Sperm cryopreservation in adolescents with cancer.

OBJECTIVE: To evaluate whether sperm banking is under-used in adolescents with cancer. STUDY DESIGN: We reviewed our 33 years long experience of semen cryopreservation and identified male subjects with cancer aged 14-21 years who banked their sperm. We hypothesized as a referring model a constant rate of referral in every age category (uniform distribution). In addition, we evaluated whether the distribution of the referrals per age varied according to historical periods and compared semen quality in the different age groups. RESULTS: Overall, 317 subjects were selected. A sharp increase with age emerged (p < 0.001): the number of cases augmented from 4 at age 14 years to 79 at age 21 years. This significant increase persisted even when focussing only on subjects older than 16-18 years. An improvement of the distribution of cases per age group emerged when limiting the analysis to the last decade of activity; however, the increase remained significant (p < 0.001). Finally, we investigated whether semen quality differed according to age groups and failed to show marked differences, suggesting that sperm banking even at the youngest ages could be of potential benefit. CONCLUSIONS: This study confirmed that sperm banking is under-used in adolescents with cancers. Future studies should better clarify the determinants of this low referral and identify interventions that can improve the situation.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

ALADDIN study: does assisted hatching of vitrified/warmed blastocysts improve live birth rate? Protocol for a multicentric randomised controlled trial.

INTRODUCTION: Recent data suggest a higher clinical pregnancy rate performing assisted hatching (AH) on previously cryopreserved embryos but fail to demonstrate significant effects on live birth rate. However, current evidence is based on studies with a small sample size and may hide a type II error. Moreover, poor attention has been given to the specific effect of AH on frozen/thawed blastocysts. To shed light on this topic, we developed the present protocol for a randomised trial to investigate the benefits of the laser-mediated partial removal of the zona pellucida in vitrified/warmed blastocysts. METHODS AND ANALYSIS: The pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN) study is a multicentric prospective comparative study with a parallel randomised controlled design aiming to investigate whether AH performed on warmed blastocysts before embryo transfer can improve live birth rate. Women allocated to the control group will undergo embryo transfer of blastocysts not previously subjected to AH. Two infertility units will be involved in the study. Enrolment of patients will last 18 months with quarterly monitoring and the entire study is foreseen to be closed in 36 months. Secondary outcomes include: proportion of transferred blastocysts/thawed blastocyst, morphological features of blastocysts before embryo transfer, implantation, biochemical pregnancy, clinical pregnancy (ultrasound visible gestational sac), miscarriage, multiple pregnancy, preterm birth (<37 weeks of gestation), obstetrical and neonatal complications and congenital anomaly rates. ETHICS AND DISSEMINATION: This protocol received a favourable ethical opinion from the Ethical Committee of IRCCS San Raffaele Scientific Institute and the Ethical Committee Area 2 Milan. Each participant will provide written consent to participate and remain encoded during the study. The trial results will be published in peer-reviewed journals and presented at conferences. TRIAL REGISTRATION NUMBER: NCT03623659; Pre-results.

The presence of dominant follicles and corpora lutea does not perturb response to controlled ovarian stimulation in random start protocols.

The advent of random start protocols to shorten the time needed to store oocytes in women with malignancies has represented an important improvement in the field of fertility preservation. However, Randomized Controlled Trials are difficult to implement in this area and available evidence that supports this approach remains modest. To shed more light on this issue, we compared the follicular development between the ovary carrying the dominant follicle or the corpus luteum and the contralateral resting ovary in 90 women who underwent random start controlled ovarian stimulation (COS). In fact, ovarian response did not differ between the two ovaries. Subgroup analyses according to the phase of the cycle at the initiation of COS, the type of malignancy, the use of letrozole and the magnitude of the ovarian response did not allow to identify any condition showing a difference in the follicular response between the active and the resting ovaries. In conclusion, follicular growth does not seem to be perturbed by the presence of a dominant follicle or a corpus luteum.

Clinical comparison between conventional and microdissection testicular sperm extraction for non-obstructive azoospermia: Understanding which treatment works for which patient.

