Rho Signaling-Directed YAP/TAZ Regulation Encourages 3D Spheroid Colony Formation and Boosts Plasticity of Parthenogenetic Stem Cells.

Cell proliferation, apoptosis and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs) that are able to proliferate maintaining pluripotency and, at the same time, to give rise to all populations belonging to the three germ layers, in response to specific stimuli. ESCs are, therefore, considered a well-established in vitro model to study the complexity of these processes. In this perspective, we previously generated parthenogenetic embryonic stem cells (ParthESC), that showed many features and regulatory pathways common to bi-parental ESCs. However, we observed that mono-parental cells demonstrate a high ability to form outgrowths and generate 3D spheroid colonies, which are distinctive signs of high-plasticity. Furthermore, preliminary evidence obtained by WTA, revealed the presence of several differentially expressed genes belonging to the Rho and Hippo signaling pathways. In the present study, we compare bi-parental ESCs and ParthESC and analyze by Real-Time PCR the differentially expressed genes. We demonstrate up-regulation of the Rho signaling pathway and an increased expression of YAP and TAZ in ParthESC. We also show that YAP remains in a dephosphorylated form. This allows its nuclear translocation and its direct binding to TEADs and SMADs, that are up-regulated in ParthESC. Altogether, these complex regulatory interactions result in overexpression of pluripotency related genes, in a global DNA hypomethylation and a histone-dependent chromatin high permissive state that may account for ParthESC high potency, possibly related to their exclusive maternal origin.

IVF outcome in poor responders failing to produce viable embryos in the preceding cycle.

This study postulated that poor-responder women failing to obtain viable embryos would represent a subgroup of subjects with extremely poor prognosis. To elucidate this aspect, women in this condition over a 4-year period were retrospectively identified and their IVF outcomes in subsequent cycles were evaluated. A total of 108 women satisfied the selection criteria and underwent at least one further IVF cycle. There were 19 women excluded because they opted for a mild approach using clomiphene citrate alone, leaving 89 women for data analyses. Four women had a live birth during this first cycle, corresponding to a delivery rate per started cycle of 4.5% (95% CI 1.5-10.0%). From a public health perspective, the mean cost per delivery was € 124,540. Younger age emerged as the unique predictive factor of success. In conclusion, women with poor ovarian response failing to obtain viable embryos have extremely low chances of success in subsequent cycles. Considering the costs and risks of IVF, the appropriateness of pursuing treatments in these women is questionable. Younger women may represent a possible exception since their chances of delivery are higher.

The vanishing endometrioma: the intriguing impact of pregnancy on small endometriotic ovarian cysts.

There is a general belief that pregnancy has a beneficial effect on endometriosis but evidence is scanty. Moreover, some recent warning reports documented progression-growth of ovarian endometriomas during pregnancy. To further elucidate this aspect, we set up a prospective cohort study. We recruited 24 women carrying endometriomas at the time of in vitro fertilization who got pregnant. Selected women were contacted about 12-18 months after oocyte retrieval and invited to refer for ultrasound. The primary aim was to evaluate the modifications of endometriotic cysts after pregnancy. Twenty-four women carrying 40 endometriomas accepted to participate. The number of cysts per patient was significantly reduced following pregnancy (p = 0.003). It was unchanged in eight cases (33%), increased in two cases (8%) and reduced in three cases (13%). In the remaining 11 women (46%), no cyst could be detected. A comparison of the size of the endometrioma could be done in 21 cysts; the mean ± SD diameter before and after pregnancy was 20 ± 9 and 18 ± 7 mm, respectively (p = 0.27). In conclusion, pregnancy has a beneficial effect on endometriomas. A consistent proportion of cysts becomes undetectable after delivery. Further studies are warranted to clarify the mechanisms determining the disappearance of these cysts.

Pregnancy outcome in women with endometriomas achieving pregnancy through IVF.

