Second stimulation in the same ovarian cycle: an option to fully-personalize the treatment in poor prognosis patients undergoing PGT-A.

PURPOSE: Our primary objective was to assess whether immediately undergoing a second stimulation in the same ovarian cycle (DuoStim) for advanced-maternal-age and/or poor-ovarian-reserve (AMA/POR) patients obtaining ≤ 3 blastocysts for preimplantation-genetic-testing-for-aneuploidies (PGT-A) is more efficient than the conventional-approach. METHODS: All AMA/POR patients obtaining ≤ 3 blastocysts after conventional-stimulation between 2017 and 2019 were proposed DuoStim, and 143 couples accepted (DuoStim-group) and were matched for the main confounders to 143 couples who did not accept (conventional-group). GnRH-antagonist protocol with recombinant-gonadotrophins and agonist trigger, intra-cytoplasmatic-sperm-injection (ICSI) with ejaculated sperm, PGT-A and vitrified-warmed euploid single-blastocyst-transfer(s) were performed. The primary outcome was the cumulative-live-birth-delivery-rate per intention-to-treat (CLBdR per ITT) within 1 year. If not delivering, the conventional-group had 1 year to undergo another conventional-stimulation. A cost-effectiveness analysis was also conducted. RESULTS: The CLBdR was 10.5% in the conventional-group after the first attempt. Only 12 of the 128 non-pregnant patients returned (165 ± 95 days later; drop-out = 116/128,90.6%), and 3 delivered. Thus, the 1-year CLBdR was 12.6% (N = 18/143). In the DuoStim-group, the CLBdR was 24.5% (N = 35/143; p = 0.01), 2 women delivered twice and 13 patients have other euploid blastocysts after a LB (0 and 2 in the conventional-group). DuoStim resulted in an incremental-cost-effectiveness-ratio of 23,303€. DuoStim was costlier and more effective in 98.7% of the 1000 pseudo-replicates generated through bootstrapping, and the cost-effectiveness acceptability curves unveiled that DuoStim would be more cost-effective than the conventional-approach at a willingness-to-pay threshold of 23,100€. CONCLUSIONS: During PGT-A treatments in AMA/POR women, DuoStim can be suggested in progress to rescue poor blastocyst yields after conventional-stimulation. It might indeed prevent drop-out or further aging between attempts.

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

RESEARCH QUESTION: Does maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified-warmed euploid single embryo transfer (SET)? DESIGN: Observational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified-warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5-25; n = 1392 performing 859 SET), underweight (BMI <18.5; n = 160 performing 112 SET) and overweight (BMI >25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. RESULTS: The m-ER per patient decreases as maternal BMI increases from 17 up to 22-23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: -0.6%, 95% confidence interval [CI]: -1.1 to -0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1-3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46-0.96, P = 0.03). CONCLUSION: These data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer.

Oocyte competence is independent of the ovulation trigger adopted: a large observational study in a setting that entails vitrified-warmed single euploid blastocyst transfer.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013-July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9-1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy.

The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.

