Seven Years of Vitrified Blastocyst Transfers: Comparison of 3 Preparation Protocols at a Single ART Center.

Introduction: Frozen-thawed embryo transfers (FET) have become a standard practice to increase cumulative pregnancy rates, however, the choice of the best preparation protocol remains a matter of debate. Design: Retrospective analysis of clinical pregnancy (CPR) and live birth rate (LBR) of FET in natural cycles (NC-FET), modified natural cycles with hCG-triggered ovulation (mNC-FET), and hormonal artificial replacement (AR-FET). Materials and Methods: For natural cycles, patients were monitored by ultrasound to evaluate the dominant follicle and by urinary LH kits (NC-FET). When the endometrial thickness reached at least 7 mm and the dominant follicle 16-20 mm, hCG was administered in absence of urinary LH surge (mNC-FET). Embryo thawing and transfer was planned 7 days after LH surge or hCG administration. For the AR-FET, oral estradiol valerate was administered from day 2 of menstrual cycle until endometrial thickness reached at least 7 mm and transfer was planned after 5 days of vaginal progesterone start. Only single vitrified blastocyst transfers were included. Results: In total 2,895 transfers were performed of which 561 (19.4%) carried out with NC-FET, 1,749 (60.4%) with mNC-FET and 585 (20.2%) with AR-FET. CPRs were 32.62, 43.05, and 37.26%, respectively. LBR were 24.06, 33.56, and 25.81%, respectively. A statistically significant (p < 0.001) higher LBR for mNC-FET vs. NC-FET (OR 0.49-0.78) and AR-FET (OR 0.47-0.74) was observed. A higher ectopic pregnancy rate (p = 0.002) was observed in NC-FET (3.28%) than in AR-FET (1.83%) and mNC-FET (0.40%). A higher abortion rate (p = 0.031) in pregnancies <12 weeks was observed in AR-FET (27.52%) than in NC-FET (19.67%) and in mNC-FET (19.39%). At Post hoc analysis only female age (OR 0.91-0.95), antimullerian hormone (AMH) (OR 1.01-1.07) and mNC-FET (OR 1.39-1.98) were statically significant prognostic factors for LBRs. Conclusions: These results demonstrate a superior CPR and LBR following FET in hCG-triggered ovulation cycles compared to NC and AR-FET, a higher ectopic pregnancy rate in NC-FET and a higher abortion rate in pregnancies <12 weeks in AR-FET. However, these data need to be confirmed in randomized and prospective studies before definitive conclusions can be drawn. Clinicaltrials.gov ID: NCT03581422.

Preimplantation Genetic Testing for Aneuploidy Improves Clinical, Gestational, and Neonatal Outcomes in Advanced Maternal Age Patients Without Compromising Cumulative Live-Birth Rate.

PURPOSE: To report the effects of blastocyst stage aneuploidy testing on clinical, gestational, and neonatal outcomes for patients of advanced maternal age undergoing IVF. METHODS: This is a single-center observational-cohort study with 2 years follow-up. The study includes a total of 2538 couples undergoing 2905 egg collections (control group), 308 (PGT-A), and 106 (drop-out group, consenting for PGT-A but withdrawing due to poor embryological outcome) RESULTS: Compared with control group, PGT-A showed improved clinical outcomes (live-birth rate per transferred embryo, LBR 40.3% vs 11.0%) and reduced multiple pregnancy rate (MPR, 0% vs 11.1%) and pregnancy loss (PL, 3.6% vs 22.6%). Drop-out group showed the worst clinical outcomes suggesting that abandoning PGT-A due to poor response to ovarian stimulation is not a favorable option. Cytogenetic analysis of product of conceptions and CVS/amniocentesis showed higher aneuploid pregnancy rates for control group regardless of embryo transfer strategy (0%, 17.9%, and 19.9%, for PGT-A, control day 5 and day 3, respectively). Multivariate analysis showed no negative impact of PGT-A-related interventions on cumulative delivery rate (26.3%, 95% CI 21.5-31.6 vs 24.0%, 95% CI 22.5-25.6 for PGT-A and control, respectively) and on neonatal outcomes. CONCLUSION: PGT-A improves clinical outcomes, particularly by reducing pregnancy loss and chromosomally abnormal pregnancy for patients of advanced maternal age, with no major impact on cumulative live-birth rate (CLBR) per egg retrieval.

Oocyte Cryopreservation in Oncological Patients: Eighteen Years Experience of a Tertiary Care Referral Center.