OBJECTIVES: The superiority of microdissection testicular sperm extraction (mTESE) over conventional TESE (cTESE) for men with non-obstructive azoospermia (NOA) is debated. We aimed to compare the sperm retrieval rate (SRR) of mTESE to cTESE and to identify candidates who would most benefit from mTESE in a cohort of Caucasian-European men with primary couple's infertility. MATERIAL AND METHODS: Data from 49 mTESE and 96 cTESE patients were analysed. We collected demographic and clinical data, serum levels of LH, FSH and total testosterone. Patients with abnormal karyotyping were excluded from analysis. Age was categorized according to the median value of 35 years. FSH values were dichotomized according to multiples of the normal range (N) (N and 1.5 N: 1-18 mIU/mL, and > 18 mIU/mL). Testicular histology was recorded for each patient. Descriptive statistics and logistic regression analyses tested the impact of potential predictors on positive SRR in both groups. RESULTS: No differences were found between groups in terms of clinical and hormonal parameters with the exception of FSH values that were higher in mTESE patients (p = 0.004). SRR were comparable between mTESE and cTESE (49.0% vs. 41.7%, p = 0.40). SRRs were significantly higher after mTESE in patients with Sertoli cell-only syndrome (SCOS) (p = 0.038), in those older than 35 years (p = 0.03) and with FSH >1.5N (p < 0.001), as compared to men submitted to cTESE. Multivariable logistic regression analysis showed that mTESE was independent predictor of positive SR in patients older than 35 years (p = 0.002) and with FSH > 1.5N (p = 0.018). Moreover, increased FSH levels (p = 0.03) and both SCOS (p = 0.01) and MA histology (p = 0.04) were independent predictors of SRR failure. CONCLUSIONS: Microdissection and cTESE showed comparable success rates in our cohort of patients with NOA. mTESE seems beneficial for patients older than 35 years, with high FSH values, or when SCOS can be predicted. Given the high costs associated with the mTESE approach, the identification of candidates most likely to benefit from this procedure is a major clinical need.

Frozen-thawed blastocyst transfer in natural cycle: feasibility in everyday clinical practice.

PURPOSE: Transfer of frozen-thawed embryos in natural cycle is gaining consensus but evidence on this approach is scanty. The aim of this study is reporting on the feasibility of this type of policy in everyday clinical practice. METHODS: We retrospectively selected all women undergoing the procedure between July 2013 and December 2014. During the study period, women were systematically scheduled for natural cycle if they referred regular menstrual cycles. Hormone replacement therapy (HRT) was conversely prescribed if the woman had irregular menstrual cycles or if the monitoring of the natural cycle failed. The analysis exclusively focussed on the first cycle per woman. RESULTS: Overall, 251 women were selected. HRT was initially chosen in 52 women, leaving 199 women suitable for the natural cycle. This procedure could be performed in 194 of these women (97%, 95% CI 95-99%). Two additional women initially allocated to HRT ultimately performed the blastocyst transfer with natural cycle. Overall, 196 were thus treated with natural cycle (78%, 95% CI 73-83%). The basal characteristics of the women who did and did not undergo natural cycles were similar with the exceptions of serum FSH (p < 0.001) and AMH (p = 0.03). The live birth rate did not also differ (34% versus 31%, p = 0.63). Characteristics of women treated with the natural cycle who did (n = 67) and did not (n = 129) achieve a live birth did not differ. CONCLUSION: Frozen-thawed blastocyst transfer in natural cycle can be successfully performed in the vast majority of women.

Obstetric and neo-natal outcomes of ICSI cycles using pentoxifylline to identify viable spermatozoa in patients with immotile spermatozoa.

Pentoxifylline (PF) represents an effective tool in stimulating motility and identifying viable spermatozoa in intracytoplasmic sperm injection (ICSI) patients presenting exclusively with immotile spermatozoa. However, its use is not universally accepted for its possible detrimental effects on oocytes, embryos or newborns. To evaluate whether PF use may affect obstetrical/neo-natal outcomes, 102 patients achieving a clinical pregnancy after a PF-ICSI in four IVF units in Spain and Italy were followed up after delivery. Neo-natal malformations were classified according to the World Health Organization International Classification of Diseases (ICD-10, range Q00-Q99). Malformation rate was compared with data published by other groups regarding children conceived by conventional IVF or ICSI reporting a 5.3% and 4.4% frequency of ICD-10 codes, respectively. Of 134 clinical pregnancies, 122 babies (82 singletons and 40 twins) were registered. Among singletons, the rates of low birthweight (≤2500 g) and preterm birth (<37 weeks) were 6.1% and12%, respectively. Regarding malformation rate per live births, 4/122 (3.3%, 95% confidence interval: 0.9-8.2%) babies with ICD-10 malformations were recorded. This is the first report on neo-natal outcomes deriving from PF-ICSI. Although based on a limited cohort, results do not suggest an increase of adverse outcomes, including malformation rates, following this procedure.

