Inter-centre reliability in embryo grading across several IVF clinics is limited: implications for embryo selection.

RESEARCH QUESTION: What is the intra- and inter-centre reliability in embryo grading performed according to the Istanbul Consensus across several IVF clinics? DESIGN: Forty Day 3 embryos and 40 blastocysts were photographed on three focal planes. Senior and junior embryologists from 65 clinics were invited to grade them according to the Istanbul Consensus (Study Phase I). All participants then attended interactive training where a panel of experts graded the same embryos (Study Phase II). Finally, a second set of pictures was sent to both embryologists and experts for a blinded evaluation (Study Phase III). Intra-centre reliability was reported for Study Phase I as Cohen's kappa between senior and junior embryologists; inter-centre reliability was instead calculated between senior/junior embryologists and experts in Study Phase I versus III to outline improvements after training (i.e. upgrade of Cohen's kappa category according to Landis and Koch). RESULTS: Thirty-six embryologists from 18 centres participated (28% participation rate). The intra-centre reliability was (i) substantial (0.63) for blastomere symmetry (range -0.02 to 1.0), (ii) substantial (0.72) for fragmentation (range 0.29-1.0), (iii) substantial (0.66) for blastocyst expansion (range 0.19-1.0), (iv) moderate (0.59) for inner cell mass quality (range 0.07-0.92), (v) moderate (0.56) for trophectoderm quality (range 0.01-0.97). The inter-centre reliability showed an overall improvement from Study Phase I to III, from fair (0.21-0.4) to moderate (0.41-0.6) for all parameters under analysis, except for blastomere fragmentation among senior embryologists, which was already moderate before training. CONCLUSIONS: Intra-centre reliability was generally moderate/substantial, while inter-centre reliability was just fair. The interactive training improved it to moderate, hence this workflow was deemed helpful. The establishment of external quality assessment services (e.g. UK NEQAS) and the avant-garde of artificial intelligence might further improve the reliability of this key practice for embryo selection.

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.

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.

Why are they not coming back? A single-center follow-up study on oncological women oocyte's storing for fertility preservation.

Introduction: Oocyte cryopreservation is a valid option for female cancer patients to preserve fertility. The number of patients undergoing fertility preservation (FP) cycles has increased over the past years. Nevertheless, the rates of patients returning to use their cryopreserved material have shown to be considerably low, ranging from 5-8%, but significant data regarding the reasons of such low return rates are scarce. Methods: This study is a single-center follow-up retrospective study evaluating the return rate of oncological women who underwent FP at a tertiary care Fertility Center and assessing the reasons influencing the patients who did not return. Data about patients who returned to attempt pregnancy were retrieved from internal registries. Non-returned patients were assessed with a standardized phone survey investigating health condition, marital status and family projects, spontaneous conceptions, and the reasons why they had not returned to use their gametes. A univariate analysis between returned and non-returned patients was performed. Results: Of the 397 patients who received counseling about FP, 171 (43.1%) underwent oocyte cryopreservation between 2001 and 2017. Nine (5%) died, and 17 (10%) were lost at follow-up. A total of 20 patients (11.7%) returned and 125 did not. In the non-returned group, 37 (29.6%) did not have a partner, 10 (8%) had a previous spontaneous conception, and 15 (12%) had recurrent malignancy at the time of follow-up. In the univariate analysis, younger age at freezing (31.8±6.2 vs. 35.2±4.7; p 0.018), lack of a partner (p 0.002), type of cancer (other than breast cancer; p 0.024) were the significant factors in the non-returned group. As for the personal reason for not coming back, patients mainly answered as follows: lack of a partner (29, 23.2%), the desire for spontaneous motherhood (24, 19.2%), previous spontaneous pregnancies after FP procedures (16, 12.8%), and still ongoing hormonal therapy for breast cancer (13, 10.4%). All patients confirmed their will to keep the storage of their oocytes. Discussion: The impact of a cancer diagnosis on a woman's maternal desire, sentimental status and life priorities should be studied more thoroughly. Studies investigating hormonal therapy suppression in breast cancer patients seeking pregnancy should be encouraged. Clinical trial registration: https://clinicaltrials.gov, identifier NCT05223764.

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.

Cryopreservation of a small number of spermatozoa in yolk-filled human zonae pellucidae.