Objective: The aim of the present study is to report our experience on elective women fertility preservation before cancer treatment. Study Design: This is a single-center retrospective observational study, including all patients who underwent elective fertility preservation before oncological treatment between January 2001 and March 2019 at our Institute. Results: Of a total of 568 women who received fertility counseling, 244 (42.9%) underwent 252 oocyte retrieval cycles after controlled ovarian stimulation for cryopreservation. The majority of patients were diagnosed with breast cancer (59.9%), followed by women affected by Hodgkin's and non-Hodgkin's lymphoma (27.4%). A minority comprised patients diagnosed with other malignancies that affected soft tissues (2.8%), ovary borderline type (2.4%), digestive system (1.6%), leukemia (1.6%), uterine cervix (1.2%). The remaining 3.1% were affected by other cancer types. The mean age of the cohort was 31.3 ± 6.4 years and the mean oocyte retrieval was 13.5± 8.4. Of 11 women who returned to attempt a pregnancy, three performed two thawed cycles. We obtained four pregnancies from 24 embryo transfers (Pregnancy Rate 36.4% for couple): two miscarriages and two live births. Overall, 95.7% of oocytes are still in storage. Conclusions: A close collaboration between Cancer and Fertility Center in a tertiary care hospital is essential to provide a good health service in oncological patients. Offering fertility preservation is no longer considered optional and must be included in every therapeutic program for women who receive an oncological diagnosis in their reproductive age. Oocyte cryopreservation appears to be a good opportunity for fertility preservation. Our results, although they are obtained in a small sample, are encouraging, even if only 4.5% of patients returned to use their gametes.

An Observational Retrospective Cohort Trial on 4,828 IVF Cycles Evaluating Different Low Prognosis Patients Following the POSEIDON Criteria.

Objective: To study the actual controlled ovarian stimulation (COS) management in women with suboptimal response, comparing clinical outcomes to the gonadotropins consume, considering potential role of luteinizing hormone (LH) addition to follicle-stimulating hormone (FSH). Design: Monocentric, observational, retrospective, real-world, clinical trial on fresh intra-cytoplasmic sperm injection (ICSI) cycles retrieving from 1 to 9 oocytes, performed at Humanitas Fertility Center from January 1st, 2012 to December 31st, 2015. Methods: COS protocols provided gonadotropin releasing-hormone (GnRH) agonist long, flare-up, short and antagonist. Both recombinant and urinary FSH were used for COS and LH was added according to the clinical practice. ICSI outcomes considered were: gonadotropins dosages; total, mature, injected and frozen oocytes; cumulative, transferred and frozen embryos; implantation rate; pregnancy, delivery and miscarriage rates. Outcomes were compared according to the gonadotropin regimen used during COS. Results: Our cohort showed 20.8% of low responders, defined as 1-3 oocytes retrieved and 79.2% of "suboptimal" responders, defined as 4-9 oocytes retrieved. According to recent POSEIDON stratification, cycles were divided in group 1 (6.9%), 2 (19.8%), 3 (11.7%), and 4 (61.5%). The cohort was divided in 3 groups, according to the gonadotropin's regimen. Women treated with FSH plus LH showed worst prognostic factors, in terms of age, basal FSH, AMH, and AFC. This difference was evident in suboptimal responders, whereas only AMH and AFC were different among treatment groups in low responders. Although a different result, in terms of oocytes and embryos detected, major ICSI outcomes (i.e., pregnancy and delivery rates) were similar among groups of COS treatment. Outcomes were significantly different among Poseidon groups. Implantation, pregnancy and delivery rates were significantly higher in Poseidon group 1 and progressively declined in other POSEIDON groups, reaching the worst percentage in group 4. Conclusions: In clinical practice, women with worst prognosis factors are generally treated with a combination of LH and FSH. Despite low prognosis women showed a reduced number of oocytes retrieved, the final ICSI outcome, in terms of pregnancy, is similarly among treatment group. This result suggests that the LH addition to FSH during COS could improve the quality of oocytes retrieved, balancing those differences that are evident at baseline. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03290911.

Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience.

