Alberto Vaiarelli

Dual ovarian stimulation and random start in assisted reproductive technologies: from ovarian biology to clinical application

Purpose of review The opportunity to use gonadotropins to stimulate the growth of antral follicles coming from different follicular waves available in different moment of the menstrual cycle allowed the implementation of innovative protocols in assisted reproductive technologies. The purpose of this review is to explore the possible advantages related to these new controlled ovarian stimulation (COS) strategies. Recent findings Women exhibit major and minor wave patterns of ovarian follicular development during the menstrual cycle, as it is in animal species. These observations led to the introduction of two new strategies for COS: the random start and the double ovarian stimulation within a single menstrual cycle. Summary The use of gonadotropin-releasing hormone antagonist COS protocols, started randomly at any day of the menstrual cycle, is today a standard procedure in those cases where obtaining oocytes is an urgent task, such as in case of fertility preservation for malignant diseases or other medical indications. On the other hand, in poor prognosis patients, double ovarian stimulation has been suggested with the aim of maximizing the number of oocytes retrieved within a single menstrual cycle and, in turn increasing the chance to obtain a reproductively competent embryo. Randomized control trials are necessary to confirm these preliminary findings.

Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles

Preimplantation genetic diagnosis and aneuploidy testing (PGD/PGS) use is constantly growing in IVF, and embryo/biopsy traceability during the additional laboratory procedures needed is pivotal. An electronic witnessing system (EWS), which showed a significant value in decreasing mismatch occurrence and increasing detection possibilities during standard care IVF, still does not guarantee the same level of efficiency during PGD/PGS cycles. Specifically, EWS cannot follow single embryos throughout the procedure. This is however critical when an unambiguous diagnosis corresponds to each embryo. Failure Mode and Effects Analysis (FMEA) is a proactive method generally adopted to define tools ensuring safety along a procedure. Due to the implementation of a large quantitative PCR (qPCR)-based blastocyst stage PGD/PGS programme in our centre, and to evaluate the potential procedural risks, a FMEA was performed in September 2014. Forty-four failure modes were identified, among which six were given a moderate risk priority number (>15) (RPN; product of estimated occurrence, severity and detection). Specific corrective measures were then introduced and implemented, and a second evaluation performed six months later. The meticulous and careful application of such measures allowed the risks to be decreased along the whole protocol, by reducing their estimated occurrence and/or increasing detection possibilities.

Abnormally fertilized oocytes can result in healthy live births: improved genetic technologies for preimplantation genetic testing can be used to rescue viable embryos in in vitro fertilization cycles

OBJECTIVE To test whether abnormally fertilized oocyte (AFO)-derived blastocysts are diploid and can be rescued for clinical use. DESIGN Longitudinal-cohort study from January 2015 to September 2016 involving IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A). Ploidy assessment was incorporated whenever a blastocyst from a monopronuclear (1PN) or tripronuclear zygote (2PN + 1 smaller PN; 2.1 PN) was obtained. SETTING Private IVF clinics and genetics laboratories. PATIENT(S) A total of 556 women undergoing 719 PGT-A cycles. INTERVENTION(S) Conventional chromosome analysis was performed on trophectoderm biopsies by quantitative polymerase chain reaction. For AFO-derived blastocysts, ploidy assessment was performed on the same biopsy with the use of allele ratios for hetorozygous SNPs analyzed by means of next-generation sequencing (1:1 = diploid; 2:1 = triploid; loss of heterozygosity = haploid). Balanced-diploid 1PN- and 2.1PN-derived blastocysts were transferred in the absence of normally fertilized transferable embryos. MAIN OUTCOME MEASURE(S) Ploidy constitution and clinical value of AFO-derived blastocysts in IVF PGT-A cycles. RESULT(S) Of the 5,026 metaphase II oocytes injected, 5.2% and 0.7% showed 1PN and 2.1PN, respectively. AFOs showed compromised embryo development (P<.01). Twenty-seven AFO-derived blastocysts were analyzed for ploidy constitution. The 1PN-derived blastocysts were mostly diploid (n = 9/13; 69.2%), a few were haploid (n = 3/13; 23.1%), and one was triploid (n = 1/13; 7.7%). The 2.1PN-derived blastocysts were also mostly diploid (n = 12/14; 85.7%), and the remainder were triploid. Twenty-six PGT-A cycles resulted in one or more AFO-derived blastocysts (n = 26/719; 3.6%). Overall, eight additional balanced-diploid transferable embryos were obtained from AFOs. In three cycles, the only balanced-diploid blastocyst produced was from an AFO (n = 3/719; 0.4%). Three AFO-derived live births were achieved: one from a 1PN zygote and two from 2.1PN zygotes. CONCLUSION(S) Enhanced PGT-A technologies incorporating reliable ploidy assessment provide an effective tool to rescue AFO-derived blastocysts for clinical use.

