Sperm donation: an alternative to improve post-ICSI live birth rates in advanced maternal age patients.

STUDY QUESTION: Can sperm donation increase live birth rates following ICSI in advanced maternal age (AMA) patients? SUMMARY ANSWER: Sperm donation increases the live birth rate in AMA ICSI cycles. WHAT IS KNOWN ALREADY: In ICSI practice, sperm donation has been predominantly applied to overcome male infertility. The involvement of paternal age and lower sperm quality in the severe reduction in fertility observed in AMA patients remains to be clarified. STUDY DESIGN, SIZE, DURATION: Retrospective multicenter cohort study including data generated between 2015 and 2019 from 755 ICSI cycles achieving a fresh embryo transfer, of which 337 were first homologous cycles (normozoospermic partner sperm and homologous oocytes) and 418 were first sperm donation cycles (donor sperm and homologous oocytes). The association of sperm origin (partner vs donor) with live birth was assessed by multivariate analysis in non-AMA (<37 years, n = 278) and AMA (≥37 years, n = 477) patients, separately, including in the model all variables previously found to be associated with live birth in a univariate analysis (number of MII oocytes recovered, number of embryos transferred, and maternal age). ICSI outcomes were compared between sperm donation and homologous cycles in overall, non-AMA and AMA patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in three fertility clinics and included 755 Caucasian patients aged 24-42 years undergoing their first homologous or sperm donation ICSI cycle achieving a fresh embryo transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The multivariate analysis revealed that sperm donation was positively associated with the likelihood of a live birth independently of all other variables tested in AMA (P = 0.02), but not in non-AMA patients. Live birth, delivery, and miscarriage rates differed substantially between sperm donation and homologous AMA cycles; live birth and delivery rates were 70-75% higher (25.4% vs 14.5% and 22.5% vs 13.5%, respectively; P < 0.01), while miscarriage occurrence was less than half (18.0% vs 39.5%; P < 0.01) in sperm donation compared to homologous AMA cycles. LIMITATIONS, REASONS FOR CAUTION: This study is limited by its retrospective nature, differences in patients profiles between sperm donation and homologous-control groups and varying proportion of donor cycles between fertility centers, although these variations have been controlled for in the statistical analysis. WIDER IMPLICATIONS OF THE FINDINGS: The findings suggest that sperm donation increases live birth rates while reducing miscarriage occurrence in AMA patients, and thus may be a valid strategy to improve ICSI outcomes in this growing and challenging patient group. STUDY FUNDING/COMPETING INTEREST(S): N/A. TRIAL REGISTRATION NUMBER: N/A.

Exploring the pros and cons of new approaches for gamete cross-border donation based on fresh and vitrified oocytes.

As highlighted by European statistics, the employment of donor oocytes is a growing option for women who cannot make use of their own gametes. As the potential recipients are continuously increasing in number, a donor programme which satisfies this demand is mandatory. Improvements in cryopreservation techniques, like oocyte and embryo vitrification, have led to the overcoming of the sequence of stimulation-retrieval-transfer both from a spatial and a temporal point of view, with the development of cryobanks of oocytes permitting crossborder donation. However, while some studies report comparable success when using vitrified and fresh oocytes we still need to investigate whether the use of fresh oocytes give higher live birth rate than cryopreserved ones, when the same number of oocytes are given. The performance of embryo cryopreservation, conversely, seems to be more reliable. A novel approach based on the shipment of frozen sperm from the recipient's country to the oocyte donor's one, where fresh oocytes are inseminated and the resulting embryos frozen and transported back to the referring IVF centre to perform a frozen embryo transfer may be a good strategy. We believe that the use of frozen embryos from fresh oocytes could be associated with a higher cumulative live birth rate per cycle, while favouring personalised oocyte recipient care with a flexible number of oocytes assigned and limiting the burden of travelling abroad.

A novel transnational fresh oocyte donation (TOD) program based on transport of frozen sperm and embryos.