Application of failure mode and effect analysis in an assisted reproduction technology laboratory.

Assisted reproduction technology laboratories have a very high degree of complexity. Mismatches of gametes or embryos can occur, with catastrophic consequences for patients. To minimize the risk of error, a multi-institutional working group applied failure mode and effects analysis (FMEA) to each critical activity/step as a method of risk assessment. This analysis led to the identification of the potential failure modes, together with their causes and effects, using the risk priority number (RPN) scoring system. In total, 11 individual steps and 68 different potential failure modes were identified. The highest ranked failure modes, with an RPN score of 25, encompassed 17 failures and pertained to "patient mismatch" and "biological sample mismatch". The maximum reduction in risk, with RPN reduced from 25 to 5, was mostly related to the introduction of witnessing. The critical failure modes in sample processing were improved by 50% in the RPN by focusing on staff training. Three indicators of FMEA success, based on technical skill, competence and traceability, have been evaluated after FMEA implementation. Witnessing by a second human operator should be introduced in the laboratory to avoid sample mix-ups. These findings confirm that FMEA can effectively reduce errors in assisted reproduction technology laboratories.

The impact of Alpha/ESHRE consensus regarding oocytes with aggregates of smooth endoplasmic reticulum (SERa) on in vitro fertilization outcome.

PURPOSE: The present study aimed to gather information on the impact of Alpha/European Society of Human Reproduction and Embryology (ESHRE) consensus regarding oocytes with aggregates of smooth endoplasmic reticulum (SERa) on in vitro fertilization outcome. In particular, we investigated if patients undergoing intracytoplasmic sperm injection (ICSI) and whose oocytes are discarded due to SERa have a higher chance of embryo transfer cancellation compared to patients without SERa oocytes. METHODS: This is a nested case-control study drawn from the cohort of women referring for in vitro fertilization with ICSI. Cases were patients showing at least one oocyte with SERa at the time of injection. Controls were subsequent patients showing no SERa oocytes and matched ratio 1:1 for age, clinical indication to in vitro fertilization (IVF), and body mass index. The main outcome was the rate of embryo transfer cancellation. RESULTS: The percentage of women experiencing a transfer cancellation (absence of suitable oocytes or viable embryos) in their ICSI cycle were significantly higher in cases (18 %) compared to controls (8 %) (p = 0.02); however, adjusted odds ratio for FSH and number of SERa oocytes, of follicles, of retrieved oocytes, and of inseminated oocytes were not statistically significant. CONCLUSIONS: We have shown that the exclusion of SERa oocytes from ICSI cycles causes an increased frequency of transfer cancellation. This effect is mostly due to the reduced number of available oocytes after exclusion of SERa oocytes.

Intrafollicular iron and ferritin in women with ovarian endometriomas.

OBJECTIVE: To evaluate whether iron contained in ovarian endometriomas can diffuse through the cyst wall and negatively affect ovarian function. DESIGN: Prospective case series. SETTING: Infertility unit in an academic setting. POPULATION: Thirty-nine infertile women with unilateral endometriomas who underwent in vitro fertilization. METHODS: Iron and ferritin assessments in pools of follicular fluids obtained from affected and contralateral intact gonads. MAIN OUTCOME MEASURES: Iron and ferritin concentrations. RESULTS: Follicular fluid iron content did not differ between the two gonads. The median [interquartile range (IQR)] follicular concentrations in the affected and unaffected ovaries were 59 (IQR 44-74) and 59 (IQR 47-73) µg/dL, respectively (p = 0.77). Conversely, ferritin concentration was significantly higher in affected gonads. The median (IQR) concentrations of ferritin in the affected and unaffected ovaries were 57 (IQR 31-146) and 33 (IQR 23-67) µg/mL, respectively (p = 0.026). When considering together the 78 studied ovaries, no significant correlations emerged between follicular iron and ferritin and variables reflecting ovarian responsiveness and oocyte developmental competence. CONCLUSIONS: Iron may diffuse from ovarian endometriomas into the adjacent ovarian tissue. However, this phenomenon does not appear to markedly affect ovarian function. Some effective biological mechanisms such as ferritin storage may effectively sequester free iron, so limiting its detrimental effects.