STUDY QUESTION: Are the reproductive outcomes (clinical, obstetric and perinatal) different between follicular phase stimulation (FPS)- and luteal phase stimulation (LPS)-derived euploid blastocysts? SUMMARY ANSWER: No difference was observed between FPS- and LPS-derived euploid blastocysts after vitrified-warmed single embryo transfer (SET). WHAT IS KNOWN ALREADY: Technical improvements in IVF allow the implementation non-conventional controlled ovarian stimulation (COS) protocols for oncologic and poor prognosis patients. One of these protocols begins LPS 5 days after FPS is ended (DuoStim). Although, several studies have reported similar embryological outcomes (e.g. fertilization, blastulation, euploidy) between FPS- and LPS-derived cohort of oocytes, information on the reproductive (clinical, obstetric and perinatal) outcomes of LPS-derived blastocysts is limited to small and retrospective studies. STUDY DESIGN, SIZE, DURATION: Multicenter study conducted between October 2015 and March 2019 including all vitrified-warmed euploid single blastocyst transfers after DuoStim. Only first transfers of good quality blastocysts (≥BB according to Gardner and Schoolcraft's classification) were included. If euploid blastocysts obtained after both FPS and LPS were available the embryo to transfer was chosen blindly. The primary outcome was the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer in the two groups. To achieve 80% power (α = 0.05) to rule-out a 15% difference in the LBR, a total of 366 first transfers were required. Every other clinical, as well as obstetric and perinatal outcomes, were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Throughout the study period, 827 patients concluded a DuoStim cycle and among them, 339 did not identify any transferable blastocyst, 145 had an euploid blastocyst after FPS, 186 after LPS and 157 after both FPS and LPS. Fifty transfers of poor quality euploid blastocysts were excluded and 49 patients did not undergo an embryo transfer during the study period. Thus, 389 patients had a vitrified-warmed SET of a good quality euploid blastocyst (182 after FPS and 207 after LPS). For 126 cases (32%) where both FPS- and LPS-derived good quality blastocysts were available, the embryo transferred was chosen blindly with a 'True Random Number Generator' function where '0' stood for FPS-derived euploid blastocysts and '1' for LPS-derived ones (n = 70 and 56, respectively) on the website random.org. All embryos were obtained with the same ovarian stimulation protocol in FPS and LPS (GnRH antagonist protocol with fixed dose of rec-FSH plus rec-LH and GnRH-agonist trigger), culture conditions (continuous culture in a humidified atmosphere with 37°C, 6% CO2 and 5% O2) and laboratory protocols (ICSI, trophectoderm biopsy in Day 5-7 without assisted hatching in Day 3, vitrification and comprehensive chromosome testing). The women whose embryos were included had similar age (FPS: 38.5 ± 3.1 and LPS: 38.5 ± 3.2 years), prevalence of male factor, antral follicle count, basal hormonal characteristics, main cause of infertility and previous reproductive history (i.e. previous live births, miscarriages and implantation failures) whether the embryo came from FPS or LPS. All transfers were conducted after warming in an artificial cycle. The blastocysts transferred after FPS and LPS were similar in terms of day of full-development and morphological quality. MAIN RESULTS AND THE ROLE OF CHANCE: The positive pregnancy test rates for FPS- and LPS-derived euploid blastocysts were 57% and 62%, biochemical pregnancy loss rates were 10% and 8%, miscarriage rates were 15% and 14% and LBRs were 44% (n = 80/182, 95% CI 37-51%) and 49% (n = 102/207, 95% CI 42-56%; P = 0.3), respectively. The overall odds ratio for live birth (LPS vs FPS (reference)) adjusted for day of blastocyst development and quality, was 1.3, 95% CI 0.8-2.0, P = 0.2. Among patients with euploid blastocysts obtained following both FPS and LPS, the LBRs were also similar (53% (n = 37/70, 95% CI 41-65%) and 48% (n = 27/56, 95% CI 35-62%) respectively; P = 0.7). Gestational issues were experienced by 7.5% of pregnant women after FPS- and 10% of women following LPS-derived euploid single blastocyst transfer. Perinatal issues were reported in 5% and 0% of the FPS- and LPS-derived newborns, respectively. The gestational weeks and birthweight were similar in the two groups. A 5% pre-term delivery rate was reported in both groups. A low birthweight was registered in 2.5% and 5% of the newborns, while 4% and 7% showed high birthweight, in FPS- and LPS-derived euploid blastocyst, respectively. Encompassing the 81 FPS-derived newborns, a total of 9% were small and 11% large for gestational age. Among the 102 LPS-derived newborns, 8% were small and 6% large for gestational age. No significant difference was reported for all these comparisons. LIMITATIONS, REASONS FOR CAUTION: The LPS-derived blastocysts were all obtained after FPS in a DuoStim protocol. Therefore, studies are required with LPS-only, late-FPS and random start approaches. The study is powered to assess differences in the LBR per embryo transfer, therefore obstetric and perinatal outcomes should be considered observational. Although prospective, the study was not registered. WIDER IMPLICATIONS OF THE FINDINGS: This study represents a further backing of the safety of non-conventional COS protocols. Therefore, LPS after FPS (DuoStim protocol) is confirmed a feasible and efficient approach also from clinical, obstetric and perinatal perspectives, targeted at patients who need to reach the transfer of an euploid blastocyst in the shortest timeframe possible due to reasons such as cancer, advanced maternal age and/or reduced ovarian reserve and poor ovarian response. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Definition of a clinical strategy to enhance the efficacy, efficiency and safety of egg donation cycles with imported vitrified oocytes.