STUDY QUESTION: Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails? SUMMARY ANSWER: The risk of inconclusive chromosomal-assessment after trophectoderm biopsy was 2.5% but a further biopsy and vitrification-warming appeared not to impair the competence of euploid blastocysts. WHAT IS KNOWN ALREADY: The increasing implementation of multicell trophectoderm biopsy has significantly reduced the risk of inconclusive diagnosis after preimplantation-genetic-testing (PGT). Yet, few reports have defined the variables that influence the risk of failure or described the technical and clinical outcomes after re-biopsy. STUDY DESIGN, SIZE, DURATION: Retrospective multicenter study involving 8990 blastocyst biopsies conducted between April 2013 and September 2017 at six IVF centers but analyzed at a single genetic laboratory. A total of 206 blastocysts were successfully re-biopsied after warming and re-expansion, then re-vitrified. And 49 of these blastocysts were diagnosed euploid and used in single-embryo-transfers (SETs). Logistic regression analyses were conducted. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 3244 PGT-for-aneuploidies (PGT-A) cycles with a freeze-all approach, vitrification and qPCR-based analysis were performed by 2687 consenting couples. DNA amplification failure (AF) or non-concurrent data resulted in inconclusive diagnoses. In case of DNA amplification, the cellularity of the biopsy was estimated according to a previously validated method. Euploid SETs were performed. Clinical pregnancy, miscarriage, live birth rates (LBR) and perinatal outcomes were monitored. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 2.5% of trophectoderm biopsies resulted in an inconclusive diagnosis (N = 228/8990). Specifically, 2% (N = 176/8990) resulted in AF and 0.5% (N = 52/8990) in non-concurrent results. The only parameters significantly associated with inconclusive diagnoses were the IVF center and the embryo age (days) at biopsy. Among samples with successful amplification, the number of cells in the biopsy and the day of biopsy were critical to limit non-concurrent results. In total, 213 blastocysts with an inconclusive diagnosis were warmed for re-analysis and the survival rate was 96.7% (N = 206/213). The euploidy rate in blastocysts biopsied twice was 51.9% (N = 107/206) and the euploid embryos were re-vitrified. Overall, 49 euploid embryos were warmed for replacement and all survived. The LBR after SET was 38.8% (N = 19/49). No minor/major obstetrical/perinatal complication was reported. LIMITATIONS, REASONS FOR CAUTION: A single aneuploidy-testing method was adopted in this retrospective analysis. A more powered report of the clinical and obstetrical/perinatal outcomes after re-biopsied and re-vitrified blastocysts euploid SET requires a larger sample size. WIDER IMPLICATIONS OF THE FINDINGS: It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Delayed childbearing and female ageing impair assisted reproductive technology outcome in survivors of male haematological cancers.

PURPOSE: To analyse the impact of female characteristics on assisted reproductive technology outcome among male haematological cancer survivors. METHODS: A retrospective analysis of 93 haematological cancer survivors attending our tertiary referral fertility centre between June 1998 and June 2017 for achieving fatherhood with assisted reproductive technology treatments. RESULTS: A progressive increase in the median female age was observed during the study period (32.2 years until the year 2007 and 36.9 years from the year 2012). Fifty-five out of 93 patients were treated with intracytoplasmic sperm injection (ICSI) (113 ovarian stimulations, 108 ICSI procedures). Cryopreserved ejaculated sperm was used in 28 couples, fresh sperm in 19, and thawed testicular sperm in 8 couples. Mean female age at ovarian stimulation was 37.0 ± 4.7 years. Twenty-six pregnancies resulted in a full-term birth (23% per started ovarian stimulation; 43.6% per couple) and 33 children were born. No significant differences were observed according to source of sperm (fresh, frozen, testicular) and multivariate analysis confirmed that maternal age was the only variable inversely related to the cumulative delivery rate, being five times lower (15.7%) when the female partner was ≥ 40 years (OR = 0.22, 95% CI 0.06-0.77) vs. 58.3% with younger women (p = 0.0037). CONCLUSIONS: Delayed childbearing and female ageing affect ICSI outcome in couples where the male is a survivor of haematological cancer. This topic should be discussed when counselling male cancer patients about fertility preservation.

No advantage of fresh blastocyst versus cleavage stage embryo transfer in women under the age of 39: a randomized controlled study.