Can we use incubators with atmospheric oxygen tension in the first phase of in vitro fertilization? A retrospective analysis.

PURPOSE: Aim of the present study was to compare two culture strategies used in our routine in vitro fertilization program. METHODS: This is a retrospective analysis. Two culture systems were used in parallel and analysed retrospectively: 1) Use of atmospheric oxygen tension (~20 %) until insemination followed by use of low (~5 %) oxygen concentration; 2) Exclusive use of low oxygen concentration. Main outcome was the utilization rate defined as the number of transferred + vitrified embryos per inseminated oocytes. Secondary outcomes were clinical pregnancy and live birth rates. RESULTS: A total of 402 in vitro fertilization cycles were analyzed. Demographic and clinical data of patients belonging to the two culture systems were not significantly different. Utilization rate, cumulative clinical pregnancy rate and cumulative live birth rate per cycle was similar using two different oxygen concentration compared to exclusive use of low oxygen tension (37 % versus 39 %; 30 % versus 30 %; 23 % versus 28 %, respectively). CONCLUSIONS: The use of a culture system with atmospheric oxygen tension from recovery of oocytes until insemination followed by culture in low oxygen gives results similar to exclusive use of low oxygen concentration.

The strategy of group embryo culture based on pronuclear pattern on blastocyst development: a two center analysis.

PURPOSE: To compare two embryo grouping strategies. METHODS: Retrospective time-course analysis in two different centres. Two culture protocols were used at the zygote stage: "Random Group" in which zygotes were randomly grouped and "Definite Group" in which zygotes were grouped based on pronuclear pattern. Embryo culture was extended to blastocyst stage. Primary and secondary outcomes were respectively the blastulation rate and the cumulative clinical pregnancy and implantation rates. RESULT(S): A similar blastulation rate [42 and 41% day (5 + 6) blastocysts] was obtained in the two groups. Conversely, after adjusting for baseline and cycle variables, cumulative pregnancy [adjusted Odds Ratio = 2.10 (95%CI: 1.08-4.07)] and implantation [adjusted Odds Ratio = 1.78 (95%CI: 1.06-2.97)] rates were significantly higher in the "Random Group" compared to the "Definite Group". CONCLUSION(S): Two strategies of group culture gave similar results in terms of blastulation rate but the random grouping of zygotes improves pregnancy and implantation rates in IVF-cycles.

IVF outcome in women with accidental contamination of follicular fluid with endometrioma content.

OBJECTIVE: To evaluate whether accidental contamination of follicular fluid with endometrioma content during oocytes retrieval affects IVF outcome. STUDY DESIGN: We set up a retrospective review of women undergoing IVF in our infertility unit during a four years period. Cases were 19 women with ovarian endometriomas who had an accidental aspiration of the endometrioma content during oocyte retrieval. Controls (n=38) were the two subsequent age-matched women with endometriomas who underwent an uncomplicated oocyte retrieval. The main outcome was to evaluate the clinical pregnancy rate in cases and in controls. RESULTS: Accidental aspiration of endometrioma content occurred in 19 out of 314 women (6.1%, 95%CI: 3.8-9.1%). The number of embryos and top quality embryos was significantly higher in exposed women (3.1±2.0 versus 1.7±1.2 and 1.9±1.5 versus 1.1±1.1, respectively) but the chances of pregnancy were lower. The adjusted RRs of clinical pregnancy and live birth were 0.63 (95%CI: 0.49-0.87, p=0.005) and 0.60 (95%CI: 0.51-086, p=0.003), respectively. CONCLUSION: Accidental contamination of the follicular fluid with endometrioma content is an uncommon but possible event. This situation may affect IVF outcome but does not justify systematic surgical removal before the cycle.

