A 10-year follow-up of reproductive outcomes in women attempting motherhood after elective oocyte cryopreservation.

STUDY QUESTION: Which reproductive treatment outcomes are observed in women who underwent elective oocyte cryopreservation (EOC) and who returned to the clinic with a desire for a child? SUMMARY ANSWER: Whether to warm oocytes or to first use fresh own oocytes for ART depends on age upon returning, but both strategies result in favorable reproductive outcomes. WHAT IS KNOWN ALREADY: Most affluent countries have observed a trend toward postponement of childbearing, and EOC is increasingly used based on the assumption that oocytes cryopreserved at a younger age may extend a woman's reproductive lifespan and mitigate her age-related fertility decline. Although most follow-up studies after EOC have focused on women who requested oocyte warming, a substantial proportion of women who do not conceive naturally will embark on fertility treatment without using their cryopreserved oocytes. Reports on reproductive outcomes in past EOC users are scarce, and the lack of reproductive treatment algorithms in this group of women hampers counseling toward the most efficient clinical strategy. STUDY DESIGN, SIZE, DURATION: This retrospective observational single-center study encompasses 843 women who had elective oocyte vitrification between 2009 and 2019 at our fertility clinic. Women who underwent fertility preservation for medical or oncological reasons were excluded. This study describes the outcomes of the diverse reproductive treatment strategies performed until May 2022 in women returning to our clinic to attempt motherhood. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using descriptive statistics, patient characteristics and data of ovarian stimulation (OS) of EOC cycles were analyzed, as well as data related to OS and laboratory data of ART in women who pursued fertility treatment with and/or without using their cryopreserved oocytes. The primary outcome was live birth rate (LBR) per patient after oocyte warming and after ART using fresh oocytes. Secondary outcomes were return rate, utilization rate of the cryopreserved oocytes, laboratory outcomes upon return, and LBR per embryo transfer. A multivariable regression model was developed to identify factors associated with the decision to thaw oocytes as the primary strategy and factors associated with ongoing pregnancy upon return to the clinic. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1353 EOC cycles (mean ± SD, 1.6 ± 0.9 per patient) were performed. At the time of EOC, the mean age was 36.5 ± 2.8 years, mean anti-Müllerian hormone (AMH) was 2.3 ± 2.0 ng/ml, and 174 (20.6%) women had a partner. On average, 13.9 ± 9.2 mature oocytes were cryopreserved. Two hundred thirty-one (27.4%) women returned to the clinic, an average of 39.9 ± 23.4 months after EOC. Upon returning, their mean age was 40.4 ± 3.1 years, mean AMH was 1.5 ± 1.5 ng/ml, and 158/231 (68.3%) patients had a partner. As a primary approach, 110/231 (47.6%) past EOC users embarked on oocyte warming, 50/231 (21.6%) had intrauterine insemination, and 71/231 (30.7%) had ART using fresh own oocytes. Cumulative LBR (CLBR) was 45.9% (106/231) notwithstanding a miscarriage rate (MR) of 30.7% (51/166) in the entire cohort. In total, 141 women performed oocyte warming at some stage in their treatment trajectory. A subset of 90/231 (39.0%) patients exclusively had oocyte warming (41.6 ± 3.0 years, with 10.0 ± 5.2 oocytes warmed per patient). 52/231 (22.5%) patients exclusively had ART using fresh own oocytes (mean age of 39.0 ± 2.8 years, with 9.9 ± 7.4 mature oocytes retrieved per patient). CLBR was 37/90 (41.1%) in the oocyte warming-only group and 25/52 (48.1%) in the OS-only group. MR/transfer was 25.0% and 29.3% in the oocyte warming-only group and the OS-only group, respectively. LIMITATIONS, REASONS FOR CAUTION: Both sample size and the retrospective design are limitations of this study. The decision to embark on a specific reproductive treatment strategy was based on patient preference, after counseling on their treatment options. This precludes direct comparison of the efficiency of reproductive treatment options in past EOC users in this study. WIDER IMPLICATIONS OF THE FINDINGS: Reporting on clinical outcomes of women who underwent EOC and returned to the clinic to embark on divergent reproductive treatment strategies is mandatory to establish guidelines for best clinical practice in this growing patient population. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Impact of cell loss after warming of human vitrified day 3 embryos on obstetric outcome in single frozen embryo transfers.