STUDY QUESTION: What is the clinical efficacy of an oocyte donation program based on the transportation of frozen semen and embryos between two countries? SUMMARY ANSWER: The transnational oocyte donation program is efficient and reliable and it could provide a first-line strategy to overcome the lack of donors in some countries. WHAT IS KNOWN ALREADY: While there is increasing need for donated oocytes, in many countries the availability of donors is still insufficient to cover the therapeutic demands, and patients are referred abroad for treatment. Since embryo cryopreservation is reliable and efficient, we propose a strategy based on frozen embryos instead of frozen oocytes to satisfy the increasing demand for cross border oocyte donation. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including 630 patients treated from December 2015 to July 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women were treated with elective vitrified-thawed embryo shipping and embryo transfer (ET) between two IVF clinics, one in Spain and one in Italy. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 2617 embryos were created for the 630 patients and the survival rate after warming was 98.5%. After the first ET the live birth rate (LBR) was 30.6%. In 476 patients (75.5%), embryos were transferred at the cleavage stage (Day 2 or 3) and the LBR was 29.2%. Vitrified blastocysts were available for 154 patients (24.5%) and the LBR was 35%. Among patients who did not achieve a pregnancy after the first frozen ET (FET), 92.5% had at least one frozen embryo for successive procedures. 213 patients underwent a second FET. The LBR at the second FET was 30%. The cumulative LBR at the end of the observation period was 39.3%. LIMITATIONS, REASONS FOR CAUTION: The study design was retrospective. A direct comparison with vitrified oocyte donors cycle and subsequent fresh ET would have permitted to compare this strategy versus the current standard based on vitrified gametes. WIDER IMPLICATIONS OF THE FINDINGS: The LBR found in our study is more than acceptable and seems to be higher than what reported with vitrified oocytes. The transnational fresh oocyte donation program may have several advantages over the shipment of vitrified oocytes: similarly to the fresh oocyte donation program it allows for personalized care in oocyte recipient, which is provided by assigning a flexible number of oocytes, and at the same time it maintains the benefit of a frozen ART program permitting scheduling flexibility. The TOD program is efficient and may be proposed as a first-line strategy for distance and inter-countries oocyte donation programs. STUDY FUNDING, COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NA.

Characterization of the miRNA regulators of the human ovulatory cascade.

Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.

Focused time-lapse analysis reveals novel aspects of human fertilization and suggests new parameters of embryo viability.

