Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development.

STUDY QUESTION: In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? SUMMARY ANSWER: Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. WHAT IS KNOWN ALREADY: Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. STUDY DESIGN, SIZE, DURATION: Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). LIMITATIONS, REASON FOR CAUTION: RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. WIDER IMPLICATIONS OF THE FINDINGS: Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. STUDY FUNDING/COMPETING INTEREST(S): Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. TRIAL REGISTRATION NUMBER: Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

Effect of seminal plasma application to the vaginal vault in in vitro fertilization or intracytoplasmic sperm injection treatment cycles-a double-blind, placebo-controlled, randomized study.

PURPOSE: To study whether intravaginal application of seminal plasma after follicle aspiration has the potential to increase implantation and clinical pregnancy rates after IVF-ET. METHODS: We conducted a prospective, double-blind, placebo-controlled randomized study of 230 patients undergoing IVF-ET cycles. 500 µL of Fresh seminal plasma from the patient's partner or culture medium (placebo) were injected in the vaginal vault just after follicle aspiration. The main outcome measured was ongoing clinical-pregnancy rate. RESULTS: After ET cancellation in ten patients due to lack of fertilization or embryo cleavage, 220 embryo transfers (103 and 117 in the study and control groups) resulted in a clinical pregnancy rate of 36.9 % and 29.1 % for the study and control groups, corresponding to a relative increase of 26.8 %. After an early pregnancy loss of 13.1 % (5/38) and 23.5 % (8/34) in the study and control groups respectively an ongoing pregnancy rate of 32.0 % (33/103) and 22.2 % (26/117) was achieved corresponding to a relative increase of 44.1 %. Multivariate logistic regression analysis adjusted for study group, age, infertility, and cycle characteristics did not demonstrate any parameter that could predict occurrence of clinical pregnancy rates after IVF-ET. CONCLUSIONS: Patients who underwent SP intravaginal insemination after oocyte pick-up reached higher implantation and clinical pregnancy rates following ET compared to controls, although the difference did not reach statistical significance. More studies and variable methodologies may clarify the potential clinical effect of SP in improving live birth rates after ART.

Congenital malformations in infants conceived following assisted reproductive technology in comparison with spontaneously conceived infants.

OBJECTIVE: To evaluate the risk for congenital malformations diagnosed at birth following assisted reproductive technology (ART) treatments compared with live births conceived spontaneously. METHODS: A retrospective cohort study including 9042 live births following ART and 213 288 spontaneously conceived (SC) live births during the period 1997-2004.The cohort was linked to the national live birth registry to determine the outcome of the pregnancies including congenital malformations. RESULTS: An increased adjusted risk for all congenital malformations was observed in ART compared with SC infants [2.4% versus 1.9%; ORadj = 1.45; 95% CI: 1.26, 1.68]. The increased risk was observed in singleton births [2.4% versus 1.8%; ORadj = 1.41; 95% CI: 1.14, 1.71] but not in the ART conceived multiple births [2.5% versus 2.6%.; ORadj = 1.15; 95% CI: 0.90, 1.46]. Significantly increased adjusted risks for nervous, circulatory, digestive and genital system malformations were evident in the ART singleton group compared to SC infants. In addition, increased risks were also observed in separate comparisons of IVF births versus SC [ORadj = 1.28; 95% CI: 1.00, 1.63] and ICSI births versus SC [ORadj = 1.56; 95% CI: 1.31, 1.84]. Data regarding pregnancy termination or congenital malformation diagnosed later in life were not included. CONCLUSION: Infants born following ART were at significantly increased risk for congenital malformations compared to live birth conceived spontaneously.

Maternal and neonatal health outcomes following assisted reproduction.