PURPOSE: Does cell loss (CL) after vitrification and warming (V/W) of day 3 embryos have an impact on live birth rate (LBR) and neonatal outcomes? METHOD: This retrospective analysis includes cleavage stage day 3 embryos vitrified/warmed between 2011 and 2018. Only single vitrified/warmed embryo transfers were included. Pre-implantation genetic screening, oocyte donation, and age banking were excluded from the analysis. The sample was divided into two groups: group A (intact embryo after warming) and group B (≤ 50% blastomere loss after warming). RESULTS: On the total embryos (n = 2327), 1953 were fully intact (83.9%, group A) and 374 presented cell damage (16.1%, group B). In group B, 62% (232/374) of the embryos had lost only one cell. Age at cryopreservation, cause of infertility, insemination procedure, and semen origin were comparable between the two groups. The positive hCG rate (30% and 24.3%, respectively, for intact vs CL group, p = 0.028) and LBR (13.7% and 9.4%, respectively, for intact vs CL group, p = 0.023) per warming cycle were significantly higher for intact embryos. However, LBR per positive hCG was equivalent between intact and damaged embryos (45.6% vs 38.5%, respectively, p = 0.2). Newborn measurements (length, weight, and head circumference at birth) were comparable between the two groups. Multivariate logistic regression showed that the presence of CL is not predictive for LB when adjusting for patients' age. CONCLUSIONS: LBR is significantly higher after transfer of an intact embryo compared to an embryo with CL after warming; however, neonatal outcomes are comparable between the two groups.

To trigger or not to trigger ovulation in a natural cycle for frozen embryo transfer: a randomized controlled trial.

STUDY QUESTION: Is the clinical pregnancy rate (CPR) following a frozen embryo transfer (FET) in a natural cycle (NC) higher after spontaneous ovulation than after triggered ovulation [natural cycle frozen embryo transfer (NC-FET) versus modified NC-FET]? SUMMARY ANSWER: The CPR did not vary significantly between the two FET preparation protocols. WHAT IS KNOWN ALREADY: Although the use of FET is continuously increasing, the most optimal endometrial preparation protocol is still under debate. For transfer in the NC specifically, conflicting results have been reported in terms of the outcome following spontaneous or triggered ovulation. STUDY DESIGN, SIZE, DURATION: In a tertiary hospital setting, subjects were randomized with a 1:1 allocation into two groups between January 2014 and January 2019. Patients in group A underwent an NC-FET, while in group B, a modified NC-FET was performed with a subcutaneous hCG injection to trigger ovulation. In neither group was additional luteal phase support administered. All embryos were vitrified-warmed on Day 3 and transferred on Day 4 of embryonic development. The primary outcome was CPR at 7 weeks. All patients were followed further until 10 weeks of gestation when the ongoing pregnancy rate (OPR) was defined by the observation of foetal cardiac activity on ultrasound scan. Other secondary outcomes included biochemical pregnancy rate, early pregnancy loss and the number of visits, blood samples and ultrasonographic examinations prior to FET. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 260 patients (130 per study arm) were randomized, of whom 12 withdrew consent after study arm allocation. A total of 3 women conceived spontaneously before initiating the study cycle and 16 did not start for personal or medical reasons. Of the 229 actually commencing monitoring for the study FET cycle, 7 patients needed to be switched to a hormonal replacement treatment protocol due to the absence of follicular development, 12 had no embryo available for transfer after warming and 37 had a spontaneous LH surge before the ovulation trigger could be administered, although they were allocated to group B. Given the above, an intention-to-treat (ITT) analysis was performed taking into account 248 patients (125 in group A and 123 in group B), as well as a per protocol (PP) analysis on a subset of 173 patients (110 in group A and 63 in group B). MAIN RESULTS AND THE ROLE OF CHANCE: Demographic features were evenly distributed between the study groups, as were the relevant fresh and frozen ET cycle characteristics. According to the ITT analysis, the CPR and OPR in group A (33.6% and 27.2%, respectively) and group B (29.3% and 24.4%, respectively) did not vary significantly [relative risk (RR) 0.87, 95% CI (0.60;1.26), P = 0.46 and RR 0.90, 95% CI (0.59;1.37), P = 0.61, respectively]. Biochemical pregnancy rate and early pregnancy loss were also found to be not statistically significantly different between the groups. In contrast, more clinic visits and blood samplings for cycle monitoring were required in the NC-FET group (4.05 ± 1.39) compared with the modified NC-FET group (3.03 ± 1.16, P = <0.001), while the number of ultrasound scans performed were comparable (1.70 ± 0.88 in group A versus 1.62 ± 1.04 in group B). The additional PP analysis was in line with the ITT results: CPR in group A was 36.4% versus 38.1% in group B [RR 1.05, 95% CI (0.70;1.56), P = 0.82]. LIMITATIONS, REASONS FOR CAUTION: The results are limited by the high drop-out rate for the PP analysis in the modified NC-FET group as more than one-third of the subjects allocated to this group ovulated spontaneously before ovulation triggering. Nonetheless, this issue is inherent to routine clinical practice and is an important observation of an event that can only be avoided by performing a very extensive monitoring that limits the practical advantages associated with modified NC-FET. Furthermore, although this is the largest randomized controlled trial (RCT) investigating this specific research question so far, a higher sample size would allow smaller differences in clinical outcome to be detected, since currently they may be left undetected. WIDER IMPLICATIONS OF THE FINDINGS: This RCT adds new high-quality evidence to the existing controversial literature concerning the performance of NC-FET versus modified NC-FET. Based on our results showing no statistically significant differences in clinical outcomes between the protocols, the treatment choice may be made according to the patient's and treating physician's preferences. However, the modified NC-FET strategy reduces the need for hormonal monitoring and may therefore be considered a more patient-friendly and potentially cost-effective approach. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was available for this study. None of the authors have a conflict of interest to declare with regard to this study. TRIAL REGISTRATION NUMBER: NCT02145819. TRIAL REGISTRATION DATE: 8 January 2014. DATE OF FIRST PATIENT'S ENROLMENT: 21 January 2014.