STUDY QUESTION: Which is the most suitable clinical strategy in egg donation IVF cycles conducted with imported donated vitrified oocytes? SUMMARY ANSWER: The importation, and allocation, of at least eight vitrified eggs per couple during an egg donation cycle is associated with a high cumulative live birth delivery rate per cycle, as well as the confident adoption of a single blastocyst transfer strategy to minimize the risk of multiple pregnancies. WHAT IS KNOWN ALREADY: IVF using donor eggs is commonly used worldwide to treat women who are unable to conceive with their own oocytes. In 2014, the Constitutional Court (n.162/2014) gave permission for gamete donation to be allowed for ART in Italy. Initially recommended as a therapeutic approach for premature ovarian insufficiency, the use of donated oocytes has become more and more common. In countries such as Italy, fresh oocyte donation is theoretically possible, but practically impossible due to the lack of donors. In fact, the Italian law does not allow reimbursement to the young women, who can only voluntarily donate their eggs. Therefore, Italian IVF centers have established several collaborations with international oocyte cryo-banks. The most popular workflow involves the importation of donated oocytes that have been vitrified. However, recent evidence has questioned the overall efficacy of such an approach. This is because detrimental effects arising from oocyte vitrification and warming might reduce the number of eggs available for insemination, with a consequential reduction in the achievable live birth rate per cycle. STUDY DESIGN, SIZE, DURATION: This was a longitudinal cohort study, conducted between October 2015 and December 2018 at two private IVF centers. Overall, 273 couples were treated (mean maternal age: 42.5 ± 3.5 years, range: 31-50 years; mean donor age: 25.7 ± 4.2, 20-35 years) with oocytes purchased from three different Spanish egg banks. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed an overall analysis, as well as several sub-analyses clustering the data according to the year of treatment (2015-2016, 2017 or 2018), the number of warmed (6, 7, 8 or 9) and surviving oocytes (≤4, 5, 6, 7, 8 or 9) and the cycle strategy adopted (cleavage stage embryo transfer and vitrification, cleavage stage embryo transfer and blastocyst vitrification, blastocyst stage embryo transfer and vitrification). This study aimed to create a workflow to maximize IVF efficacy, efficiency, and safety, during egg donation cycles with imported vitrified oocytes. The primary outcome was the cumulative live birth delivery rate among completed cycles (i.e. cycles where at least a delivery of a live birth was achieved, or no embryo was produced/left to transfer). All cycles, along with their embryological, obstetric and neonatal outcomes, were registered and inspected. MAIN RESULTS AND THE ROLE OF CHANCE: The survival rate after warming was 86 ± 16%. When 6, 7, 8 and 9 oocytes were warmed, 94, 100, 72 and 70% of cycles were completed, resulting in 35, 44, 69 and 59% cumulative live birth delivery rates per completed cycle, respectively. When ≤4, 5, 6, 7, 8 and 9 oocytes survived, 98, 94, 85, 84, 66 and 68% of cycles were completed, resulting in 16, 46, 50, 61, 76 and 60% cumulative live birth delivery rates per completed cycle, respectively. When correcting for donor age, and oocyte bank, in a multivariate logistic regression analysis, warming eight to nine oocytes resulted in an odds ratio (OR) of 2.5 (95% CI: 1.07-6.03, P = 0.03) for the cumulative live birth delivery rate per completed cycle with respect to six to seven oocytes. Similarly, when seven to nine oocytes survived warming, the OR was 2.7 (95% CI: 1.28-5.71, P < 0.01) with respect to ≤6 oocytes. When cleavage stage embryos were transferred, a single embryo transfer strategy was adopted in 17% of cases (N = 28/162); the live birth delivery rate per transfer was 26% (n = 43/162), but among the pregnancies to term, 28% involved twins (n = 12/43). Conversely, when blastocysts were transferred, a single embryo transfer strategy was adopted in 96% of cases (n = 224/234) with a 30% live birth delivery rate per transfer (N = 70/234), and the pregnancies to term were all singleton (n = 70/70). During the study period, 125 babies were born from 113 patients. When comparing the obstetric outcomes for the cleavage and blastocyst stage transfer strategies, the only significant difference was the prevalence of low birthweight: 34 versus 5%, respectively (P < 0.01). However, several significant differences were identified when comparing singleton with twin pregnancies; in fact, the latter resulted in a generally lower birthweight (mean ± SD: 3048 ± 566 g versus 2271 ± 247 g, P < 0.01), a significantly shorter gestation (38 ± 2 versus 36 ± 2 weeks, P < 0.01), solely Caesarean sections (72 versus 100%, P = 0.02), a higher prevalence of low birthweight (8 versus 86%, P < 0.01), small newborns for gestational age (24 versus 57%, P = 0.02) and preterm births (25 versus 86%, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This retrospective study should now be confirmed across several IVF centers and with a greater sample size in order to improve the accuracy of the sub-analyses. WIDER IMPLICATIONS OF THE FINDINGS: Single blastocyst transfer is the most suitable approach to achieve high success rates per procedure, thereby also limiting the obstetric complications that arise from twin pregnancies in oocyte donation programs. In this regard, the larger the cohort of imported donated vitrified oocytes, the more efficient the management of each cycle. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Luteal phase after conventional stimulation in the same ovarian cycle might improve the management of poor responder patients fulfilling the Bologna criteria: a case series.