OBJECTIVES: Conventional sperm freezing procedures need the addition of a relatively large volume of cryoprotectant. The dilution of extremely poor sperm suspensions from ejaculate or testicular tissue may make the recovery of viable spermatozoa difficult at the moment of the intracytoplasmic sperm injection (ICSI) procedure. The cryopreservation of a few spermatozoa in empty zonae pellucidae is an interesting solution for crypto-azoospermic infertile men. We have modified this procedure by filling empty human zonae with TEST Yolk Buffer, an optimal cryoprotective medium, in order to analyse the number of zonae lost after thawing, the number of recovered spermatozoa per zona after thawing, and the sperm motility rate before freezing and after thawing. MATERIALS AND METHODS: Fifty empty human zonae pellucidae previously filled with TEST Yolk Buffer were injected with 750 motile spermatozoa from ten infertile men (15 spermatozoa per zona). Sterile straws containing two zonae each were frozen following a two-phase protocol. RESULTS: All of the zonae and 445/750 spermatozoa (59%) were recovered. The mean number (+ SD) of spermatozoa per zona was 8.9 +/- 1.9 (range: 5-12). The recovery rate of motile spermatozoa was 73% (327/445), with a mean number of motile spermatozoa per zona of 6.5 +/- 1.7 (range: 3-10). CONCLUSIONS: The cryopreservation of a small number of motile spermatozoa within empty zonae pellucidae using TEST Yolk Buffer as a freezing medium is possible without any major loss of spermatozoa and with an appreciable maintenance of sperm motility. This procedure seems to avoid: i) uncertain sperm retrieval after a laborious and time-consuming search on the day of oocyte aspiration; ii) the need for a repeated testicular biopsy; and iii) the need for heterologous insemination or oocyte cryopreservation (11).

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.

Results of in vitro fertilization in Italy after the introduction of a new law.

OBJECTIVE: To investigate the consequences of a law introduced in Italy in 2004 that forbids the fertilization or injection of more than three oocytes for assisted reproduction and does not allow any embryo selection or cryopreservation. DESIGN: Retrospective observational analysis. SETTING: Subfertile patients enrolled in an assisted reproduction program. PATIENT(S): Before the introduction of the law there were 1,179 cycles and after its enactment there were 1,860 cycles in 1,619 subfertile couples. INTERVENTION(S): Ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI) attempts. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rate. RESULT(S): Pregnancy rates (PR) per cycle (24.34% vs. 23.11%), per retrieval (28.64% vs. 25.65%), per transfer (31.37% vs. 27.74%), and the take-home babies per started cycle (19.1% vs. 18%) was not significantly different between the two periods. After introduction of the law, the PR significantly decreased in patients whose total motile sperm count was <1 x 10(6) (40.85% vs. 23.62%) and in patients receiving two embryos (35.71% vs. 23.53%). This difference was mostly the result of a reduced PR in patients <36 years old receiving two unselected embryos (41.16% vs. 30.90%). This result was, however, obscured by the higher proportion of patients <36 years (3.9% vs. 45.12%) receiving three embryos after the enactment of the law, which lead to a significantly higher PR (28.73% vs. 37.56%) and a consequent significantly higher triplet rate (0.58% vs. 4.71%). CONCLUSION(S): Women in whom elective transfer of two embryos was allowed before passing the law and couples with a severe male infertility factor had significantly reduced success rates. Although the overall PR did not change after the new law, if the transfer of frozen embryos is not considered, this was mainly the result of a higher number of embryos transferred into women <36 years old. This study shows how the negative effects of the new law are obscured by the transfer of a higher number of embryos in younger patients, resulting in a higher PR at the cost of a significantly higher triplet rate.

Normal birth after transfer of cryopreserved human embryos generated by microinjection of cryopreserved testicular spermatozoa into cryopreserved human oocytes.

OBJECTIVE: To report the first birth after transfer of cryopreserved embryos generated by intracytoplasmic sperm injection of cryopreserved testicular spermatozoa into cryopreserved human oocytes. DESIGN: Case report. SETTING: Tertiary center for reproductive technology. PATIENT(S): A 36-year-old woman with primary infertility of 3 years' duration and a 37-year-old man with congenital bilateral absence of the vas deferens. INTERVENTION(S): Cryopreservation of human embryos after oocytes and sperm thawing. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): A healthy, normal female infant with a birth weight of 2,950 g was born by cesarean section at 38 weeks' gestation, with normal 6-month follow-up. CONCLUSION(S): Embryo cryopreservation can lead to successful results, even with the use of cryopreserved gametes.