicle count are at risk of ovarian hyperstimulation syndrome (OHSS) following gonadotrophin stimulation for IVF. This study developed an FSH dosage algorithm based upon several risk factors and targeting women with the potential for OHSS so that the dose of hormone could be minimized. This significantly reduced the need for increased monitoring and incidence of treatment for OHSS, as well as reducing the treatment cycles where all the embryos were frozen. These benefits were achieved without diminishing the pregnancy rate.