Single and double embryo transfer provide similar live birth rates in frozen cycles.

Research question: Do live birth rates (LBRs) differ in frozen cycles of women who received single versus double embryo transfer?Design: Retrospective cohort study including women who underwent their first frozen embryo transfer (FET) in a tertiary referral University Hospital between 2009-2014.Results: 3601 patients were included in the analysis with 1936 (53.8%) having a single embryo transfer (SET) and 1665 (46.2%) having a double embryo transfer (DET). Overall, 657/3601 (18.24%) had a live birth. LBR were similar between SET and DET either for cleavage [100/757 (13.1%) versus 153/1032 (14.8%), p = .33] or blastocyst stage FET [256/1179 (21.7%) versus 148/633 (23.4%), p = .4). Ongoing pregnancy rates were comparable between DET and SET [316/1665 (18.9%) versus 359/1936 (18.5%)]. Multiple delivery rates were significantly higher in women with DET compared to SET [53/316 (16.7%) versus 7/359 (1.9%), p < .001]. Multivariate logistic regression analysis allowing adjustment for relevant confounders showed that the number of embryos transferred in the frozen cycle was not related to LBR.Conclusions: This is the largest study providing evidence that both SET and DET may result in similar LBR, albeit multiple pregnancy rates are significantly lower in case of SET. Therefore, SET should be the main strategy in women undergoing FET.

Frozen embryo transfer: a review on the optimal endometrial preparation and timing.

STUDY QUESTION: What is the optimal endometrial preparation protocol for a frozen embryo transfer (FET)? SUMMARY ANSWER: Although the optimal endometrial preparation protocol for FET needs further research and is yet to be determined, we propose a standardized timing strategy based on the current available evidence which could assist in the harmonization and comparability of clinic practice and future trials. WHAT IS KNOWN ALREADY: Amid a continuous increase in the number of FET cycles, determining the optimal endometrial preparation protocol has become paramount to maximize ART success. In current daily practice, different FET preparation methods and timing strategies are used. STUDY DESIGN, SIZE, DURATION: This is a review of the current literature on FET preparation methods, with special attention to the timing of the embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Literature on the topic was retrieved in PubMed and references from relevant articles were investigated until June 2017. MAIN RESULTS AND THE ROLE OF CHANCE: The number of high quality randomized controlled trials (RCTs) is scarce and, hence, the evidence for the best protocol for FET is poor. Future research should compare both the pregnancy and neonatal outcomes between HRT and true natural cycle (NC) FET. In terms of embryo transfer timing, we propose to start progesterone intake on the theoretical day of oocyte retrieval in HRT and to perform blastocyst transfer at hCG + 7 or LH + 6 in modified or true NC, respectively. LIMITATIONS REASONS FOR CAUTION: As only a few high quality RCTs on the optimal preparation for FET are available in the existing literature, no definitive conclusion for benefit of one protocol over the other can be drawn so far. WIDER IMPLICATIONS OF THE FINDINGS: Caution when using HRT for FET is warranted since the rate of early pregnancy loss is alarmingly high in some reports. STUDY FUNDING/COMPETING INTEREST(S): S.M. is funded by the Research Fund of Flanders (FWO). H.T. and C.B. report grants from Merck, Goodlife, Besins and Abbott during the conduct of the study. TRIAL REGISTRATION NUMBER: Not applicable.