BACKGROUND: There is a growing consensus that ovarian endometriomas should not be systematically removed in women selected for IVF. However, some recent evidence suggested that the presence of these cysts may negatively affect the course of pregnancy. METHODS: We set up a multicenter retrospective cohort study, including two infertility units. We analyzed data from patients achieving singleton clinical pregnancies through IVF comparing the pregnancy outcome between 78 pregnant women with endometriomas at the time of IVF and 156 patients who achieved pregnancy through IVF without endometriomas. RESULTS: The number of live births in women with and without endometriomas were 61 (78%) and 130 (83%), respectively (P = 0.39). The adjusted odds ratio (OR) of live birth in affected cases was 0.79 [95% confidence interval (CI): 0.38-1.68]. No differences were observed in late pregnancy and neonatal outcomes between the two groups. In particular, the rate of preterm birth and small-for-gestational age (SGA) was similar. The adjusted ORs were 0.47 (95% CI: 0.14-1.54) and 0.56 (95% CI: 0.12-2.56), respectively. CONCLUSIONS: Women with endometriomas achieving pregnancy through IVF do not seem to be exposed to a significant increased risk of obstetrical complications.

Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study.

BACKGROUND: An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons. METHODS: We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed. RESULTS: In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7%). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1%, respectively. A total of 128 deliveries were obtained (26.3% per cycle and 29.4% per transfer) for 450 patients (28.4%) and 147 babies were live born from 929 embryos transferred (15.8%). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95% confidence interval (CI): 0.88-0.98], number of vitrified oocytes (OR: 1.08, 95% CI: 1.01-1.17) and the day of transfer (OR: 1.97, 95% CI: 1.14-3.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4% DR, respectively). When fewer oocytes are available in women aged >38 years, results are dramatically reduced (12.6 versus 27.5% DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1% DR; data from one center only). CONCLUSIONS: Oocyte vitrification is an efficient and reliable approach, with consistent results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection.

Clomiphene citrate versus high doses of gonadotropins for in vitro fertilisation in women with compromised ovarian reserve: a randomised controlled non-inferiority trial.

BACKGROUND: The aim of the present randomised controlled non-inferiority trial is to test whether in women with compromised ovarian reserve requiring in vitro fertilisation, a protocol of ovarian stimulation using exclusively clomiphene citrate performs similarly to a regimen with high doses of gonadotropins. METHODS: Women with day 3 serum FSH > 12 IU/ml on at least two occasions or previous poor response to hyper-stimulation were recruited at four Italian infertility units. Selected women were allocated to clomiphene citrate 150 mg/day from day 3 to day 7 of the cycle (n=145) or to a short protocol with GnRH agonist 0.1 mg and recombinant FSH 450 IU daily (n=146). They were randomised by means of a computer-generated list into two groups. The study was not blinded. The main outcome of the study was the delivery rate per started cycle. RESULTS: The study was interrupted after the scheduled two years of recruitment before reaching the sample size. 148 women were allocated to clomiphene citrate and 156 to the short protocol with high doses of gonadotropins; 124 and 125 participants were analysed in the groups, respectively. Women allocated to high doses of gonadotropins retrieved more oocytes and had a higher probability to perform embryo-transfer. However, the chances of success were similar. The delivery rate per started cycle in women receiving clomiphene citrate and high-dose gonadotropins was 3% (n=5) and 5% (n=7), respectively (p=0.77). The mean estimated cost per delivery in the two groups was 81,294 and 113,107 Euros, respectively. No side-effects or adverse events were observed. CONCLUSIONS: In women with compromised ovarian reserve selected for in vitro fertilisation, ovarian stimulation with clomiphene citrate or high-dose gonadotropins led to similar chances of pregnancy but the former is less expensive. TRIAL REGISTRATION: Trial registered on http://www.clinicaltrials.gov (NCT01389713).

IVF and endometriosis-related symptom progression: insights from a prospective study.

