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.

Thyroid axis dysregulation during in vitro fertilization in hypothyroid-treated patients.

BACKGROUND: While there is a large body of evidence showing a significant impact of controlled ovarian hyperstimulation (COH) on thyroid function in euthyroid patients undergoing in vitro fertilization (IVF), information on the effect of this treatment on thyroid axis equilibrium in hypothyroid-treated patients is insufficient. The goal of this prospective study was to investigate serum thyroid-stimulating hormone (TSH) modifications in hypothyroid-treated patients during IVF. METHODS: Hypothyroid-treated women selected for IVF between November 2010 and December 2011 were considered for study entry. They were eligible if serum TSH tested the month preceding the IVF cycle was 0.4-2.5 mIU/L. Additional inclusion criteria were as follows: (1) a certified diagnosis of clinical or subclinical hypothyroidism; (2) consumption of at least 25 µg of levothyroxine daily; (3) serum free triiodothyronine and free thyroxine tested the month preceding the IVF cycle within the reference range; (4) no previous IVF cycles; (5) regular menstrual cycles; and (6) day 3 serum follicle-stimulating hormone <12 IU/mL and anti-Müllerian hormone >0.5 ng/mL. Serum TSH was tested at three time points: between day 1 and day 8 of the cycle during the month preceding the start of controlled ovarian hyperstimulation (COH), at the time of human chorionic gonadotropin (hCG) administration and at 16 days after hCG administration. RESULTS: Seventy-two women met our selection criteria. The serum levels of TSH at basal assessment, at the time of hCG administration, and at 16 days after hCG administration were 1.7 ± 0.7, 2.9 ± 1.3, and 3.2 ± 1.7 mIU/L, respectively. All pairwise comparisons were statistically significant. Serum TSH exceeded the threshold of 2.5 mIU/L in 46 subjects at the time of hCG administration (64%, [CI: 53-75%]) and in 49 subjects 16 days after hCG administration (68%, [CI: 57-79%]). CONCLUSIONS: Serum TSH increased considerably during COH in adequately treated hypothyroid women undergoing IVF. We suggest strictly monitoring these women during IVF cycles and, if necessary, promptly adjusting the levothyroxine dose. This is the most pragmatic approach but, to date, it is not supported by clinical evidence. Further studies aimed at clarifying the most suitable therapeutic strategy are thus warranted.

Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation.

Converting adult cells from one cell type to another is a particularly interesting idea for regenerative medicine. Terminally differentiated cells can be induced to de-differentiate in vitro to become multipotent progenitors. In mammals these changes do not occur naturally, however exposing differentiated adult cells to synthetic molecules capable of selectively reverting cells from their lineage commitment to a more plastic state makes it possible to re-address their fate. Only scattered information are available on the morphological changes and ultrastructural remodeling taking place when cells convert into a different and specific type. To better clarify these aspects, we derived human granulosa cell (GC) primary cultures and analyzed the morphological changes taking place in response to the exposure to the epigenetic modifier 5-azacytidine (5-aza-CR) and to the treatment with VEGF, as a stimulus for inducing differentiation into muscle cells. Ultrastructural modifications and molecular marker expression were analyzed at different intervals during the treatments. Our results indicate that the temporary up regulation of pluripotency markers is accompanied by the loss of GC-specific ultrastructural features, mainly through autophagocitosis, and is associated with a temporary chromatin decondensation. After exposure to VEGF the induction of muscle specific genes was combined with the appearance of multinucleated cells with a considerable quantity of non-spatially organized filaments. The detailed analysis of the morphological changes occurring in cells undergoing lineage re-addressing allows a better understanding of these process and may prove useful for refining the use of somatic cells in regenerative medicine and tissue replacement therapies.

Incidence of elevation of serum thyroid-stimulating hormone during controlled ovarian hyperstimulation for in vitro fertilization.