OBJECTIVE: To assess the clinical contribution of luteal-phase stimulation (LPS) to follicular-phase stimulation (FPS) in a single ovarian cycle (DuoStim) for poor responder patients fulfilling the Bologna criteria. DESIGN: Observational study (years 2015-2017) including women satisfying ≥2 of the following characteristics: maternal age ≥40 years and/or ≤3 oocytes retrieved after previous conventional stimulation and/or reduced ovarian reserve (i.e., antral follicle count <7 follicles or antimüllerian hormone <1.1 ng/mL). The LPS was started regardless of the outcome of the FPS. SETTING: Private in vitro fertilization center. PATIENT(S): A total of 100 of 297 patients fulfilling the Bologna criteria chose to undergo DuoStim. INTERVENTION(S): The FPS and LPS with the same antagonist protocol and agonist trigger, intracytoplasmic sperm injection with ejaculated sperm, preimplantation genetic testing for aneuploidies, and vitrified-warmed euploid single blastocyst transfer. MAIN OUTCOME MEASURE(S): The contribution of LPS to the cumulative live birth rate (CLBR) per intention-to-treat (ITT). RESULT(S): Patients (100) underwent FPS (maternal age, 42.1 ± 1.4 y; previous in vitro fertilization cycles with ≤3 collected oocytes, 0.7 ± 0.9; antral follicle count, 3.8 ± 1.2 follicles; and antimüllerian hormone, 0.56 ± 0.3 ng/mL). Ninety-one patients completed DuoStim. All patients were included in the analysis. More oocytes were obtained after LPS with similar developmental and chromosomal competence as paired FPS-derived ones. The CLBR per ITT increased from 7% after FPS to 15% after DuoStim. Conversely, the CLBR per ITT among the 197 patients that chose a conventional controlled ovarian stimulation strategy was 8%, as only 17 patients who were not pregnant returned for a second stimulation after the first attempt (drop-out rate, 81%). CONCLUSION(S): The LPS-derived oocytes increased the CLBR per ITT in a single ovarian cycle in patients fulfilling the Bologna criteria. The DuoStim strategy is promising to manage this thorny population of patients, especially to avoid discontinuation after a first failed attempt.

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.

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.

Progesterone for preparation of the endometrium for frozen-thawed blastocyst transfer in vitro fertilization cycles: a prospective study on patients' opinions on a new subcutaneous formulation.

We aimed to evaluate patients' perspectives on a progesterone subcutaneous formulation for endometrial preparation for frozen-thawed blastocyst transfer. In this prospective study, women with at least one experience with vaginal progesterone, undergone endometrial preparation with oral estradiol valerate and daily subcutaneous progesterone administered from the fifth day before the transfer until the day of the beta-hCG test. Patients completed three questionnaires, at enrollment (Q1), for gathering information on the experience with vaginal treatment and expectations about the subcutaneous route and then at the time of the transfer (Q2) and eight days later (Q3). Main outcome measures were patients' opinions on comfort, ease of use, convenience, overall satisfaction, level of anxiety and pain associated with the administration of subcutaneous progesterone in comparison with their previous experience. Sixty-nine women completed the questionnaires. All vaginal versus subcutaneous comparisons were significantly in favor of the subcutaneous route. When comparing patients' expectations at Q1 with patients' opinions at Q2 and Q3, all evaluations, except for one, demonstrated that the patient's positive expectation was confirmed after 5 and 13 days of treatment. In conclusion, in women with previous experience with vaginal progesterone, the subcutaneous route was associated with significantly increased acceptance.