Luteal phase anovulatory follicles result in the production of competent oocytes: intra-patient paired case-control study comparing follicular versus luteal phase stimulations in the same ovarian cycle

Abstract STUDY QUESTION Are the mean numbers of blastocysts obtained from sibling cohorts of oocytes recruited after follicular phase and luteal phase stimulations (FPS and LPS) in the same ovarian cycle similar? SUMMARY ANSWER The cohorts of oocytes obtained after LPS are larger than their paired-FPS-derived cohorts and show a comparable competence, thus resulting in a larger mean number of blastocysts. WHAT IS KNOWN ALREADY Three theories of follicle recruitment have been postulated to date: (i) the ‘continuous recruitment’ theory, (ii) the ‘single recruitment episode’ theory and (iii) the ‘wave’ theory. Yet, a clear characterization of this crucial biological process for human reproduction is missing. Recent advances implemented in in vitro fertilization (IVF), such as blastocyst culture, aneuploidy testing and vitrification, have encouraged clinicians to maximize the exploitation of the ovarian reserve through tailored stimulation protocols, which is crucial especially for poor prognosis patients aiming to conceive after IVF. LPS has been already successfully adopted to treat poor prognosis or oncological patients through Duostim, LPS-only or random-start ovarian stimulation approaches. Nevertheless, little, and mainly retrospective, evidence has been produced to support the safety of LPS in general. Feasibility of the LPS approach would severely question the classic ‘single recruitment episode’ theory of follicular development. STUDY DESIGN, SIZE, DURATION This case-control study was conducted with paired follicular phase- and luteal phase-derived cohorts of oocytes collected after stimulations in the same ovarian cycle (DuoStim) at two private IVF clinics between October 2015 and December 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included 188 poor prognosis patients undergoing DuoStim with preimplantation genetic testing for aneuploidies (PGT-A). FPS and LPS were performed with the same daily dose of recombinant-gonadotrophins in an antagonist protocol. Blastocyst culture, trophectoderm biopsy, vitrification and frozen-warmed euploid single blastocyst transfers were performed. The primary outcome was the mean number of blastocysts obtained per oocyte retrieval from paired-FPS- and LPS-derived cohorts (required sample size = 165 patients; power = 90%). Mean blastulation and euploidy rates were monitored, along with the number of oocytes, euploid blastocysts and clinical outcomes. MAIN RESULTS AND THE ROLE OF CHANCE Significantly fewer blastocysts were obtained after FPS than LPS (1.2 ± 1.1 vs. 1.6 ± 1.6, P < 0.01), due to fewer oocytes collected (3.6 ± 2.1 vs. 4.3 ± 2.8, P < 0.01) and a similar mean blastocyst rates per retrieval (33.1% ± 30.3% vs. 37.4% ± 30.8%, P = NS). The number of oocytes collected were correlated (R = 0.5, P < 0.01), while the blastocyst rates were uncorrelated among paired-FPS- and LPS-derived cohorts. Overall, a significantly lower chance of producing blastocyst(s) was reported after FPS than after LPS: 67.6% (n = 127/188, 95%CI: 60.3–74.1) vs. 77.1% (n = 145/188, 95%CI: 70.3–82.8; P = 0.05). The mean euploidy rates per retrieval were similar between FPS- and LPS-derived cohorts of oocytes (13.6% ± 22.8% vs. 16.3% ± 23.4%, P = NS). Therefore, on average fewer euploid blastocysts (0.5 ± 0.8 vs. 0.7 ± 1.0, P = 0.02) resulted from FPS. Similar ongoing-pregnancy/delivery rates were reported, to date, after FPS- and LPS-derived euploid single blastocyst transfers: 42.4% (n = 28/66, 95%CI: 30.5–55.2) vs. 53.8% (n = 35/65, 95%CI: 41.1–66.1; P = NS). LIMITATIONS, REASONS FOR CAUTION More studies need to be conducted in the future to confirm the safety of LPS, especially in terms of ovarian and follicular environment, as well as the clinical, peri-natal and post-natal outcomes. Here, we showed preliminary data suggesting a similar ongoing implantation/delivery rate (>22 weeks) between FPS- and LPS-derived euploid blastocysts, that need to be extended in the future, to populations other than poor prognosis patients and using approaches other than DuoStim together with a constant monitoring of the related peri-natal and post-natal outcomes. WIDER IMPLICATIONS OF THE FINDINGS These data, from a paired study design, highlight that LPS-derived oocytes are as competent as FPS-derived oocytes, thereby adding some evidence to support the use of LPS for poor prognosis and oncological patients and to question the ‘single recruitment episode’ theory of follicle recruitment. These findings also encourage additional studies of the basics of folliculogenesis, with direct clinical implications for the management of ovarian stimulation in IVF. TRIAL REGISTRATION None. STUDY FUNDING/COMPETING INTEREST(S) No external funds were used for this study and there are no conflicts of interest.