BACKGROUND: A possible and neglected concern in women with endometriosis undergoing IVF is the potential risk of progression of the disease. We set up a prospective study mainly aimed at evaluating the impact of IVF on endometriosis-related symptoms. MATERIALS AND METHODS: Women with surgical or echographic diagnosis of endometriosis and selected for IVF were included. In the month preceding the IVF attempt and at a second evaluation 3-6 months after the cycle, women who did not get pregnant underwent clinical assessment and transvaginal ultrasonography. Each patient was requested to complete a questionnaire on the presence, severity and modifications of endometriosis-related symptoms before and after the IVF cycle. RESULTS: Overall, 64 patients completed the study protocol. The Biberoglu-Behrman Scores and the Verbal Rate Scales for dysmenorrhea, dispareunia and chronic pelvic pain did not worsen after the procedure. Other endometriosis-related symptoms also did not change. There was no modification in size and number of endometriomas and deep peritoneal nodules. The number (%) of women reporting general improvement and worsening were 14 (22%) and 7 (11%), respectively. CONCLUSIONS: IVF does not expose women to a consistent risk of endometriosis-related symptoms progression.

Unoperated ovarian endometriomas and responsiveness to hyperstimulation.

BACKGROUND: There is a growing consensus that ovarian reserve is reduced after surgical excision of ovarian endometriomas. However, it remains to be fully clarified whether this damage precedes or follows surgery. In order to further elucidate this aspect, we evaluated ovarian responsiveness to hyperstimulation in women selected for IVF with unilateral unoperated endometriomas. The main aim of this study was to compare the number of developing follicles in the affected ovary with that in the contralateral intact gonad as a control. METHODS: Patients selected for IVF who were diagnosed with one or more monolateral endometriomas (diameter <4 cm) and who did not undergo previous ovarian surgery were retrospectively identified. We compared the number of follicles (diameter ≥ 11 mm) and the number of co-dominant follicles (diameter >15 mm) on the day of hCG administration in the affected and intact ovaries. RESULTS: Among the 84 women selected, the median interquartile range (IQR) number of follicles ≥ 11 mm in the affected and intact ovaries was 5 (3-7) and 5 (3-8), respectively (P= 0.36). The median (IQR) number of co-dominant follicles in the affected and intact ovaries was 3 (2-4) and 3 (2-5), respectively (P= 0.48). The number of co-dominant follicles was lower in the affected ovary in 36 cases (43%, 95% confidence interval: 32-53%). We also failed to identify any statistically significant difference between the two ovaries when evaluating data according to the number of cysts, their dimension, the dose of gonadotrophins used or the number of oocytes retrieved. CONCLUSIONS: In women selected for IVF, the presence of an endometrioma does not markedly affect responsiveness to hyperstimulation.

Fibroids not encroaching the endometrial cavity and IVF success rate: a prospective study.

BACKGROUND The impact of fibroids, not encroaching the endometrial cavity, have on the rate of success of IVF is still controversial. Recent meta-analyses suggest a detrimental effect of intramural lesions but not subserosal lesions. However, they also emphasize the need for further evidence. In order to elucidate this, we designed a prospective cohort study to compare the rate of success of IVF in women with and without fibroids. METHODS Exposed women were those with asymptomatic intramural or subserosal fibroids with a diameter below 50 mm and who were selected for IVF. Unexposed women were those free of fibroids, who were matched to cases by age and number of previous IVF cycles. All recruited patients underwent hystero-sonography to rule out intra-cavitary lesions. RESULTS There were 119 cases and 119 controls recruited. The number of clinical pregnancies in women with and without fibroids was 28 (24%) and 22 (19%), respectively (P= 0.43). The adjusted odds ratio (OR) for pregnancy in affected women was 1.38 [95% confidence interval (CI): 0.73-2.60]. The number of deliveries was 22 (18%) and 16 (13%), respectively (P= 0.38). The adjusted OR was 1.45 (95% CI: 0.71-2.94). Similar results emerged when focusing exclusively on women carrying intramural lesions (n= 80 couples). There was no significant relationship between clinical outcome and either the number or size of the fibroids. CONCLUSIONS In asymptomatic patients selected for IVF, small fibroids not encroaching the endometrial cavity did not impact on the rate of success of the procedure.

Effects of two vitrification protocols on the developmental potential of human mature oocytes.