Ovarian endometriomas and oocyte quality: insights from in vitro fertilization cycles.

OBJECTIVE: To evaluate whether the presence of endometriomas affects ovarian function. DESIGN: Prospective cohort study. SETTING: Infertility unit of an academic setting. PATIENT(S): Twenty-nine women undergoing an IVF cycle. INTERVENTION(S): Prospective evaluation of women with unoperated unilateral endometriomas undergoing IVF. The affected and contralateral intact gonads were compared in terms of responsiveness and oocyte quality. MAIN OUTCOME MEASURE(S): Oocyte developmental competence. RESULTS: Ovarian responsiveness and oocyte quality did not significantly differ between the affected and intact gonads. The number of codominant follicles, the number of oocytes retrieved, and the number of suitable oocytes in affected and intact gonads were 3.7 ± 2.4 and 4.1 ± 1.7, 4.2 ± 3.1 and 4.7 ± 2.5, and 3.1 ± 2.6 and 3.5 ± 2.3, respectively. The number of viable embryos and the number of high-quality embryos were 1.8 ± 2.1 and 1.8 ± 1.4 and 1.0 ± 1.7 and 0.8 ± 0.7, respectively. The fertilization rate in the affected and intact gonads was 64% and 64%, respectively. The cleavage rate was 58% and 51%, respectively. The rate of high-quality embryos was 31% and 21%, respectively. CONCLUSION(S): In women undergoing IVF, the presence of ovarian endometriomas does not affect oocyte developmental competence.

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).

Developmental potential of human oocytes after slow freezing or vitrification: a randomized in vitro study based on parthenogenesis.

The aim of the this study was to compare the in vitro developmental competence of parthenogenetically activated oocytes cryopreserved with slow-freezing or vitrification. Supernumerary metaphase II oocytes obtained during in vitro fertilization procedures were randomized to slow freezing or vitrification procedure. After thawing or devitrification, oocytes were parthenogenetically activated and cultured. Survival, activation, development rate, and cell number during culture were compared. The 2 groups showed no significant differences between the rates of parthenogenetic activation, development, good quality parthenotes and blastomere number on day 2 of culture. However, parthenotes from the devitrified oocytes continued cleaving till day 3 in a significantly low proportion (27% vs. 42%). On day 3, the mean number of blastomeres was also lower in vitrification group compared to slow-freezing (4.8 + 1.9 vs. 5.8 + 1.7). In conclusion, parthenogenesis highlights a reduced potential of vitrified oocytes to cleave on day 3 compared with oocytes from slow-freezing.

In vitro development of human oocytes after parthenogenetic activation or intracytoplasmic sperm injection.

OBJECTIVE: To compare directly in vitro developmental competence between parthenogenetically activated and intracytoplasmic sperm injection (ICSI)-fertilized oocytes. DESIGN: For each patient, three metaphase II oocytes were randomized to the ICSI procedure, while n-3 were allocated to parthenogenetic activation. SETTING: University hospital infertility unit. PATIENTS: Thirty-eight patients, aged 35.2 +/- 3.3 years (mean +/- SD) selected for ICSI. INTERVENTIONS: After 1 hour from denudation, oocytes were either fertilized by ICSI (n = 114) or chemically activated (n = 104). Fertilized and activated oocytes were cultured for up to 3 and 5 days, respectively. MAIN OUTCOME MEASURES: Development rate, cell number, and morphological grade during culture. RESULTS: The two groups showed no significant differences between rates of fertilization and parthenogenetic activation, development, and blastomere number on days 2 and 3 of culture. However, parthenotes showed a lower morphological grade, and a significantly lower proportion went on cleaving to day 3, when only activated rather than total numbers of oocytes were considered. On day 5 after activation, nine oocytes (8.6%) reached the blastocyst stage, representing 12.9% of parthenotes. CONCLUSIONS: Since most parameters examined in this study were similar between activated and fertilized oocytes, parthenogenetic activation may be a useful tool for the preclinical evaluation of experimental procedures.