OBJECTIVE: To evaluate the rate of euthyroid women encountering an elevation of serum TSH above the threshold of 2.5 mIU/L during controlled ovarian hyperstimulation (COH) for IVF. STUDY DESIGN: Six-month prospective cohort study on 175 consecutive euthyroid women undergoing their first IVF cycle. Serum TSH assessments were performed before COH, at the time of hCG administration and at +16 days after hCG administration. Women were eligible if serum TSH tested the month preceding the IVF cycle was 0.4-2.5 mIU/L. A history of thyroid disorders was an exclusion criterion. RESULTS: Serum concentrations of TSH at the three scheduled assessments were 1.5±0.5, 2.2±1.0 and 2.1±1.1 mIU/L, respectively. A statistically significant increase occurred between basal levels and levels at the time of hCG administration (p<0.001). Afterwards, levels remained stable (p=0.49). Serum TSH at the time of hCG administration exceeded the threshold of 2.5 mIU/L in 61 subjects, corresponding to 35% (95% CI: 28-42%). At +16 days after hCG administration, this event was observed in 47 subjects (27%, 95% CI: 21-34%). Baseline characteristics of women who did and did not exceed the threshold were similar apart from basal serum TSH, which was higher in the former group. The OR was 7.6 (95%CI: 2.9-20.2) per mIU/L (p<0.001). Cycle outcome and pregnancy rate were also similar. CONCLUSION: Serum TSH exceeds the threshold of 2.5 mIU/L during COH in one out of three women who are euthyroid prior to enter an IVF cycle. Further evidence is warranted to elucidate the clinical relevance of our findings.

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.

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.

In vitro fertilization outcomes in treated hypothyroidism.

BACKGROUND: Levothyroxine has been shown to enhance pregnancy outcomes in women with hypothyroidism requiring in vitro fertilization (IVF). However, the precise magnitude of these benefits remains to be determined. In particular, it has yet to be clarified whether levothyroxine may fully overcome the detrimental effects of hypothyroidism or, conversely, whether affected women remain at reduced prognosis for pregnancy outcomes. METHODS: Patients who underwent IVF-intracytoplasmic sperm injection (ICSI) over a 3-year period were reviewed. Cases were deemed eligible if they were diagnosed with clinical or subclinical hypothyroidism and were receiving levothyroxine. Controls were two subsequently age-matched euthyroid women for every case. Both cases and controls were selected only if serum thyrotropin was ≤2.5 mIU/L. RESULTS: In total, 137 women with treated hypothyroidism and 274 controls were included. Baseline characteristics of the two study groups were similar with the exception of body mass index, which was slightly higher among the cases (22.9±3.9 vs. 21.9±3.3 kg/m2, p=0.013). Most IVF-ICSI cycle outcome variables were also similar, with the exception of a higher rate of cancellation for poor response (3.6% vs. 0.7%, p=0.04), a longer duration of stimulation (10.9±2.2 vs. 10.1±2.0 days, p=0.001), a higher proportion of women failing to obtain viable embryos (17% vs. 7%, p=0.006), and a lower fertilization rate (75% vs. 86%, p=0.017) among cases. Conversely, the clinical pregnancy rate per started cycle, the implantation rate, and the live birth rate per started cycle did not differ; they were 36% and 34% (p=0.93), 28% and 22% (p=0.11), and 30% and 27% (p=0.50) in cases and controls, respectively. Subgroup analyses comparing women with (n=79) and without (n=58) thyroid autoimmunity and comparing women who were diagnosed with overt hypothyroidism (n=70) or subclinical hypothyroidism (n=67) failed to identify relevant differences. CONCLUSIONS: In our population, IVF-ICSI outcome was not significantly hampered in women with adequately treated hypothyroidism. The magnitude of the detected differences in cycle outcome was mild, and we failed to document any differences for the most relevant outcomes, i.e., pregnancy rate, implantation rate, and delivery rate. In conclusion, adequate levothyroxine treatment maintaining thyrotropin serum levels below 2.5 mIU/L may overcome the detrimental effects of hypothyroidism.

In vitro fertilization outcome in women with unoperated bilateral endometriomas.