The aim of the present study was to compare an 'open' vitrification protocol to a 'closed' vitrification protocol for mature human oocytes. A prospective comparison between fresh and sibling vitrified oocytes and a retrospective comparison between the two vitrification protocols were performed. For recruited patients undergoing an IVF cycle, two or three fresh oocytes were inseminated with intracytoplasmic sperm injection (ICSI) and the remaining three or more oocytes were vitrified according to manufacturer's instructions with a 'closed' or an 'open' vitrification system. After an unsuccessful fresh cycle, oocytes were warmed and inseminated with ICSI. Embryological parameters were recorded and compared between fresh and sibling vitrified oocytes (intrapatient) as well as between the two vitrification techniques (interpatient). Oocytes vitrified with the 'closed' system showed significantly lower fertilization and cleavage rates and a reduction in the quantity and quality of obtained embryos compared with fresh sibling oocytes (P<0.001). On the contrary, the same parameters were similar between fresh and sibling oocytes vitrified using the 'open' system. The retrospective comparison between the two vitrification protocols also showed a significant increase in clinical pregnancy rate and a reduced proportion of cancelled cycles using the 'open' system (P<0.01).

Recurrent endometrioma and ovarian reserve: biological connection or surgical paradox?

OBJECTIVE: Cumulative evidence supports the view that ovarian endometriomas originate from ovulatory events and that the ovarian reserve is reduced following surgery. On these bases, we have hypothesized that the risk of recurrence may be related to the residual ovarian reserve of the operated ovary. STUDY DESIGN: We retrospectively selected 45 women scheduled for in vitro fertilization who previously underwent surgical excision of monolateral endometriomas and compared ovarian responsiveness in those who did (n = 24) and did not (n = 21) have a recurrent endometrioma. RESULTS: In the intact ovaries, the mean ± SD number of codominant follicles in women with and without recurrences was 3.5 ± 1.7 and 3.7 ± 2.2, respectively (P = NS). In the affected ovaries, the mean ± SD number of follicles in gonads with and without recurrences was 2.5 ± 2.3 and 1.1 ± 1.5, respectively (P < .05). CONCLUSION: Ovarian responsiveness is higher in gonads that developed recurrent endometriomas.

Use of parthenogenetic activation of human oocytes as an experimental model for evaluation of polar body based PGD assay performance.

PURPOSE: To develop an experimental model to assess the feasibility of polar body preimplantation genetic diagnosis without requiring oocyte fertilization. METHODS: First polar body was removed from donated oocytes and second polar body was biopsied after parthenogenetic activation. Molecular analysis on both polar bodies involved a fluorescent multiplex polymerase chain reaction of short-tandem repeat markers, closely linked to genes of interest. Main outcome measures were: allele segregation through polar bodies and haploid nucleus, recombination rates between alleles and frequency of Allele Drop Out. RESULTS: Twenty-six out of 39 oocytes extruded a second polar body after activation. Ninety-two percent of the first polar bodies and 20 out of 26 (77%) second polar bodies were successfully amplified. Eighty percent of first polar bodies were heterozygous for CFTR and 55% for HBB. Analysis of second polar bodies predicted the genotype of the oocytes in case of heterozygous first polar body, and validated results in homozygous cases. Frequency of allele drop out was 4%. CONCLUSIONS: Our model confirms that polar body preimplantation genetic diagnosis for single gene disorders can be evaluated using parthenogenetic oocytes and offers an option to set up procedures without requiring oocyte fertilization.

Rate of severe ovarian damage following surgery for endometriomas.

BACKGROUND: There is growing and consistent evidence showing that ovarian reserve is affected following surgical excision of ovarian endometriomas. Of particular concern is the risk of severe ovarian damage leading to unresponsiveness to ovarian hyperstimulation. In this study, we aimed to determine the rate of this complication. METHODS: Ninety-three women underwent surgery for monolateral endometriomas were recruited. Patients who underwent IVF were selected and, in all cases, follicular growth was monitored by serial transvaginal ultrasonography. The main outcome measure was the rate of ovaries remaining silent when stimulated after surgery for endometriomas. RESULTS: Absence of follicular growth was observed in 12 operated ovaries although this event never occurred in the contralateral gonad (P < 0.001). The frequency (95% confidence interval) of severe ovarian damage following surgery was 13% (7-21%). CONCLUSIONS: Severe ovarian damage, occurring in gonads operated on for ovarian endometriomas, is not a rare event.