Impact of Maternal Age on Oocyte and Embryo Competence

The overall success of human reproduction, either spontaneously or after IVF, is highly dependent upon maternal age. The main reasons for age-related infertility include reduced ovarian reserve and decreased oocyte/embryo competence due to aging insults, especially concerning an increased incidence of aneuploidies and possibly decreased mitochondrial activity. Age-related chromosomal abnormalities mainly arise because of meiotic impairments during oogenesis, following flawed chromosome segregation patterns such as non-disjunction, premature separation of sister chromatids, or the recent reverse segregation. In this review, we briefly discuss the main mechanisms putatively impaired by aging in the oocytes and the deriving embryos. We also report the main strategies proposed to improve the management of advanced maternal age women in IVF: fertility preservation through oocyte cryopreservation to prevent aging; optimization of the ovarian stimulation and enhancement of embryo selection to limit its effects; and oocyte donation to circumvent its consequences.

Double Stimulation in the Same Ovarian Cycle (DuoStim) to Maximize the Number of Oocytes Retrieved From Poor Prognosis Patients: A Multicenter Experience and SWOT Analysis

A panel of experts known as the POSEIDON group has recently redefined the spectrum of poor responder patients and introduced the concept of suboptimal response. Since an ideal management for these patients is still missing, they highlighted the importance of tailoring the ovarian stimulation based on the chance of each woman to obtain an euploid blastocyst. Interestingly, a novel pattern of follicle recruitment has been defined: multiple waves may arise during a single ovarian cycle. This evidence opened important clinical implications for the treatment of poor responders. For instance, double stimulation in the follicular (FPS) and luteal phase (LPS) of the same ovarian cycle (DuoStim) is an intriguing option to perform two oocyte retrievals in the shortest possible time. Here, we reported our 2-year experience of DuoStim application in four private IVF centers. To date, 310 poor prognosis patients completed a DuoStim protocol and underwent IVF with blastocyst-stage preimplantation-genetic-testing. LPS resulted into a higher mean number of oocytes collected than FPS; however, their competence (i.e., fertilization, blastocyst, euploidy rates, and clinical outcomes after euploid single-embryo-transfer) was comparable. Importantly, the rate of patients obtaining at least one euploid blastocyst increased from 42.3% (n = 131/310) after FPS to 65.5% (n = 203/310) with the contribution of LPS. A summary of the putative advantages and disadvantages of DuoStim was reported here through a Strengths–Weaknesses–Opportunities–Threats analysis. The strengths of this approach make it very promising. However, more studies are needed in the future to limit its weaknesses, shed light on its putative threats, and realize its opportunities.

Intrauterine Insemination in Unexplained Female Infertility

Unexplained infertility (UI) is diagnosed when the standard work-up reveals normal semen analysis, normal uterus with bilateral tubal patency, and regular ovulatory cycles. The true prevalence is still debatable but it is generally accepted to affect about 20 % of infertile couples. The treatment of UI is empirical since the true reasons of the reproductive dysfunction(s) are unknown. Expectant management (EM) with proper instructions to time intercourse could be suggested as first step in the treatment algorithm, followed by intrauterine inseminations (IUIs), with or without controlled ovarian stimulation (COS). After failed cycles of IUI, assisted reproductive technologies as conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) are the proposed subsequent steps. The gonadotrophin-stimulated IUI cycles are considered a better option than natural cycles IUI. A recent Cochrane review showed that the addition of COS to IUI treatment improves live-birth rates (LBRs) in couples with UI. Likewise, a small but still statistically significant increase in pregnancy rate was found with IUI when compared with timed intercourse (TI) in stimulated cycles. The overall success rate in couples with UI is high. However, the criteria for deciding when to suggest IUI or IVF need to be investigated in randomized controlled trials.