STUDY QUESTION: Can focused application of time-lapse microscopy (TLM) lead to a more detailed map of the morphokinetics of human fertilization, revealing novel or neglected aspects of this process? SUMMARY ANSWER: Intensive harnessing of TLM reveals novel or previously poorly characterised phenomena of fertilization, such as a cytoplasmic wave (CW) preceding pronuclear formation and kinetics of pronuclear chromatin polarization, thereby suggesting novel non-invasive biomarkers of embryo quality. WHAT IS KNOWN ALREADY: In recent years, human preimplantation development has been the object of TLM studies with the intent to develop morphokinetic algorithms able to predict blastocyst formation and implantation. Regardless, our appreciation of the morphokinetics of fertilization remains rather scarce, currently including only times of polar body II (PBII) emission, pronuclear appearance and fading, and first cleavage. This is not consistent with the complexity and importance of this process, calling for further TLM studies aimed at describing previously unrecognized or undetected morphokinetic events and identifying novel developmental biomarkers. STUDY DESIGN, SIZE, DURATION: The study involved a retrospective observation by TLM of the fertilization process in 500 oocytes utilized in consecutive ICSI cycles carried out in 2016. A maximum of five fertilized oocytes per patients were included in the analysis to reduce possible patient-specific biases. Oocytes of patients with different diagnoses of infertility where included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Microinjected oocytes where assessed by a combined TLM-culture system (Embryoscope). Oocytes that were not amenable to TLM assessment, due to excess of residual corona cells or inadequate orientation for the observation of PBII emission, were not analysed. We identified and monitored 28 parameters relevant to meiotic resumption, pronuclear dynamics, chromatin organization, and cytoplasmic/cortical modifications. Times (T) were expressed as mean ± SD hours post-insemination (p.i.) and analysed, where appropriate, by Paired T Student or Fisher's exact tests. MAIN RESULTS AND ROLE OF CHANCE: PBII emission was occasionally followed (4.3% of cases) by the transient appearance of a protrusion of the cell surface, the fertilization cone (FC), probably resulting from interaction of the male chromatin with the oocyte cortex. Pronuclear formation was always preceded by a radial CW originating from the initial position of the male pronucleus (PN) and extending towards the oocyte periphery. The appearance of the CW followed a precise sequence, occurring always 2-3 h after PBII emission and shortly before PN appearance. Male and female PN appeared virtually simultaneously at approximately 6.2 h p.i. However, while the female PN always formed cortically and near the site of emission of the PBII, the initial position of the male PN was cortical, intermediate, or central (15.2%, 31.2% and 53.6%, respectively). PN juxtaposition involved rapid and straight movement of the female PN towards the male PN. In addition, the initial position of male PN formation was predictive of the position of PN juxtaposition. It was also observed that nucleolar precursor bodies (NPBs) aligned along the juxtaposition area and this happened considerably earlier for the female PN (8.2 ± 2.6 vs.11.2 ± 4.1 h, P = 0.0001). Although it occurred rarely, displacement of juxtaposed PN to the cortex was strongly associated (P < 0.0001) with direct cleavage into three blastomeres at the first cell division. The times of PN breakdown and first cleavage showed a very consistent trend, occurring earlier or progressively later depending on whether initial male PN positioning was central, intermediate or cortical, respectively. Finally, time intervals between discrete fertilization events were strongly associated with embryo quality on Day 3. For example, longer intervals between disappearance of the cytoplasmic halo and PN breakdown were highly predictive of reduced blastomere number and increased fragmentation (P = 0.0001). LARGE SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: Some of the morphokinetic parameters assessed in this study may require better definition to reduce inter-operator annotation variability. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, overall, these data represent the most detailed morphokinetic description of human fertilization. Many of the illustrated parameters are novel and may be amenable to further elaboration into algorithms able to predict embryo quality, as suggested by the findings presented in this study. STUDY FUNDING/COMPETING INTERESTS: None.

Effect of different gonadotrophin priming on IVM of oocytes from women with normal ovaries: a prospective randomized study.

This study was designed to determine if the efficiency of in-vitro maturation (IVM) in women with normal ovaries can be improved by gonadotrophin administration. 400 women were randomly allocated in four groups: group A, non-primed cycles; group B, human chorionic gonadotrophin (HCG)-primed cycles; group C, FSH-primed cycles; and group D, FSH- plus HCG-primed cycles. There were significant differences in the IVM rate among the groups. In groups where HCG was used, the overall maturation rate was higher (57.9% in group B and 77.4% in group D; 48.4% in group A and 50.8% in group C) and the percentage of total available metaphase II-stage oocytes was higher (60.4% in group B and 82.1% in group D; 48.4% in group A and 50.8% in group C). The overall clinical pregnancy rate per transfer (CPR) was 18.3% and the implantation rate (IR) was 10.6%. There was a difference in CPR among the groups: group D (29.9%) versus group A (15.3%), P = 0.023; group D versus group B (7.6%), P < 0.0001; group D versus group C (17.3%), P = 0.046. The results of this study are clearly in favour of FSH plus HCG priming. FSH priming and HCG priming alone showed no significant effects on clinical outcome.

IVM--the first choice for IVF in Italy.

In March 2004, a new law was introduced in Italy to regulate assisted reproduction; at present it is impossible to use more than a maximum of three oocytes per IVF cycle, nor can embryos or prezygotes (2PN cells) be selected or cryopreserved. The prohibitions introduced by the new law have, on the one hand, reduced the expectations of success of current techniques and, on the other hand, stimulated clinicians and embryologists to work on new therapeutic strategies so as to offer the highest chances of success with the lowest risks. In-vitro maturation (IVM) of oocytes fits very well with these new requirements: ovarian stimulation is avoided and the handling of spare oocytes is facilitated. The IVM protocol is an intriguing alternative to conventional IVF techniques, since it removes the side-effects of drug stimulation, especially ovarian hyperstimulation syndrome, and it also reduces the costs of the entire procedure, both in terms of 'time consumption' and 'patient/society costs for drugs'. In the authors' IVF centre the IVM technique has been used for more than a year, with significant success in terms of maturation and fertilization rates, percentage of embryo transfers, number of pregnancies and, finally, healthy babies born.