Vitrified-warmed blastocyst transfer on the 5th or 7th day of progesterone supplementation in an artificial cycle: a randomised controlled trial.

Prospective studies comparing different durations of progesterone supplementation before transfer of vitrified-warmed blastocysts in an artificial cycle are lacking. However, in oocyte donation programmes, the sporadic available evidence demonstrates considerable differences in clinical pregnancy rates according to the duration of progesterone administration. This randomised controlled trial (RCT), included 303 patients undergoing a frozen-thawed embryo transfer (FET) of one or two vitrified-warmed blastocyst(s) in an artificial cycle. Randomisation was performed when the endometrial thickness reached ≥7 mm after oestrogen supplementation. One hundred and fifty two patients in group A received 7 d of vaginal micronised progesterone tablets and 151 patients in group B received 5 d of micronised vaginal progesterone before FET. No differences were seen in clinical pregnancy rate between both groups: 42/152 (27.6%) in group A versus 49/151 (32.5%) in group B. Although no statistically significant difference was observed in clinical pregnancy rates, our study was powered to detect an absolute difference of 16%. In this regard, we cannot exclude that smaller, clinically relevant differences might exist and our study did not have the power to detect this. Patients were also not blinded for the intervention, causing a potential bias.

Neonatal health including congenital malformation risk of 1072 children born after vitrified embryo transfer.

STUDY QUESTION: Does vitrification of Day 3 and Day 5 embryos adversely affect birth outcomes of singletons and twins in comparison with peers born after fresh embryo transfer? SUMMARY ANSWER: Neonatal health parameters, including the prevalence of congenital malformations, in singletons and twins born after embryo vitrification are similar to or slightly better than after fresh embryo transfer. WHAT IS ALREADY KNOWN: Although vitrification, rather than slow-freezing, of embryos is routine practice nowadays, convincing evidence regarding the safety for the offspring is sparse. Literature data comprise results from mostly small-sized studies or studies including only Day 3 or only Day 5 vitrified embryo transfers. Overall, better or comparable perinatal outcomes, in terms of higher birthweight and lower risk for small-for-gestational age or for low birthweight, have been reported for singletons born after vitrified embryo transfer compared with fresh embryo transfer. According to the single available study with sufficient sample size, the congenital malformation rate was found to be comparable after vitrified and fresh embryo transfers. STUDY DESIGN, SIZE, DURATION: Data were collected from 960 cycles after transfer of embryos vitrified on Day 3 (n = 457) or Day 5 (n = 503) and from 1644 cycles after fresh embryo transfer on Day 3 (n = 853) or Day 5 (n = 791), performed between 2008 and 2013 at the Centre for Reproductive Medicine of the university hospital UZ Brussel. Outcome measures were neonatal health in terms of birthweight, small-for-gestational age, prematurity rate, perinatal death and major/minor/total malformation rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Perinatal health parameters of 11 stillborns and 1061 live borns (827 singletons and 234 twins) in the vitrified group and of 28 stillborns and 1838 live borns (1374 singletons and 464 twins) in the fresh embryo group are reported. Within 3 months after birth, children in the two study groups were assessed clinically with special attention to congenital malformations by a paediatrician blinded to the type of embryo transfer. Data were analysed by multiple linear and logistic regression, adjusted for treatment variables and maternal characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Mothers to infants in the vitrified group were on average slightly older and more often suffering from pregnancy-related hypertensive disorders than mothers to infants in the fresh transfer group. Singletons born after vitrification showed a higher birthweight standard deviation score (SDS) (-0.4 versus -0.7; 95% confidence interval (CI): 0.0-0.3, P = 0.001) and a lower small-for-gestational age rate (AOR: 0.55; 95% CI: 0.34-0.90) in comparison with peers born after fresh embryo transfer. Preterm birth rate and perinatal death rate were comparable between the two groups (AOR: 0.91; 95% CI: 0.57-1.43 and AOR: 0.97; 95% CI: 0.40-2.36). In twins, neonatal outcomes including birthweight SDS, small-for-gestational age and prematurity rates were comparable in the vitrified and the fresh groups, when adjusted for confounders. Furthermore, the rate of major congenital malformations in live borns was comparable between the vitrified group and the fresh group, both in singletons (2.6 versus 2.8%; AOR: 0.91; 95% CI: 0.47-1.78) and in twins (2.4 versus 2.7%; AOR: 0.51; 95% CI: 0.05-5.72). Also, the total malformation rate in the vitrified group (3.4%; 95% CI: 2.4-4.8) did not differ from the rate in the fresh embryo group (3.9%; 95% CI: 3.1-5.0). The embryonic stage at vitrification or fresh transfer (cleavage-stage embryo or blastocyst) did not influence the birth characteristics or malformation rate. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study is the rather small twin group. Therefore, the outcome results for twins should be interpreted cautiously. WIDER IMPLICATIONS OF THE FINDINGS: This study provides evidence that transfer of vitrified Day 3 and Day 5 embryos does not adversely affect the neonatal health of the offspring in comparison with transfer of fresh embryos. Furthermore, neonatal outcomes were not different after transfer of vitrified blastocysts compared with transfer of vitrified cleavage-stage embryos. STUDY FUNDING/COMPETING INTERESTS: Educational grants for establishing and organizing the data collection have come from IBSA, Ferring, Organon, Shering-Plough and Merck. Merck Belgium funded the data collection for outcomes after vitrification between 2012 and 2015. All co-authors, except M.B., declared no conflict of interest. M.B. has received consultancy fees from Organon, Serono Symposia and Merck.