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.

Pre-implantation genetic testing in ART: who will benefit and what is the evidence?

Pre-implantation genetic diagnosis for aneuploidy testing (PGD-A) is a tool to identify euploid embryos during IVF. The suggested populations of patients that can benefit from it are infertile women of advanced maternal age, with a history of recurrent miscarriages and/or IVF failures. However, a general consensus has not yet been reached.After the clinical failure of its first version based on cleavage stage biopsy and 9 chromosome-FISH analysis, PGD-A is currently performed by 24 chromosome screening techniques on trophectoderm (TE) biopsies. This approach has been clearly demonstrated to involve a higher clinical efficiency with respect to the standard care, in terms of sustained pregnancy rate per transfer and lower miscarriage rate. However, data about PGD-A efficacy calculated on a per intention-to-treat basis, as well as an analysis of its cost-effectiveness, are still missing.TE biopsy is a safe and extensively validated approach with low biological and technical margin of error. Firstly, the prevalence of mosaic diploid/aneuploid blastocysts is estimated to be between 0 and 16 %, thus largely tolerable. Secondly, all the comprehensive chromosome screening (CCS) technologies adapted to, or designed to conduct PGD-A are highly concordant, and qPCR in particular has been proven to show the lowest false positive error rate (0.5 %) and a clinically recognizable error rate per blastocyst of just 0.21 %.In conclusion, there is a sufficient body of evidence to support the clinical application of CCS-based PGD-A on TE biopsies. The main limiting factor is the need for a high-standard laboratory to conduct blastocyst culture, biopsy and vitrification without impacting embryo viability.

Follicular versus luteal phase ovarian stimulation during the same menstrual cycle (DuoStim) in a reduced ovarian reserve population results in a similar euploid blastocyst formation rate: new insight in ovarian reserve exploitation.

OBJECTIVE: To compare the euploid blastocyst formation rates obtained after follicular phase (FP) versus luteal phase (LP) stimulation performed in the same menstrual cycle in a preimplantation genetic diagnosis for aneuploidy testing (PGD-A) program in patients with reduced ovarian reserve. DESIGN: Prospective paired noninferiority observational study. SETTING: Private infertility program. PATIENT(S): Forty-three reduced ovarian reserve patients undergoing a PGD-A. INTERVENTION(S): Both FP and LP stimulations using follicle-stimulating hormone and luteinizing hormone in combination with gonadotropin-releasing hormone (GnRH) antagonist starting on day 2 of the cycle and 5 days after the first oocyte retrieval, respectively, where GnRH agonist was used for both FP and LP ovulation triggering; a trophectoderm biopsy quantitative polymerase chain reaction-based PGD-A strategy; and single euploid blastocyst transfers during a subsequent natural cycle. PRIMARY OUTCOME MEASURE: euploid blastocyst rate per injected metaphase 2 (MII) oocyte; secondary outcome measures: number of cumulus-oocyte complexes (COCs), MII oocytes, and blastocysts. RESULT(S): Patients with an antimüllerian hormone level of <1.5 ng/mL, antral follicle count of <6 follicles, and/or <5 oocytes retrieved in a previous cycle were included. No statistically significant differences were found in the number of retrieved COCs (5.1 ± 3.4 vs. 5.7 ± 3.3), MII oocytes (3.4 ± 1.9 vs. 4.1 ± 2.5), or biopsied blastocysts per stimulated cycle (1.2 ± 1.2 vs. 1.4 ± 1.7) from FP versus LP stimulation, respectively. No differences were observed in the euploid blastocyst rate calculated either per biopsied blastocyst (46.9% vs. 44.8%) or injected MII oocyte (16.2% vs. 15.0%). CONCLUSION(S): Stimulation with an identical protocol in the FP and LP of the same menstrual cycle resulted in a similar number of blastocysts in patients with reduced ovarian response. The LP stimulation statistically significantly contributed to the final transferable blastocyst yield, thus increasing the number of patients undergoing transfer per menstrual cycle.

Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study.