Laparoscopic excision of ovarian dermoid cysts with controlled intraoperative spillage. Safety and effectiveness.

OBJECTIVE: To compare the operative and postoperative course in patients undergoing laparoscopy for dermoid cyst to that observed in subjects with other types of ovarian masses and of patients undergoing laparotomy for teratomas. STUDY DESIGN: Retrospective analysis. From 1994 to 1996, 49 women underwent laparoscopic cystectomy for dermoid cysts. The operative and postoperative course was compared to that of 190 patients undergoing operative laparoscopy for other adnexal masses and to that of 43 patients undergoing laparotomy for dermoid cysts from 1992 to 1996. The cysts were aspirated to reduce spillage and removed via a laparoscopic bag inserted in a 10-mm trocar. Culdotomy was never used. The abdominal cavity was abundantly flushed during the procedure and before closure. RESULTS: Dermoid cystectomy was successfully performed laparoscopically in 47 of 49 cases. Spillage occurred in 43 cases (88%), and postoperative fever occurred in 3 (6.1%). No case of peritonitis was recorded. Significant differences between laparoscopy and laparotomy were observed in the rate of bilaterality (4% vs. 25%), spillage (88% vs. 9%) and mean hospital stay (37 vs. 83 hours). When laparoscopic excision of dermoid cysts and other masses was compared, we did not observe any significant difference in operative time or complication rates, apart from transient fever. CONCLUSION: Laparoscopy is safe and effective for dermoid cysts and allows shorter hospitalization than laparotomy. As observed for other benign cysts, laparoscopy should become the technique of choice for the removal of most, if not all, ovarian dermoid cysts.

[Spontaneous pregnancy in "sterile couples"]. / Le gravidanze spontanee nelle "coppie sterili".

The aim of the study is to evaluate the results of tests performed in "infertile" couples that have obtained spontaneous pregnancy. 953 couples were studied from 1986 to 1991; 108 spontaneous pregnancies occurred (11.1%). All patients were evaluated according to standard procedures recommended by literature. "Female factor" as cause of infertility was present in 41.5%; "cervical factor" was present in 19.4%; "male factor" was present in 48.1% while in 14.8% of the couples evaluated no pathology was showed. In this series, spontaneous pregnancy occurred within 4 year of infertility in more than 90%; no significant difference was present between primary and secondary infertility. A considerable incidence of pathologies was observed in couples that obtained spontaneous pregnancy. Our data suggest that common investigation for infertility may have not a real prognostic value.

[Induction of puberty and maintenance of secondary sexual characteristics in males with thalassemia major]. / Induzione della pubertà e mantenimento delle caratteristiche sessuali secondarie in maschi portatori di talassemia major.

18 males with beta-thalassaemia major were studied; mean age was 17.6 flAF 4.3, 8 of 18 males evaluated were prepubertal (Tanner 1), 5 were early and mid pubertal (Tanner 2-3) and 5 were full pubertal (Tanner 5). All patients (pts) underwent GnRH test and 61% showed positive response. 8 pts. were treated with hCG; 2 were azoospermic, 6 were aspermic. The starting dose was 1000 IU twice weekly; dose adjustments were made basing on testosterone levels and clinical features. Clinical examination, testosterone assays, semen analysis were performed every 3 months during the treatment period. Therapy was started 18-48 months ago and it is still in progress. Within 12 months of treatment 4 pts progressed to Tanner stage 4, 3 pts to Tanner stage 5, 1 showed no response. Semen analysis showed an improvement in 6 pts: after 12 months of therapy 1 pts was aspermic, 5 were azoospermic, 1 was oligozoospermic. Our data showed that hCG administration is effective in inducing and maintaining secondary sexual character.