Do ARTs affect the incidence of monozygotic twinning?

STUDY QUESTION: Does the manipulation of gametes or embryos during ARTs increase the risk for monozygotic twinning (MZT)? SUMMARY ANSWER: Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. WHAT IS KNOWN ALREADY: Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. STUDY DESIGN, SIZE, DURATION: All clinical pregnancies after single ET carried out in our centre between 2004 and 2013 (n = 6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. MAIN RESULTS AND THE ROLE OF CHANCE: The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29-0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36-5.34; aOR 2.05, 95% CI 1.29-3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. LIMITATION, REASONS FOR CAUTION: This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. WIDER IMPLICATION OF THE FINDINGS: Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. STUDY FUNDING/COMPETING INTERESTS: No external funding was used for this study. There are no conflicts of interest.

What is the optimal duration of progesterone administration before transferring a vitrified-warmed cleavage stage embryo? A randomized controlled trial.

STUDY QUESTION: What is the impact on clinical pregnancy rates when vitrified cleavage stage Day 3 embryos, warmed and cultured overnight to Day 4, are transferred on the 3rd or 5th day of progesterone administration in an artificial cycle? SUMMARY ANSWER: Clinical pregnancy rates are similar when transferring a vitrified-warmed cleavage stage Day 3 embryo after overnight culture on the 3rd or 5th day of progesterone administration. WHAT IS KNOWN ALREADY: In artificially prepared cycles, progesterone supplementation is generally started 3 days before embryo transfer, although the optimal length of exposure to progesterone before frozen embryo transfer (FET) has not been established. However, in a natural cycle, serum progesterone levels start to rise before ovulation, due to the LH-stimulated production by the peripheral granulosa cells. Hence, it could be postulated that progesterone supplementation before embryo transfer in an artificial cycle should start earlier or even later. STUDY DESIGN, SIZE, DURATION: Prospective, randomized controlled trial, encompassing 300 patients who had embryos frozen on Day 3 and who underwent FET in an artificial cycle. Between 1 November 2012 and 31 December 2014, 300 patients were allocated to one of two groups as soon as endometrial thickness reached ≥7 mm on ultrasound after estrogen supplementation. A computer-generated randomization list was used, not concealed to the physicians. Each patient was enrolled into the study only once. FET was performed on the fifth day of progesterone supplementation in Group A, whereas in Group B, FET was performed on the third day of vaginal micronized progesterone administration. Embryos were thawed the day before transfer and after overnight culture, one or two Day 4 embryos were transferred. PARTICIPANTS/MATERIALS, SETTING, METHODS: One hundred and fifty patients in Group A received 5 days of vaginal micronized progesterone tablets and one hundred and fifty patients in Group B received 3 days of micronized progesterone vaginally before FET. In Group A, 13 patients did not have an embryo transfer, compared with 12 patients in Group B. MAIN RESULTS AND THE ROLE OF CHANCE: Clinical pregnancy rates did not differ significantly between both groups (37/137 (27.0%) in Group A versus 26/138 (18.8%) in Group B (OR 1.6 (CI 0.9-2.82), ITALIC! P = 0.11). However, early pregnancy loss was significantly higher in Group B (32/58 (55.2%)) compared with Group A (21/58 (36.2%)) (OR 0.46 (CI 0.22-0.97), ITALIC! P = 0.04). LIMITATIONS, REASONS FOR CAUTION: Although no statistically significant difference was seen in the primary outcome, the study may have been underpowered to detect smaller differences. The study was also not blinded and patients were aware of the exact duration of progesterone supplementation. WIDER IMPLICATIONS OF THE FINDINGS: This is the first randomized controlled trial to show that duration of progesterone administration in an artificially prepared FET cycle may modulate cycle outcome and that too short progesterone supplementation might be deleterious. STUDY FUNDING/COMPETING INTERESTS: No external finance was involved in this study. All authors declare to have no conflict of interest with regard to this trial. TRIAL REGISTRATION NUMBER: The trial was registered at clinicaltrials.gov (NCT01940653). TRIAL REGISTRATION DATE: 9 September 2013. DATE OF FIRST PATIENT'S ENROLLMENT: 1 November 2012.