STUDY QUESTION: Is an elective single-embryo transfer (eSET) policy an efficient approach for women aged >35 years when embryo selection is enhanced via blastocyst culture and preimplantation genetic screening (PGS)? SUMMARY ANSWER: Elective SET coupled with enhanced embryo selection using PGS in women older than 35 years reduced the multiple pregnancy rates while maintaining the cumulative success rate of the IVF programme. WHAT IS KNOWN ALREADY: Multiple pregnancies mean an increased risk of premature birth and perinatal death and occur mainly in older patients when multiple embryos are transferred to increase the chance of pregnancy. A SET policy is usually recommended in cases of good prognosis patients, but no general consensus has been reached for SET application in the advanced maternal age (AMA) population, defined as women older than 35 years. Our objective was to evaluate the results in terms of efficacy, efficiency and safety of an eSET policy coupled with increased application of blastocyst culture and PGS for this population of patients in our IVF programme. STUDY DESIGN, SIZE, DURATION: In January 2013, a multidisciplinary intervention involving optimization of embryo selection procedure and introduction of an eSET policy in an AMA population of women was implemented. This is a retrospective 4-year (January 2010-December 2013) pre- and post-intervention analysis, including 1161 and 499 patients in the pre- and post-intervention period, respectively. The primary outcome measures were the cumulative delivery rate (DR) per oocyte retrieval cycle and multiple DR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Surplus oocytes and/or embryos were vitrified during the entire study period. In the post-intervention period, all couples with good quality embryos and less than two previous implantation failures were offered eSET. Embryo selection was enhanced by blastocyst culture and PGS (blastocyst stage biopsy and 24-chromosomal screening). Elective SET was also applied in cryopreservation cycles. MAIN RESULTS AND THE ROLE OF CHANCE: Patient and cycle characteristics were similar in the pre- and post-intervention groups [mean (SD) female age: 39.6 ± 2.1 and 39.4 ± 2.2 years; range 36-44] as assessed by logistic regression. A total of 1609 versus 574 oocyte retrievals, 937 versus 350 embryo warming and 138 versus 27 oocyte warming cycles were performed in the pre- and post-intervention periods, respectively, resulting in 1854 and 508 embryo transfers, respectively. In the post-intervention period, 289 cycles were blastocyst stage with (n = 182) or without PGS (n = 107). A mean (SD) number of 2.9 ± 1.1 (range 1-4) and 1.4 ± 0.8 (range 1-3) embryos were transferred pre- and post-intervention, respectively (P < 0.01) and similar cumulative clinical pregnancy rates per transfer and per cycle were obtained: 26.8, 30.9% and 29.7, 26.3%, respectively. The total DR per oocyte retrieval cycle (21.0 and 20.4% pre- and post-intervention, respectively) defined as efficacy was not affected by the intervention [odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.7-1.1; P = 0.23]. However, a significantly increased live birth rate per transferred embryo (defined as efficiency) was observed in the post-intervention group 17.0 versus 10.6% (P < 0.01). Multiple DRs decreased from 21.0 in the preintervention to 6.8% in the post-intervention group (OR = 0.3. 95% CI = 0.1-0.7; P < 0.01). LIMITATIONS, REASONS FOR CAUTION: In this study, the suitability of SET was assessed in individual women on the basis of both clinical and embryological prognostic factors and was not standardized. For the described eSET strategy coupled with an enhanced embryo selection policy, an optimized culture system, cryopreservation and aneuploidy screening programme is necessary. WIDER IMPLICATIONS OF THE FINDINGS: Owing to the increased maternal morbidity and perinatal complications related to multiple pregnancies, it is recommended to extend the eSET policy to the AMA population. As shown in this study, enhanced embryo selection procedures might allow a reduction in the number of embryos transferred and the number of transfers to be performed without affecting the total efficacy of the treatment but increasing efficiency and safety. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: None.

Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.