This study assessed the risk for maternal complications in women and neonatal outcomes in children conceived following assisted reproductive treatment as compared with spontaneously conception and also separately evaluated conventional IVF and intracytoplasmic sperm injection (ICSI). The prospective cohort included 1161 women with singleton pregnancies: 561 who conceived following assisted reproduction (223 following IVF and 338 following ICSI) and 600 who conceived spontaneously. No differences were observed in pregnancy complications (including spontaneous abortion, pregnancy-induced hypertension, gestational diabetes and Caesarean delivery) except for significantly increased risk for excess vaginal bleeding in assisted reproduction pregnancies (21.4% versus 12.9%; OR 1.67, 95% CI 1.18-2.37), which was prominent in women who reported polycystic ovary syndrome. Neonates born following assisted reproduction had increased risk for prematurity (10.6% versus 5.3%; OR 1.72, 95% CI 1.04-2.87), and IVF, but not ICSI, was associated with significantly increased risk for prematurity (OR 2.36, 95% CI 1.28-4.37) and low birthweight (OR 1.89, 95% CI 1.03-3.46). In conclusion, this study observed only an increased risk for excess vaginal bleeding as a pregnancy-associated complication in singleton pregnancies following assisted compared with spontaneous conception. However, singleton neonates born following IVF, but not ICSI, were at increased risk for prematurity.

Presence of IL-18 in testicular tissue of fertile and infertile men.

Recently, IL-18 was identified in human testes. Moreover, an inverse correlation was found between the levels of IL-18 and the number and motility of spermatozoa. We examined the presence of IL-18 protein in normal and impaired spermatogenesis. Testicular tissue specimens were taken from 25 nonobstructive azoospermic patients undergoing testicular sperm extraction and from autopsies of three healthy controls. The presence of IL-18 in human testicular cells was examined by immunohistochemical staining of paraffin-embedded sections, using a specific antibody for human IL-18. In testicular tissue of healthy controls as well as in study cases, presence of IL-18 was identified in somatic, mitotic, meiotic and post-meiotic cells in correlation with their presence. In all patients, Leydig cells were less intensively stained. Mitotic cells were immunostained in the control group and less intensively in hypospermatogenesis and maturation arrest subgroups. Primary spermatocytes were in general most efficiently stained. The expression of IL-18 mRNA (as examined by real-time PCR analysis) showed significantly lower expression in testicular tissues with impaired spermatogenesis when compared to normal tissues. We report the first study demonstrating the presence of IL-18 in human testicular tissue at the protein level. The presence of this cytokine in somatic as well as in different types of germ cells may suggest its involvement in the regulation of the spermatogenic process and steroidogenesis under physiological and pathological conditions.

Surrogate in vitro fertilization outcome in typical and atypical forms of Mayer-Rokitansky-Kuster-Hauser syndrome.

BACKGROUND: The genital malformations in Mayer-Rokitansky-Küster-Hauser syndrome (MRKH) are frequently accompanied by associated malformations whose forms were recently classified as typical (isolated uterovaginal aplasia/hypoplasia) and atypical (the addition of malformations in the ovary or renal system). The aim of this study was to compare the surrogate IVF performance of women with typical and atypical forms including their chances of achieving pregnancy. METHODS: The follow-up data on a total of 102 cycles of surrogate IVF in 27 MRKH patients treated in our department between 2000 and 2010 were analysed. Twenty patients with the typical form who underwent 72 IVF cycles were compared with seven patients with the atypical form who underwent 30 IVF cycles. The various examined parameters of these intended mothers were age, hormonal profile during controlled ovarian hyperstimulation and laboratory outcome. RESULTS: The mean number of gonadotrophin ampoules needed for stimulation and treatment duration was significantly higher in the atypical form (3600 ± 1297IU for 13 ± 2.3 days versus 2975 ± 967 IU for 11.6 ± 1.6 days, P≤ 0.01). Serum estradiol and progesterone levels measured on the hCG administration day were similar. A significantly higher mean number of follicles 12.6 ± 6 versus 8.9 ± 5.4, P≤ 0.03, metaphase II (MII) oocytes 8.7 ± 5.1 versus 6.7 ± 4.8, P≤ 0.05, fertilizations 6 ± 3.6 versus 4.4 ± 3.3, P≤ 0.03 and cleaving embryos 5.7 ± 3.8 versus 4.1 ± 3.3, P≤ 0.01 were available in patients with the typical form compared with those with the atypical form, respectively. There was no significant difference in fertilization rate, cleavage rate or the mean number of transferred embryos. Embryo quality of the transferred ones and pregnancy rate per cycle were also similar between the two groups. CONCLUSIONS: Women with the typical form of MRKH needed fewer gonadotrophins and for a shorter duration for ovarian hyperstimulation. The mean number of follicles, oocytes, MII oocytes, fertilizations and cleaving embryos was higher among women with the typical form. Pregnancy rates were similar since the available number and quality of transferred embryos to the surrogate mother were not affected.

Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients.

BACKGROUND: Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model. METHODS: From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed. RESULTS: Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%. CONCLUSIONS: Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

In vitro maturation of human germinal vesicle-stage oocytes: role of epidermal growth factor-like growth factors in the culture medium.

BACKGROUND: In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side effects of gonadotrophin stimulation for IVF. The pregnancy rates from oocytes matured in vitro are still lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Recently, it was demonstrated that LH exerts its action on ovulation, at least in part, through stimulation of the production of the epidermal growth factor family members amphiregulin (Areg) and epiregulin (Ereg) in pre-ovulatory follicles, and they, in turn, serve as paracrine mediators of LH. We aimed to investigate the effect of supplementation of the medium with Areg and Ereg on the maturation rate of immature oocytes. METHODS: A total of 105 sibling human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation were cultured in a complex defined medium either with or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/ml) for 24 h. RESULTS: Significantly more oocytes reached the metaphase II stage at 24 h in media supplemented with Areg and Ereg (75.5 versus 36.5%, P < 0.001). In vitro matured oocytes retrieved from the two subgroups had no statistically significant difference in fertilization and cleavage rates or morphology scores. Overall, a significantly higher number of Day 2 (52.8 versus 26.9% P < 0.01) and Day 3 (45.2 versus 23%, P < 0.05) embryos originated from GV oocytes cultured in the Areg- and Ereg-enriched medium. CONCLUSIONS: Supplementation of the maturation medium with Areg and Ereg improves the maturation of human GV oocytes in vitro.

EGF-like growth factors as LH mediators in the human corpus luteum.

BACKGROUND: This study aims to investigate the role of epidermal growth factor-like ligands, amphiregulin (Ar) and epiregulin (Ep), in regulation of apoptosis in luteinized human granulosa cells. METHODS: Luteinized human granulosa cells were obtained from women undergoing IVF treatment. Ar and Ep mRNA levels were measured by real-time RT-PCR. The rate of apoptosis was measured by TUNEL. Progesterone levels were measured using radioimmunoassay. Ar- and Ep-induced activation of signaling cascades and Ar protein levels were detected by western blotting. RESULTS: LH stimulation of luteinized human granulosa cells induced biosynthesis of Ar and Ep mRNA in a time-dependent manner. The blockade of MEK (by U0126) reduced the expression of LH-induced Ar and Ep biosynthesis. Incubation of the cells with Ar and Ep completely abolished the increase in apoptosis rate induced by serum starvation, and concomitantly caused a pronounced increase in progesterone production. Stimulation of the cells with Ar and Ep also activated the ERK and AKT signaling cascades. Finally, we demonstrated that the pro-survival effect of Ar and Ep is partially dependent on their ability to induce progesterone production. CONCLUSIONS: Ar and Ep serve as pro-survival LH mediators in the human corpus luteum.

Peritoneal macrophage depletion by liposomal bisphosphonate attenuates endometriosis in the rat model.

BACKGROUND: Activation of macrophages is central to the implantation of endometriosis (EM). We examined the hypothesis that macrophage depletion by intraperitoneal (IP) injection of liposomal alendronate (LA) could result in EM attenuation in a rat model, thus supporting the notion of the pivotal role of macrophages in EM pathology. METHODS: In this study, 90 rats were subjected to an EM model and were divided randomly into seven groups: five groups were treated by 4x once-weekly IP injections of LA (0.02, 0.1, 1, 5 or 10 mg/kg) and the other two groups received saline injections (control) or empty liposomes. Sham-operated rats also received empty liposomes. Depletion of circulating monocytes was determined by flow cytometry analyzes of blood specimens. Four weeks after the initial surgery, the number, size and weight of implants were recorded, adhesions were graded, macrophage infiltration was assessed and the peritoneal fluid was analyzed for monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor alpha (TNFalpha). RESULTS: Monocyte depletion following IP LA administration resulted in an inhibitory effect on the initiation and growth of EM implants, as expressed by implantation rate, adhesion scoring, implants' size and weight (>0.1 mg/kg LA, P < 0.05). Reduced numbers of infiltrating macrophages were observed in implants of the 1 mg/kg LA group. Peritoneal fluid MCP-1 levels were negatively correlated with LA dose (P < 0.001), whereas no significant correlation could be found for TNFalpha. CONCLUSIONS: Macrophage depletion using IP LA has been shown to effectively inhibit the initiation and growth of EM implants, in a rat EM model. The clear dose-response effect may be viewed as a confirmation of the validity of the concept and encourages further study.