Frozen-thawed embryo transfers in natural cycles with spontaneous or induced ovulation: the search for the best protocol continues.

STUDY QUESTION: Is natural cycle frozen-thawed embryo transfer (NC-FET) associated with better clinical pregnancy rates (CPR) when compared to modified natural cycle frozen-thawed embryo transfer (mNC-FET)? SUMMARY ANSWER: NC-FET is associated with a higher CPR compared to mNC-FET. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the impact of hCG triggering on clinical outcomes after frozen-thawed embryo transfer (FET), and information on the effect of luteal phase support (LPS) is lacking. STUDY DESIGN, SIZE, DURATION: This retrospective study included all (n = 2353) consecutive cycles with FET of vitrified cleavage and blastocyst stage embryos warmed between January 2010 and April 2015 in a tertiary centre. The FET cycles were grouped by type as follows: NC (n = 501), NC + LPS (n = 828) or mNC + LPS (n = 1024). Artificial cycles were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed mixed-effect multilevel multivariable regression analysis to account for the clustering of FETs using embryos derived from the same patient and/or ovarian stimulation cycle. Adjustment for the following potential confounders was also performed: female age at oocyte retrieval, number of oocytes retrieved, fresh cycle pregnancy outcome, embryo transfer rank, number of embryos transferred, embryo stage and grade and endometrial thickness. Bonferroni adjustment for multiple comparisons was performed whenever indicated. MAIN RESULTS AND THE ROLE OF CHANCE: The unadjusted CPR per cycle was significantly higher in the NC-FET group (46.9%) when compared with the mNC-FET + LPS groups (29.7%, P < 0.001) but not the NC-FET + LPS group (39.9%, P = 0.069). The lower clinical performance of mNC-FET + LPS remained significant even after adjusting for potential confounders [adjusted odds ratio (95% CI) compared to the NC-FET groups: 2.18 (1.64-2.90) and 1.67 (1.31-2.12) for the NC-FET and NC-FET + LPS groups, respectively]. A sensitivity analysis restricting the sample only to the first FET performed by the couple in our centre was also performed. The predicted CPR in this multivariable logistic regression model remained significantly higher in the NC-FET (53.9%) and NC-FET + LPS (44.9%) groups when compared to mNC-FET + LPS (34.2%, all Bonferroni-adjusted pairwise comparisons with P ≤ 0.01). LIMITATIONS, REASONS FOR CAUTION: The interpretation of the findings of this study is limited by the retrospective nature of the analysis and the potential for unmeasured confounding. WIDER IMPLICATIONS OF THE FINDINGS: The use of mNC-FET, using hCG triggering or progesterone supplementation, should be reconsidered in light of the potential negative effect on pregnancy outcome. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts.