PURPOSE: Is there a difference in implantation and pregnancy rates between embryos transferred electively at cleavage or blastocyst stage in infertile women ≤ 38 years with at least four zygotes on day 1 post retrieval? METHODS: A randomized clinical trial was conducted in a single tertiary care hospital with a sample size of 194 patients in each arm for a total population of 388 women. Patients less than 39 years of age with more than three fertilized oocytes and less than four previous assisted reproductive technology (ART) attempts were inclusion criteria. RESULTS: The two groups were similar for age, years of infertility, indication to treatment, basal antimüllerian hormone and FSH, number of previous ART cycles, primary or secondary infertility, type of induction protocol, days of stimulation, total gonadotrophin dose, and estradiol (E2) and progesterone (P) levels at trigger. No statistically significant differences were found in terms of number of retrieved oocytes, inseminated oocytes, fertilization rate, canceled transfers (7.73% in blastocyst and 3.61% in cleavage stage group), and cycles with frozen embryos and/or oocytes. Although a higher number of fertilized oocytes were in the blastocyst stage group (6.18 ± 1.46 vs 5.89 ± 1.54, p = 0.052), a statistically greater number of embryos/randomized cycle were transferred at cleavage stage (1.93 ± 0.371) compared with the number of transferred blastocysts (1.80 ± 0.56), probably due to the number of embryos not reaching blastocyst stage (3.09%). The implantation rate (28.37 vs 25.67%), pregnancy rate per cycle (36.06 vs38.66%), transfer (39.66 vs 40.11%), spontaneous abortions (19.72% vs 12.00%), delivery rate per cycle (27.84 vs 32.99%), and transfer (30.17 vs 34.22%) were not significantly different between the blastocyst and cleavage stage groups. The twin delivery rate was higher in the blastocyst stage group, although not significant (42.59 vs 28.12%). The mean numbers of frozen blastocyst (2.30 ± 1.40 vs 2.02 ± 1.00) and frozen oocytes (7.09 ± 3.55vs 6.79 ± 3.26) were not significantly different between the two groups. CONCLUSIONS: Fresh blastocyst-stage transfer versus cleavage-stage transfer did not show any significant difference in terms of implantation and pregnancy rate in this selected group of patients. A high twin delivery rate in both groups (35.59%) was registered, and although not significant, they were higher in the blastocyst transfer group (42.59 vs 28.12%). Our conclusion supports considering single embryo transfer (SET) policy, even in cleavage stage in patients younger than 39 years with at least four zygotes. TRIAL REGISTRATION: ClinicalTrials.gov registration number NCT02639000.

Obstetric outcome and incidence of congenital anomalies in 2351 IVF/ICSI babies.

PURPOSE: The aim of this study was to provide a comprehensive follow-up of fetal and perinatal outcome and the incidence of congenital anomalies in babies born after fresh embryo transfers compared to those conceived spontaneously in infertile couples. METHODS: Retrospective comparative analysis of all clinical pregnancies from fresh cleavage-stage embryo transfer cycles (IVF and ICSI) compared with infertile patients who conceived spontaneously in the same time period (control). Congenital anomalies were classified following the European Surveillance of Congenital Anomalies (EUROCAT) classification. RESULTS: A total of 2414 assisted reproductive technology (ART) pregnancies were compared to 582 spontaneous conceptions in the control infertile group representing 2306 deliveries. No significant differences were found in pregnancy outcome between the two groups (delivery rate, abortion rate, ectopic pregnancies, medical abortions for fetal anomalies, single and twins mean gestational age, and weight at delivery). A significant difference (p < 0.001) was found in the twin (21.3 vs 2.3 %) and triplet rates (2.3 vs 0 %). A total of 2351 babies were delivered in the ART group and 449 in the control group. A total of 90 babies (3.8 %) were diagnosed with a major congenital anomaly in the ART group and 15 (3.3 %) in the control group (p = ns). The overall rate of major congenital anomalies (105/2800) in ART and spontaneous pregnancies in infertile couples was significantly higher when compared to the EUROCAT 2.0 versus 3.75 % (p = 0.0002). DISCUSSION: Babies born after ART treatments and from spontaneous conception in infertile couples had rates of congenital anomalies higher than those recorded by the EUROCAT. However, the rates of anomalies were not different within the infertile population whether conceived by ART or spontaneously. These data suggest that the diagnosis of infertility in itself is the common denominator for the increase in the rates of anomalies seen in both ART and spontaneous conceptions.

Artificial shrinkage of blastocysts prior to vitrification improves pregnancy outcome: analysis of 1028 consecutive warming cycles.

PURPOSE: This study aims to compare implantation, pregnancy, and delivery rates in frozen transfer cycles with blastocysts that were vitrified either with artificial shrinking (AS group) or without (NAS group). METHODS: Retrospective comparative study of artificial shrinking of blastocysts prior to vitrification and frozen embryo transfer cycles in infertile patients undergoing frozen embryo transfer (FET) was done at the Humanitas Fertility Center between October 2009 and December 2013. Main outcome measure(s) were implantation (IR), pregnancy (PR), and delivery rates (DR) between the two groups. RESULTS: A total of 1028 consecutive warming blastocyst transfer cycles were considered. In 580 cycles (total of 822 blastocysts), artificial shrinking was performed prior to vitrification (AS group), while in the remaining 448 cycles (total of 625 blastocysts), the artificial shrinking was not performed (NAS group). There were no differences in patient age (36.4 ± 3.7 vs. 36.3 ± 3.9) and number of embryos transferred (1.41 ± 0.49 vs. 1.38 ± 0.50) between groups. The IR, PR, and DR in the AS group were significantly higher (p < 0.05) than in the NAS group (29.9 vs. 23.0 %, 36.3 vs. 27.9 %, and 26.5 vs. 18.1 %, respectively). CONCLUSIONS: Performing AS of blastocysts prior to vitrification appears to improve implantation, pregnancy, and delivery rates probably related to a decreased risk of ultrastructural cryodamages, plausible when cryopreserving expanded blastocysts.