STUDY QUESTION: What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients? SUMMARY ANSWER: Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for PGS. WHAT IS KNOWN ALREADY: In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented. STUDY DESIGN, SIZE, DURATION: This is a prospective longitudinal cohort study performed between October 2009 and August 2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding blastomeres and trophectoderm (TE) samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples undergoing IVF cycles with PGS where the female partner was older than 40 years and with a good response to controlled ovarian stimulation (>10 MII oocytes retrieved) were enrolled into the study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor infertility (motile sperm count of <500 000/ml after preparation of a fresh ejaculate). The PBs, blastomere and TE samples were sequentially biopsied and analyzed by array comparative genomic hybridization (aCGH). The analysis of chromosome segregation patterns was performed to infer the origin of aneuploidy and to investigate the diagnostic accuracy of both PB and cleavage-stage PGS strategies. MAIN RESULTS AND THE ROLE OF CHANCE: Twenty-one sets of complete data (PB1/PB2/blastomere/TE) including 84 aCGH experiments showed a pattern of multiple meiotic errors typically caused by sister chromatid separation errors and predominantly arising in the second meiotic division. Twenty-two of the 24 (91.7%) errors in the first meiotic division arose as a consequence of premature sister chromatid predivision. In half of these cases, the second meiotic division resulted in a balancing chromosome segregation event producing a normal female complement for that chromosome in the resulting embryo. Overall, only 62 out of 78 (79.5%) of the abnormal meiotic segregations had errors in the either one or both PBs consistent with the aneuploidies observed in their resulting embryos. Ten of the 21 (47.6%) embryos had aneuploidies other than female meiotic-derived ones, most of which detected on Day 3 and confirmed on Day 5 or 6 of embryo development (20/25) with chromosomal loss being three times more frequent than gains. Notably, as high as 20% of female-derived aneuploidies detected on PBs and confirmed on Day 3 were rescued at the blastocyst stage, mainly as a result of diploidization of trisomic chromosomes. On a per chromosome basis, the sensitivity in predicting blastocyst chromosomal complement was significantly lower for PB approach, 61.7%, compared with blastomeres analysis, 86.4% (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The study was limited to the analysis of oocytes and embryos from AMA patients. Thus, these findings apply only to this patient group. Comparisons with other patient populations including patients with different indications for PGS should be made in future research. In addition, higher resolution and/or more accurate chromosomal screening tests could be used in future studies to corroborate the current findings. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide critical insights into the mechanisms causing errors during female meiosis and the preimplantation embryo development period to improve the design and treatment outcome of PGS.

The worldwide frozen embryo reservoir: methodologies to achieve optimal results.

Cryopreservation of the human embryo has been successfully achieved at the zygote (day 1), cleavage (day 2/3), and blastocyst (day 5) stages; however, each stage presents specific advantages and disadvantages. During the past decades, two major methods have been applied: slow freezing (equilibrium procedure) and vitrification (nonequilibrium procedure). The overwhelming majority of published data prove that the latest vitrification methods induce less cellular trauma and are a more effective cryopreservation technique of human embryos than any other versions of slow freezing. For this reason, fragmented and slow-cleaving embryos that normally would not be recommended may be revaluated for cryopreservation by using the vitrification method. Furthermore, if laser-assisted necrotic blastomere removal is associated with the slow-freezing/thawing procedure, good clinical results can be obtained. Finally, the most proper embryo cleavage stage at which to perform cryopreservation has to be assessed according to clinical indications and laboratory experience.

Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program.

BACKGROUND: Recent advancement of minimum volume vitrification methods has resulted in a dramatic increase in the efficiency of the process. The aim of this study was to estimate the cumulative reproductive outcome of a cohort of infertile couples undergoing ICSI and oocyte vitrification in restrictive legal conditions, where only a limited number of oocytes could be inseminated per cycle and embryo selection and cryopreservation were forbidden. METHODS: In this prospective longitudinal cohort study, the cumulative ongoing pregnancy rates obtained by the insemination of fresh and vitrified oocytes from the same cohort were calculated as primary outcome measures. Moreover, the effect of basal and cycle characteristics on clinical outcomes were assessed. RESULTS: Between September 2008 and May 2009, 182 ICSI cycles were performed where oocyte vitrification was possible. A total of 104 first and 11 second oocyte warming cycles were then performed in non-pregnant patients of the same cohort. The overall ongoing pregnancy rates obtained in the fresh, and first and second warming cycles were 37.4, 25.0 and 27.3%, respectively. The overall cumulative ongoing clinical pregnancy rate observed per stimulation cycle was 53.3%. Maternal age was the only characteristic found to influence the reproductive outcome, with an inverse correlation between the age >40 and the ongoing pregnancy rates (P = 0.04, by Cox regression analysis). CONCLUSIONS: High cumulative ongoing pregnancy rates can be obtained with transfers of embryos derived from fresh and cryopreserved oocytes in a typical infertile population. Female age significantly affects outcomes in this system.

Embryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study.

BACKGROUND: A successful oocyte cryopreservation programme is of utmost importance where a limited number of oocytes can be inseminated per cycle, to overcome legal and ethical issues related to embryo storage, for oocyte donation programmes and for fertility preservation (especially for cancer patients). Vitrification has been recently proposed as an effective procedure for this purpose. METHODS: In order to validate the effectiveness of oocyte vitrification a non-inferiority trial was started on sibling metaphase II (MII) oocytes. To demonstrate the non-inferiority based on an absolute difference of 17% in the fertilization rate per sibling oocyte, a minimum of 222 oocytes were required. After oocyte denudation, MII oocytes with normal morphology were randomly allocated to fresh ICSI insemination or to vitrification procedure. If pregnancy was not obtained a subsequent ICSI cycle was performed with warmed oocytes of the same cohort. In both groups, three oocytes were inseminated per cycle by ICSI procedure. Primary end-points were fertilization rates calculated per warmed and per injected oocytes. Secondary end-points were zygote and embryo morphology. RESULTS: A total of 244 oocytes were involved in this study. Of the 120 fresh sibling oocytes inseminated, 100 were fertilized (83.3%). Survival rate of sibling vitrified oocytes was 96.8% (120/124 oocytes). Fertilization rate after ICSI was 76.6% (95/124) per warmed oocyte and 79.2% (95/120) per survived/inseminated oocyte. No statistical difference in fertilization rates was observed between the two groups when calculated per sibling oocytes (absolute difference -6.73%; OR: 0.65; 95% CI = 0.33-1.29; P = 0.20) and per inseminated oocyte (absolute difference -4.17%; OR: 0.76; 95% CI = 0.37-1.53; P = 0.50). Embryo development was also similar in both treatment groups up till Day 2. The percentage of excellent quality embryos was 52.0% (52/100) in the fresh group and 51.6% (49/95) in the vitrification group (absolute difference -0.43%; OR: 0.98; 95% CI = 0.53-1.79; P = 0.9). The mean age of the 40 patients included in this study was 35.5 +/- 4.8 years (range 26-42). Fifteen clinical pregnancies were obtained in the vitrification cycles of 39 embryo transfers performed (37.5% per cycle, 38.5% per embryo transfer), with an implantation rate of 20.2% (19/94). Three spontaneous miscarriages occurred (20%). Twelve pregnancies are ongoing (30.0% per cycle, 30.8% per embryo transfer) beyond 12 weeks of gestation. CONCLUSIONS: Our results indicate that oocyte vitrification procedure followed by ICSI is not inferior to fresh insemination procedure, with regard to fertilization and embryo developmental rates. Moreover, ongoing clinical pregnancy is compatible with this procedure, even with a restricted number of oocytes available for insemination. The promising clinical results obtained, in a population of infertile patients, need to be confirmed on a larger scale. CLINICAL TRIALS REGISTRATION NUMBER: iSRCTN60158641.

Outlook: Roles of FSH and LH during the follicular phase: insight into natural cycle IVF.

A mounting interest in natural cycle IVF has challenged the medical community to better understand the mechanisms controlling the follicular phase and ovulation in particular, in an effort to optimize this procedure and its outcome. For practical reasons, the advancement of the follicular phase in the menstrual cycle is commonly timed according to the onset of last menses. However, this precludes knowing when the follicular phase truly begins and hampers the possibility of optimizing timing of late follicular-phase events, notably, the triggering of ovulation. Clinicians, therefore, use surrogate markers of follicular maturation, such as oestrogen production and follicular size. Because it is impossible to identify the low-amplitude intercycle basal FSH signal, efforts have reverted toward controlling when it takes place, either with exogenous oestrogen or with oral contraceptives. In the late follicular phase, the occurrence of LH surge results from a balance between the opposite effects of rising oestrogen concentrations, which favour the LH surge, and the opposing effects mediated by the gonadotrophin surge-attenuating factor, a peptide of ovarian origin. This review looks into the mechanisms that control these two hinges of the follicular phase, the basal FSH signal and LH surge, in the context of optimizing natural cycle IVF.