STUDY QUESTION: What is the effect of artificial shrinkage by laser-induced collapse before vitrification on the implantation potential after transfer of vitrified-warmed blastocysts? SUMMARY ANSWER: The artificial shrinkage by laser-induced collapse did not significantly increase the implantation rate per transferred collapsed blastocyst (37.6%) compared with non-collapsed blastocysts (28.9%) [odds ratio (OR): 1.48, 95% confidence interval (CI): 0.78-2.83]. WHAT IS KNOWN ALREADY: Retrospective studies have demonstrated that artificial shrinkage of the blastocyst prior to vitrification can have a positive effect on blastocyst survival after warming. A recent study found a similar survival rate but higher implantation rate for collapsed blastocysts. So far, no randomized controlled trial has been conducted to investigate the implantation potential of collapsed blastocysts. STUDY DESIGN, SIZE, DURATION: Prospective randomized trial. Patients were recruited from December 2011 until April 2014 and warming cycles were included until July 2014. Patients were randomized in the fresh cycle if blastocysts were available for vitrification and were allocated to the study or control arm according to a computer-generated list. In the study group, blastocysts underwent laser-induced collapse before vitrification. In the control group, blastocysts were vitrified without collapsing. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 443 patients signed informed consent and 270 patients had blastocysts vitrified. One-hundred and thirty-five patients were allocated to the study group and 135 to the control group. Sixty-nine patients from the study group and 69 from the control group returned for at least one warming cycle in which 85 and 93 blastocysts were warmed in the first cycle, respectively. Primary outcome was implantation rate per embryo transferred in the first warming cycle. Secondary outcomes were survival and transfer rates, blastocyst quality after warming, clinical pregnancy rate and implantation rate per warmed blastocyst. Blastocysts were vitrified-warmed one by one using closed vitrification and one or two blastocysts were transferred per warming cycle. MAIN RESULTS AND THE ROLE OF CHANCE: We calculated that the group sample sizes of 80 embryos in the collapse group and 80 embryos in the control group were needed to achieve 80% power to detect a difference between the group proportions of +20% with P < 0.05. In the study group, 69 first warming cycles resulted in 69 transfers with 1.2 blastocysts (n = 85) transferred. In the control group, an average of 1.3 blastocysts (n = 83) were transferred in 67 out of 69 warming cycles. Implantation rates per embryo transferred in the first warming cycle were not different between both groups (38 versus 29%, OR: 1.48; 95% CI: 0.78-2.83), neither was the implantation rate per warmed embryo (38 versus 26%, OR: 1.74; 95% CI: 0.92-3.29). When all warming cycles were considered (n = 135 in each group), survival rate after collapse was significantly higher compared with the control group (98.0 versus 92.0%, OR: 4.25; 95% CI: 1.19-15.21). Furthermore, a higher percentage of high-quality blastocysts (36.3 versus 23.5%, OR: 1.86; 95% CI: 1.12-3.08) and hatching blastocysts (19.2 versus 5.4%, OR: 4.18; 95% CI: 1.84-9.52) were found compared with the control group. LIMITATIONS, REASONS FOR CAUTION: The study lasted more than 2.5 years since fewer patients than expected returned for a warming cycle because of the high ongoing pregnancy rates in the fresh IVF/ICSI cycle. WIDER IMPLICATIONS OF THE FINDINGS: Although no significant higher implantation rate was found after collapse, the better survival and post-warm embryo quality convinced us to recognize a clinical benefit of artificial shrinkage and to implement it in routine vitrification practice. TRIAL REGISTRATION NUMBER: NCT01980225, www.clinicaltrials.gov. The first patient was included November 2011 and the study was registered October 2013.

Cryopreserved embryo transfer in an artificial cycle: is GnRH agonist down-regulation necessary?

The use of GnRH agonist downregulation in artificial endometrium priming cycles for cryopreserved embryo transfer was retrospectively investigated to establish whether higher live birth rates resulted. Six hundred and ninety-nine patients underwent 1129 artificial endometrium priming cycles for the transfer of cryopreserved embryos between 1 July 2009 and 1 June 2012. Hormonal supplementation with (group A, n = 280 cycles) or without (group B, n = 849 cycles) GnRH agonist co-treatment was given. Live birth rates were comparable between the two groups per started cycle (14.9% [41/275] in group A versus 15.1% [127/839] in group B) or per embryo transfer (17.5% [41/234] in group A versus 17.6% [127/723] in group B). After logistic regression analysis, the only variables that were significantly associated with live birth rates were day of embryo transfer (OR 0.69; 95% CI 0.48 to 0.98) for day 3 versus day 5 embryos, the number of embryos transferred (OR 2.13; 95% CI 1.58 to 2.86) for two embryos versus one embryo transferred and the endometrial thickness on the day of embryo transfer (OR 1.15; 95% CI 1.05 to 1.25). Live birth rates after cryopreserved embryo transfer in artificial cycles did not increase when a GnRH agonist was administered.

Deliveries of normal healthy babies from embryos originating from oocytes showing the presence of smooth endoplasmic reticulum aggregates.