Procedure for rapid oocyte selection based on quantitative analysis of cumulus cell gene expression.

PURPOSE: To develop a procedure for the analysis of gene expression in cumulus cells during the interval between ovum pick up and insemination to select the best oocytes for fertilization. METHODS: Five RNA extraction methods, three reverse transcription procedures followed by Real-time quantitative PCR and one single-step mRNA quantification kit were tested to measure the expression of five genes in cumulus cells. RESULTS: Two RNA extraction kits gave the best combination of efficiency and purity. One reverse transcription procedure gave the best speed and efficiency. The single-step kit required more biological material than would be available from single cumulus oocyte complexes (COCs). CONCLUSIONS: Our test identified a combination of RNA extraction and reverse transcription procedures that enables the level measurement of 5 selected cumulus cell transcripts within 4 h. Using this combination it was possible to obtain a reliable quantification of gene expression in 44 out of 46 individual COCs collected from seven patients.

Endometriotic ovarian cysts negatively affect the rate of spontaneous ovulation.

BACKGROUND: A growing body of evidence suggests that ovarian reserve is damaged after excision of ovarian endometriomas. However, it may not be excluded that gonadal damage is at least partly caused by the very presence of an endometrioma per se, thus preceding surgery. To clarify this aspect, we set up a prospective study in women with monolateral endometriomas in order to assess the rate of ovulation in affected ovaries. MATERIALS AND METHODS: Seventy women with monolateral endometriomas who had not undergone previous adnexal surgery underwent serial ecographic examinations to determine the side of ovulation. RESULTS: Ovulation occurred in the affected ovary in 22 cases (31%; 95% CI: 22-43%). Assuming that the expected rate of ovulation in both ovaries in healthy women is similar, this difference is statistically significant (P = 0.002). CONCLUSION: The physiological mechanisms leading to ovulation are deranged in ovaries with endometriomas.

The long pentraxin 3 is a soluble and cell-associated component of the human semen.

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition receptor, involved in several processes ranging from innate resistance and inflammation to clearance of apoptotic cells and organization of hyaluronic acid-rich extracellular matrices. PTX3 is also a novel marker in several pathological conditions of infectious, inflammatory, or autoimmune origin. This study was designed to assess whether PTX3 is expressed in the male reproductive tract and whether PTX3 interacts with human spermatozoa influencing their function. Here we show for the first time by immunohistochemistry that PTX3 is expressed in the male genital tract in perivascular connective tissue, in endothelial cells, in the interstitium, and in the cytoplasm of prostatic epithelial glandular cells; PTX3 was detectable in seminal plasma in variable levels, which correlated with the percentage of normal spermatozoa. Moreover, PTX3 binds to spermatozoa, in particular with immotile cells, localizing in the neck and in the subacrosomial region. Finally, recombinant PTX3 did not interfere with sperm motility.

Is the dimension of ovarian endometriomas significantly modified by IVF-ICSI cycles?

Information regarding the growth and development of endometriomas during IVF-intracytoplasmic sperm injection (ICSI) cycles is lacking. In this study, the aim was to estimate the influence of IVF-ICSI on the dimension of these cysts and to assess whether this treatment contributes to the manifestation of new endometriomas in patients already affected. Women were eligible if they had been diagnosed with ovarian endometrioma(s). Recruited patients who failed to become pregnant were scanned again 3-6 months later to evaluate the modification of the dimension of the cysts and/or the manifestation of new endometriomas. Forty-eight women completed the study protocol. The median (interquartile range) values for the volume of the cysts before and after the IVF-ICSI cycle were 3.9 (2.9-7.9) ml and 4.9 (2.4-9.9) ml, respectively (Wilcoxon rank test for paired data, not significant). The development of a new endometrioma was documented in one case (2.1%, 95% confidence interval 0.1-11.1%). Fear about the growth of ovarian endometriomas in women who have to undergo IVF-ICSI is not justified.