Follicular fluid levels of the long pentraxin PTX3.

OBJECTIVE: Pentraxin-3 (PTX3) is a long pentraxin that plays a key role in female fertility as a structural and essential constituent of the cumulus oophorus extracellular matrix. Despite considerable evidence supporting this role of PTX3 in mice, data in humans are scanty. The aim of the present study was (1) to evaluate follicular fluid concentrations of PTX3; (2) to test the hypothesis that levels of the molecule correlate with oocyte characteristics (corona radiata, aspect of the cumulus, nuclear maturity, and fertilization); and (3) to evaluate the possibility that peripheral concentration of PTX3 may be of clinical help in monitoring ovarian hyperstimulation. METHODS: ELISA was used to determine PTX3 concentration. Levels of PTX3 were tested in 96 follicles. RESULTS: The mean +/- SD and the median (interquartile range) were 17.9 +/- 18.3 and 12.1 (6.5-23.6) ng/mL, respectively. Levels of the molecule did not appear to be normally distributed. At the day of ovum pick-up, levels of PTX3 were 6.3-fold higher in follicular fluid than in peripheral blood (95% CI, 3.6-9.0). No statistically significant difference emerged linking follicular fluid concentration of PTX3 and oocyte quality. In a series of ten women, plasma concentration of PTX3 did not vary during ovarian hyperstimulation, resulting in levels of 1.0 +/- 0.5 at the 3rd day of the menstrual cycle and 1.0 +/- 0.6 ng/mL at the day of oocyte retrieval. CONCLUSIONS: Results from the present study support the following conclusions: (1) elevated levels of soluble PTX3 can be found in follicular fluid; (2) follicular fluid concentration of PTX3 cannot by used as a marker of oocyte quality; and (3) plasma concentration of the molecule is not influenced by ovarian hyperstimulation.

Damage to ovarian reserve associated with laparoscopic excision of endometriomas: a quantitative rather than a qualitative injury.

OBJECTIVE: Retrospective studies suggest that laparoscopic excision of endometriomas is associated with a reduced responsiveness to ovarian hyperstimulation. In this study, we prospectively evaluated ovarian response to hyperstimulation in women selected for in vitro fertilization and intracytoplasmic sperm injection cycles who previously underwent laparoscopic enucleation of a monolateral endometrioma. STUDY DESIGN: Operated and contralateral intact ovaries of the same patient were compared in terms of number of follicles, number of oocytes retrieved, fertilization rate, and rate of high-quality embryos. RESULTS: Thirty-eight subjects were included. A reduced number of dominant follicles, oocytes, embryos, and high-quality embryos was observed in the operated gonad. The mean percentage of reduction was 60% (95% confidence interval 38-81%), 53% (95% confidence interval 30-75%), 55% (95% confidence interval 28-81%), and 52% (95% confidence interval 17-87%), respectively. Fertilization rate and rate of good-quality embryos were similar. CONCLUSION: Laparoscopic excision of endometriomas is associated with a quantitative but not a qualitative damage to ovarian reserve.

Reproductive prognosis in male patients with azoospermia at the time of cancer diagnosis.

OBJECTIVE: Fertility status evaluation in patients found azoospermic at the time of malignancy diagnosis. DESIGN: Case series follow-up. SETTING: University Hospital Sperm Banking service. PATIENT(S): Male cancer patients found azoospermic between 1986 and June 2000. INTERVENTION(S): Patients were interviewed about their reproductive history and were invited for a free semen analysis. MAIN OUTCOME MEASURE(S): Pregnancy rate (PR) and percentage of patients with viable spermatozoa in the ejaculate. RESULT(S): Sixty-five patients were enrolled; 16 were lost at follow-up, 7 died, and 42 were followed to the end of the study. The median (range) time of follow-up for the 42 remaining patients was 9 years (2-14 years). Seventeen patients had wanted to father a child; 12 had a child (71%, 95% confidence interval [CI] 48%-88%). Semen analysis results were available in 11 patients. Resumption of spermatogenesis was documented in 9 cases (82%, 95% CI 53%-97%). CONCLUSION(S): Spontaneous fertility recovery is frequent in patients who are azoospermic at the time of cancer diagnosis.