The possible association between IVF and breast cancer incidence.

BACKGROUND: The possible association between ovulation-inducing drugs and breast cancer development has been debated. Our aim was to evaluate the incidence of breast cancer in a cohort of women exposed to in vitro fertilization (IVF). METHODS: A retrospective cohort analysis was performed by linkage of the computerized database of all women treated at the IVF Unit at Assaf Harofeh Medical Center between 1986 and 2003, and the Israeli National Cancer Registry. The standardized incidence ratio (SIR) was computed as the ratio between the observed number of breast cancer cases and the expected cases, adjusted for age and continent of birth, in the general population. Tumor characteristics of the IVF patients were studied by reviewing original medical records. RESULTS: 35 breast carcinomas were diagnosed among 3,375 IVF-treated women, compared to 24.8 cases expected (SIR = 1.4; 95% CI 0.98-1.96). Age >or=40 years at IVF treatment (SIR = 1.9; 95% CI 0.97-3.30), hormonal infertility (SIR = 3.1; 95% CI 0.99-7.22), and >or=4 IVF cycles (SIR = 2.0; 95% CI 1.15-3.27) were found to be risk factors to develop breast cancer compared to the general population. Multivariate analysis revealed that women who underwent >or=4 IVF cycles compared to those with one to three cycles were at risk to develop breast cancer, although not significantly (SIR = 1.9; 95% CI 0.95-3.81). Of IVF-treated women 85% had ER(+) tumors and 29% had positive family history. CONCLUSIONS: A possible association between IVF therapy and breast cancer development was demonstrated, especially in women >or=40 years of age. These preliminary findings need to be replicated in other cohort studies.

Morphology assessment and fluorescence in situ hybridization of the same spermatozoon using a computerized cell-scanning system.

BACKGROUND: Poor sperm morphology is statistically associated with an increase in the incidence of chromosome abnormalities. Our aim was to examine the possible correlation between chromosomal aberrations and sperm morphology in the same cell. METHODS: 12349 spermatozoa from 7 teratozoospermic and one globozoospermic patients, and from 3 fertile donors were analyzed using a system which scans for cell morphology and chromosomal ploidy in the same cell using digital technology. RESULTS: Chromosomal aberrations were detected in 5.3% of teratozoospermic cases and in 6.7% in the globozoospermic patient compared with 1.6% in donors (P < 0.0001). Chromosomal aberrations were more common in abnormally formed sperm compared with normal spermatozoa: 4.5% vs 1.3% in the teratozoospermic group and 2.0% vs 0.3% in the control group (NS), especially frequent among sperm with two heads or two tails (52.1-77.2%) or extreme head deformations (10.6-11.1%) irrespective of grouping, and in mild amorphous heads in the globozoospermic patients (20.2%). The frequency of chromosomal aberrations in morphologically normal sperm was comparable whether derived from teratozoospermic or normospermic patients. CONCLUSIONS: The computerized cell-scanning system demonstrated the relationship between chromosomal aberrations and sperm morphology in the same spermatozoon. The incidence of chromosomal aberrations was positively linked to abnormal sperm morphology, the more severe the abnormality, the higher the incidence of aneuploidy.

PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation.

LH and prostaglandin E(2) (PGE(2)) share many similar effects on the pre-ovulatory follicle. They can induce independently cumulus expansion, the resumption of meiosis and progesterone production. However, cyclooxygenase-2 (COX-2) inhibitors were found to hinder most of the LH-induced effects. Recently, EGF-like growth factors amphiregulin (Ar) and epiregulin (Ep) were found to be produced in response to LH stimulation and to induce cumulus expansion and oocyte maturation. We aimed at evaluating whether PGE(2) induces Ar and Ep syntheses in human granulosa cells and whether the inhibition of PGE(2) production by selective COX-2 inhibitor, nimesulide, affects LH-induced Ar and Ep biosynthesis. Ar and Ep mRNA levels increased following PGE(2) stimulation, in a dose- and time-dependent manner, which resembled those of LH. The blockade of protein kinase A (PKA) (by H89) and mitogen-activated protein kinase (MAPK) (by UO126) reduced the expression of PGE(2)-induced Ar and Ep biosynthesis. Although the stimulation of the cells with LH in the presence of nimesulide did not change the progesterone levels, it resulted in a significant reduction of Ar and Ep biosynthesis. In conclusion, PGE(2) may mimic LH action, at least in part, by the induction of Ar and Ep biosynthesis, which involves cAMP/PKA and MAPK pathways. The negative effect of nimesulide on the ovulatory process may be due to the reduction of Ar and Ep biosynthesis, which implies a possible collaborative role between PGE(2) and LH on their induction.

Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

EGF-like factor epiregulin and amphiregulin expression is regulated by gonadotropins/cAMP in human ovarian follicular cells.

Epiregulin and amphiregulin are growth factors involved in cancer development, but their potential role in signaling in the gonads is still obscure. We report here that basal expression of these growth factors is evident in human granulosa cells obtained from women treated for in vitro fertilization, when examined by RT-PCR using RNA isolated from primary cultures of ovarian granulosa cells. Expression of these factors was elevated concomitantly with elevation of progesterone production in these cells upon stimulation with luteinizing hormone (LH), and to a lesser extent with follicle stimulating hormone (FSH), both essential stimulants for ovulation and luteinization. Epiregulin and amphiregulin gene expression was dose- and time-dependent when measured subsequent to LH stimulation. Moreover, forskolin, which activates adenylate cyclase, was as efficient as LH in stimulating expression of these growth factors. It is suggested that upregulation of the epiregulin and amphiregulin expression is part of the signal transduction pathway which leads to ovulation and luteinization in the human ovary.

The outcome of ICSI of immature MI oocytes and rescued in vitro matured MII oocytes.

BACKGROUND: The use of immature oocytes is limited to cases where these are the only available oocytes, and they are usually only microinjected with sperm after having undergone maturation in vitro. This study compares the outcome of injection of sperm into metaphase I oocytes immediately after their denudation (MI) performed 2 h after their retrieval, with the outcome of injection of sperm into rescued in vitro matured metaphase II (IVM MII) oocytes after their short incubation in routine laboratory conditions. METHODS: ICSI was performed on MI oocytes, rescued IVM MII oocytes and on MI oocytes that were incubated but failed to extrude their first polar body (arrested IVM MI). Fertilization and cleavage rates were compared with those achieved in mature metaphase II oocytes (MII). RESULTS: ICSI of MI oocytes showed impaired performance compared with ICSI of rescued IVM MII oocytes and MII oocytes, in terms of oocyte degeneration rate (11 versus 6 versus 4%; P < 0.0001), fertilization rate (28 versus 44 versus 68%; P < 0.0001) and multipronucleated fertilization (10 versus 4 versus 4%; P < 0.01). The cleavage rate was lower in rescued IVM MII oocytes compared with MII oocytes (86 versus 95%; P < 0.01). Arrested IVM MI oocytes showed similar results to those of MI oocytes but had a lower cleavage rate (72 versus 96%; P < 0.01). CONCLUSIONS: The injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes.

Coasting-what is the best formula?

Coasting is a method to decrease the incidence of ovarian hyperstimulation syndrome (OHSS), which involves withdrawing exogenous gonadotrophins until the serum estradiol (E(2)) level decreases. The application of this strategy, as it appears in the literature, has been variable, with heterogeneous criteria for initiating and ending the coasting process and as a result, reports of efficacy are inconsistent. In attempt to establish a recommended protocol for coasting we reviewed and analysed 10 relevant studies, found by a Medline search. Based on the data collected, coasting should be initiated when the serum E(2) concentration exceeds 3000 pg/ml, but not unless the leading follicles reach a diameter of 15-18 mm. Its duration should be limited to <4 days, thus, preventing the decrease in implantation and pregnancy rates that occur after longer periods of coasting. Administration of hCG should be withheld until serum E(2) falls below 3000 pg/ml. Based on the published data, these suggested guidelines result in an acceptably low incidence of severe OHSS (<2%) and provide satisfactory fertilization and pregnancy rates (55-71% and 36.5-63% respectively). A multicentre randomized prospective study would help to confirm the effectiveness of this approach.

Factors influencing the outcome of ICSI in patients with obstructive and non-obstructive azoospermia: a comparative study.