OBJECTIVE: To evaluate IVF outcome in women with unoperated bilateral endometriomas. DESIGN: Multicenter retrospective cohort study. SETTINGS: Two infertility units. PATIENT(S): Thirty-nine women with bilateral endometriomas matched with 78 unexposed control subjects. INTERVENTION(S): Analysis of data from patients who underwent in vitro fertilization (IVF)-intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Ovarian responsiveness and oocyte quality. RESULT(S): Responsiveness to ovarian hyperstimulation was significantly reduced in women with bilateral endometriomas. The total numbers of developing follicles in case and control subjects were 9.6 ± 3.3 and 14.1 ± 6.8, respectively. The numbers of oocytes retrieved were 7.1 ± 3.2 and 9.8 ± 5.5, respectively. Conversely, oocyte retrieval was not hampered by the presence of the ovarian endometriomas. The rates (interquartile range) of oocytes retrieved per total number of developing follicles in case and control subjects were 77% (57%-88%) and 71% (63%-79%), respectively. Moreover, the quality of the retrieved oocytes did not differ. The fertilization rates (IQR) were 67% (56%-100%) and 70% (57%-100%), respectively. The rates (IQR) of top quality embryos per oocyte used were 33% (25%-50%) and 33% (20%-43%), respectively. The implantation rates were 22% and 23%, respectively. The clinical pregnancy rate and the delivery rate also did not differ. CONCLUSION(S): Although the presence of bilateral endometriomas at the time of IVF affects responsiveness to hyperstimulation, the quality of the oocytes retrieved and the chances of pregnancy are not affected.

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).

Blood cell mitochondrial DNA content and premature ovarian aging.

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

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.

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.

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.

Prevalence and incidence of depressive and anxious symptoms in couples undergoing assisted reproductive treatment in an Italian infertility department.

OBJECTIVE: We have conducted a longitudinal observational study in order to evaluate the prevalence and the incidence of depressive and anxious symptoms in women and men seeking infertility treatment and to analyze associated factors or risk factors for these kinds of disorders. STUDY DESIGN: A total of 1000 consecutive couples that visited our center for the first time were asked to join this study. Depressive and anxious symptoms were assessed with self-rating Zung Depression Scale (ZDS) and Zung Anxiety Scale (ZAS) questionnaires. A second assessment was planned at the time of ß HCG dosage (or at the moment of cycle suspension). A standard questionnaire was used to investigate socio-demographic information and the psychological aspects of couples undergoing in vitro fertilization (IVF) treatment. The fertility history and outcome of IVF treatment were collected from patients' medical records. RESULTS: 14.7% of women had anxious symptoms and 17.9% depressive symptoms, whereas 4.5% of men had anxious symptoms and 6.9% depressive symptoms. Women with depressive and anxious symptoms were younger, more often had an anxious partner and had a longer history of infertility. Men with depressive and anxious symptoms more frequently had a temporary job, they had an anxious partner and they were more frequently at the first in vitro fertilization (IVF) cycle. The incidence of depressive and/or anxious symptoms was 18.5% in women and 7.4% in men. Age and previous IVF treatments seem not to be associated with incidence of depressive or anxious symptoms. CONCLUSION: Both the prevalence and incidence of depressive and/or anxious symptoms in couples undergoing IVF treatment were worthy of note and should not be underestimated. More attention must be paid to psychological aspects in young women and in couples with a long history of infertility or previous failure treatments. Having an anxious partner was associated with anxious and depressive symptoms. For this reason, both males and females might benefit from psychological intervention, which could also help them to be supportive to each other.

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.

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.

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.

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).

Hyperstimulation during IVF cycles does not modify dimensions of small subserosal and intramural leiomyomas.

To evaluate the influence of IVF on the dimension of leiomyomas, we selected 72 women with small intramural or subserosal leiomyomas and assessed the size of these lesions before and after the treatment cycle. The mean ± SD diameters of the tumors at these two time points were 20.5 ± 9.5 and 20.6 ± 10.2 mm, respectively, thus supporting the conclusion that IVF does not influence the growth of these lesions.