Effect of sex and estrogens on neuronal activation in an animal model of migraine.

OBJECTIVE: In this study, we evaluated the influence of sex and estrogen treatment on nitroglycerin (NTG)-induced neuronal activation in the rat brain. BACKGROUND: Systemic NTG activates cerebral nuclei of rat involved in nociceptive transmission, as well as in neuroendocrine and autonomic functions. These changes are considered relevant for migraine, since NTG consistently induces spontaneous-like attacks in migraineurs. METHODS: Intact and castrated male and female rats, and castrated female rats treated with estradiol benzoate (or placebo) were injected with NTG and sacrificed after 4 hours. Rats were perfused, and their brains were processed for Fos protein, a marker of neuronal activation. RESULTS: Data showed a reduced expression of NTG-induced Fos protein in the paraventricular nucleus (PVH), supraoptic nucleus (SON), and nucleus trigeminalis caudalis (SPVC) of male rats in comparison with female rats. Furthermore, in castrated female rats, NTG-induced neuronal activation was reduced in PVH, SON, central nucleus of the amygdala (AMI), nucleus tractus solitarius (NTS), area postrema (AP), and SPVC, while in castrated male rats Fos expression was reduced uniquely in the SPVC. Chronic administration of estrogens restored Fos protein expression in PVH, SON, AMI, NTS, AP, and SPVC in castrated female rats. CONCLUSION: These data provide a support for the existence of a sexual dimorphism in NTG-induced neuronal activation, and they prompt a specific model for evaluating and modulating the influence of estrogens upon the cerebral structures implicated in the pathophysiology of migraine.

Human oocyte ultravitrification with a low concentration of cryoprotectants by ultrafast cooling: a new protocol.

OBJECTIVE: To evaluate the efficacy of a new ultravitrification technique with a low concentration of cryoprotectants. DESIGN: Ultravitrification research. SETTING: Private assisted reproduction center. PATIENT(S): Oocytes donated voluntarily with the aim of research. INTERVENTION(S): Ultravitrification with different protocols of 100 mature oocytes and 100 immature oocytes divided in four groups to determine which is the adequate cryoprotectant concentration and the appropriate cooling solution. MAIN OUTCOME MEASURE(S): Human oocytes survival rate with low concentration of cryoprotectants by ultravitrification technique. RESULT(S): We obtained higher survival rates with slush nitrogen than with liquid nitrogen (92% vs. 56%) and better results with 2 M of cryoprotectants than with 1.5 M (92% vs. 60%). The best protocol was 2 M PrOH + 0.5 M sucrose + slush nitrogen with a mature oocytes survival rate of 92% (23 of 25) and immature of 88% (22 of 25). CONCLUSION(S): This ultravitrification technique is a new option to preserve human oocytes that avoids the use of a high cryoprotectant concentration while obtaining a high survival rate with a concentration of cryoprotectants typical of slow freezing.

Menstrual cycle-related changes in plasma oxytocin are relevant to normal sexual function in healthy women.

Circulating levels of the neuro-hypophysial nonapeptide oxytocin increase during sexual arousal and orgasm in both men and women. A few studies have evaluated the effect of the menstrual cycle on plasma oxytocin in normally cycling, sexually active, healthy fertile women using or not using contraceptive pills. In 20 ovulating women and 10 women taking an oral contraceptive (group 1 and group 2, respectively), sexual function, hormonal profile, and plasma oxytocin (OT) were evaluated throughout the menstrual cycle. In group 1, plasma OT was significantly lower during the luteal phase in comparison with both the follicular and ovulatory phases. Plasma oxytocin was significantly correlated with the lubrication domain of the Female Sexual Function Index (FSFI) during the luteal phase and showed a trend towards statistical significance during the follicular phase. In group 2, plasma OT did not show any significant fluctuation throughout the menstrual cycle, even though a significant correlation was evident with both the arousal and the lubrication domain of the FSFI during the assumption of the contraceptive pill. These findings suggest that plasma OT fluctuates throughout the menstrual cycle in normally cycling healthy fertile women with adequate sexual activity but not taking any oral contraceptive pill. Moreover, plasma OT levels significantly relates to the genital lubrication in both women taking and not taking oral contraceptive pill apparently confirming its role in peripheral activation of sexual function.