BACKGROUND: Factors influencing success of sperm retrieval in azoospermic patients and outcome of ICSI were evaluated. METHODS AND RESULTS: Uni- and multifactorial analysis were performed using logistic and stepwise analysis, following surgical sperm retrieval by percutaneous epididymal sperm aspiration (55 cycles) or testicular sperm extraction (142 cycles) in 52 and 123 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) respectively. ICSI cycles using fresh or cryopreserved-thawed sperm were included. Sperm were retrieved to allow ICSI in 100 and 41% of OA and NOA patients, with no significant correlation with patients' age or FSH level. Occurrence of pregnancy was significantly correlated with female age (90th quantile: 38 years), number of oocytes retrieved (10th quantile: five oocytes) and number of oocytes injected (10th quantile: four oocytes). Sperm origin (epididymal versus testicular), status (fresh or thawed), male partner's age, and serum FSH had no significant effect upon implantation rate, pregnancy rate per embryo transfer or spontaneous miscarriage rate. CONCLUSIONS: In OA patients ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, the lack of efficient non-invasive parameters to predict sperm retrieval in NOA suggests that elective surgical sperm retrieval may be offered to these patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative. Female factors such as age and ovarian reserve have significant impact upon clinical success rates.

Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia.

BACKGROUND: It is unclear whether or not testicular sperm extraction (TESE) should be repeated for patients in whom no sperm were found during their first TESE attempt. METHODS AND RESULTS: The outcome of repeated TESE was evaluated in patients with non-obstructive azoospermia (NOA) after failing to obtain sperm in their first extraction attempt, or having used all available cryopreserved testicular tissue. Out of 83 patients with NOA, patients repeated TESE two (n = 22), three (n = 8), four (n = 6) and five (n = 3) times. Distribution of main testicular histology included germ cell aplasia (55%), maturation arrest (29%) and germ cell hypoplasia (16%). The first TESE yielded mature sperm for ICSI in 39% of patients (sp+), and failed in the remaining 61% (sp-). A second TESE yielded mature sperm in 1/4 from the sp- group and in 16/18 from the sp+ group. At the third, fourth and fifth trials, 8/8, 5/6 and 3/3 of the original sp+ patients were sp+ again respectively. Compared with the outcome of the first trial, all further trials did not differ statistically in the rate of fertilization (54 versus 49%), implantation (9.5 versus 5.4%), or clinical pregnancy/cycle (19 versus 15%). No pregnancies were achieved among the three patients after their fifth TESE. Pregnancies occurred in all histological groups, except maturation arrest. CONCLUSIONS: The outcome of repeated TESE cycles, up to the fourth trial, justifies the procedure.

Outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa in patients with non-mosaic Klinefelter's syndrome.

BACKGROUND: Recently, intracytoplasmic sperm injection (ICSI) of testicular spermatozoa retrieved surgically from patients with non-mosaic Klinefelter's syndrome resulted in several deliveries. The aim of this study was to evaluate the outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa in these patients. METHODS AND RESULTS: Following informed consent regarding the genetic risks of their potential offspring, mature testicular spermatozoa were found in five out of 12 (42%) patients who underwent testicular sperm extraction, and ICSI was performed while excess tissue was cryopreserved. The mean age of the patients was 28.7 +/- 3.6 (range 23-36 years). Their baseline FSH was elevated (mean 38.3 +/- 11.4; range 22-58 mIU/ml). All patients had small testicles of 2-4 ml in volume. The outcome of ICSI using fresh or cryopreserved-thawed testicular spermatozoa during five cycles in each group, was compared. No statistical significant difference was found in the two pronuclear fertilization rate (66 versus 58%), embryo cleavage rate (98 versus 90%) and embryo implantation rate (33.3 versus 21.4%) for fresh or cryopreserved sperm accordingly. The clinical outcome after using fresh testicular sperm included two singleton pregnancies (one delivered and one ongoing) and a triplet pregnancy resulting in a twin delivery (after reduction of an 47,XXY embryo). After using cryopreserved-thawed testicular spermatozoa, two pregnancies were obtained resulting in one delivery of twins and one early spontaneous abortion. CONCLUSIONS: Outcome of ICSI using cryopreserved-thawed testicular spermatozoa of patients with non-mosaic Klinefelter's syndrome is comparable with that following the use of fresh spermatozoa. The genetic implications for the future offspring should be explained to the patients.