Blood loss during transvaginal oocyte retrieval.

BACKGROUND/AIMS: Limited information is available regarding the quantity of blood loss associated with uncomplicated transvaginal oocyte retrieval. The aim of the present study was evaluating the quantity of such a loss. METHODS: One hundred and fifty consecutive women undergoing oocyte retrieval were recruited. They underwent blood test assessment and ultrasonographic transvaginal evaluation at three different times: (1) immediately before initiating oocyte retrieval, (2) 4-6 h later, and (3) 72 h later. RESULTS: At 4-6 h after oocyte retrieval, the red blood cell count and the hemoglobin concentration were significantly reduced, whereas pelvic free fluid had significantly increased. The estimated median (Interquartile range) blood loss was 72 (-8/162) ml. None of the recruited women was found to have a hemoglobin reduction >2 g/day or an increase in the pelvic free fluid >200 ml or a calculated blood loss >500 ml (0.0%, 95% CI: 0.0-2.4%). No significant worsening from baseline was observed at the 72 h evaluation. CONCLUSIONS: The quantity of blood loss following oocyte retrieval is clinically unremarkable in the vast majority of women.

IVF-ICSI outcome in women operated on for bilateral endometriomas.

BACKGROUND: The influence of previous conservative surgery for endometriomas on IVF-ICSI outcome is debated. Conflicting information emerging from the literature may be consequent to the fact that endometriomas are mostly monolateral. The contralateral intact ovary may adequately supply for the reduced function of the affected one. To clarify this point, we assess IVF-ICSI outcome in women operated on for bilateral endometriomas. METHODS: Women selected for IVF-ICSI cycles who previously underwent bilateral endometriomas cystectomy were matched (1:2) for age and study period with patients who did not undergo prior ovarian surgery. RESULTS: Sixty-eight cases and 136 controls were recruited. Women operated on for bilateral endometriotic ovarian cysts had a higher withdrawal rate for poor response (P < 0.001). In these patients, despite the use of higher doses of gonadotrophins, the number of follicles (P = 0.006), oocytes retrieved (P = 0.024) and embryos obtained (P = 0.024) were significantly lower. The clinical pregnancy rate per started cycle in cases and controls was 7% and 19% (P = 0.037) and the delivery rate per started cycle was 4% and 17%, respectively (P = 0.013). CONCLUSIONS: IVF outcome is significantly impaired in women operated on for bilateral ovarian endometriomas.

The clomiphene citrate challenge test (CCCT) in women with elevated basal FSH: biological significance and predictive value.

OBJECTIVE: The rationale of the clomiphene citrate challenge test (CCCT) is that day 10 serum FSH is influenced by the quality of the recruited oocytes. Biological evidence supporting this assumption is, however, lacking. The aim of this study is to investigate the relationship between results from the CCCT and the quantity and the quality of the recruited oocytes. STUDY DESIGN: Patients selected for in vitro fertilization (IVF) and who were found to have elevated basal FSH (n=114) underwent an IVF cycle using follicles developing during CCCT. Subsequently, a subgroup of patients (n=89) underwent a second cycle receiving high doses of gonadotropins. The main outcome considered was the transfer of viable embryos. RESULTS: During the CCCT cycle, the area under the receiver operating characteristics (ROC) curves for day 3 and day 10 serum FSH to predict embryo-transfer was 0.48 (95% CI, 0.37-0.60) and 0.74 (95% CI, 0.63-0.82), respectively. In the subsequent cycle, the area under the ROC curves for the two variables was 0.58 (0.43-0.72) and 0.58 (0.43-0.72), respectively. CONCLUSIONS: CCCT effectively mirrors the quantity and the quality of the recruited oocytes but its predictive value is low.