STUDY QUESTION: Should oocytes showing the presence of smooth endoplasmic reticulum aggregates (SER) be considered for embryo transfer? SUMMARY ANSWER: The present study shows that embryos derived from metaphase II oocyte with visible SER (SER+MII) have the capacity to develop normally and may lead to newborns with no major malformations. WHAT IS KNOWN ALREADY: It has been reported that the presence of SER in the cytoplasm of oocytes has a negative impact on embryo development, and is associated with a decreased clinical outcome and an increased risk of congenital anomalies. Therefore, it has been recommended that embryos derived from SER-positive oocytes should not be transferred. STUDY DESIGN, SIZE, DURATION: Consecutive ICSI cycles with at least one SER+MII oocyte were retrospectively analyzed regarding embryological and pregnancy outcome and compared with ICSI cycles showing only oocytes without SER (SER-MII). PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 394 SER-positive (SER+) cycles and 6845 SER-negative (SER-) cycles were analyzed. The Student's t-test, one-way analysis of variance test and χ(2) test were used for statistical analysis. P value of <0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Comparable fertilization rates were observed in SER+ (76.2%) and SER- (73.5%) cycles. In case of blastocyst culture, the cycle efficiency was lower in SER+ than in SER- cycles (mean 42.2 versus 62.8%, P < 0.001). The pregnancy and clinical pregnancy (CP) rates per embryo transfer (ET) were comparable for SER+ and SER- cycles (37.6 versus 37.8% and 33.0 versus 32.4%, respectively). In the SER+ cycles, the fertilization rates of SER+MII and SER-MII (72.9 versus 77.0%), as well as the capacity to develop into good-quality embryos on Days 3 (62.3 versus 63.7%) and 5 (45.4 versus 47.4%), were similar. In the 364 SER+ cycles, the ETs were subdivided in: ET with only SER+MII (n = 31; 8.5%), ET with only SER-MII (n = 235; 64.5%) and ET with mixed SER+ and SER-MII (n = 98; 26.9%). The pregnancy (25.8, 37.4 and 41.8%, respectively) and CP rates (22.6, 32.4 and 37.9%, respectively) were not different between the three subgroups. Among the cycles with known outcome, there was no difference in the rate of major malformations between SER+ cycles (5.3%) and SER- cycles (2.1%). Moreover, no major malformations were reported from the live borns definitely originating from SER+MII embryos. In addition, three newborns, from single ET with frozen-thawed embryos originating from SER+MII oocytes, were delivered and presented no major malformation. LIMITATIONS, REASONS FOR CAUTION: Taking into account the previous publications and our neonatal data, a follow-up of the children born after ET with embryos originating from SER+ cycles is encouraged. WIDER IMPLICATION OF THE FINDINGS: More studies should be performed to investigate the origin and effect of SER aggregates on the molecular status of oocytes and embryos. STUDY FUNDING/COMPETING INTEREST(S): No external funding was either sought or obtained for this study and there are no potential competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos.

STUDY QUESTION: Is the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos? SUMMARY ANSWER: Vitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight. WHAT IS KNOWN ALREADY: After slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage. STUDY DESIGN, SIZE, DURATION: Survival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed. MAIN RESULTS AND ROLE OF CHANCE: Survival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification. LIMITATIONS, REASONS FOR CAUTION: This analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos. WIDER IMPLICATIONS OF THE FINDINGS: Although it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed. STUDY FUNDING/COMPETING INTEREST(S): none.

Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification.

PURPOSE: To compare two vitrification methods and two warming methods for human oocyte vitrification using a high security closed device in terms of survival, fertilization and embryo development. METHODS: For vitrification, oocytes were (1) immediately placed in equilibration solution or (2) they were gradually exposed to the cryoprotectants. For warming, oocytes were placed (1) in a 25 µl preheated (37 °C) thawing solution droplet that was put at room temperature for 1 min once the oocytes were inside or (2) in a 150 µl droplet for 1 minute at 37 °C. RESULTS: Survival and preimplantation development were significantly lower when warming was performed in a small preheated droplet. There was no significant difference in survival and embryo development between the gradual or direct exposure to cryoprotectants. CONCLUSIONS: Using this high security closed vitrification device a 90 % survival rate can be achieved when the oocytes are immediately warmed in a large volume at 37 °C.

Closed blastocyst vitrification of biopsied embryos: evaluation of 100 consecutive warming cycles.

BACKGROUND: The aim of this study was to analyse the outcome of closed blastocyst vitrification of embryos biopsied at the cleavage stage. METHODS: Vitrification of supernumerary blastocysts was performed using the closed CBS-VIT High Security straws. Warming cycles (n = 100) for patients with preimplantation genetic diagnosis (PGD) and/or aneuploidy screening in the fresh cycle were analysed. The outcome parameters were morphological survival and transfer rates after warming, clinical pregnancy rate and implantation rate (with fetal heart beat). Clinical outcome was compared with two control groups of (i) vitrified/warming transfer cycles without embryo biopsy and (ii) fresh Day 5 transfer of biopsied embryos. RESULTS: In total, 131 blastocysts were warmed with a morphological survival of 83.2% (109/131) and a transfer rate of 79.4% (104/131). Day 5 blastocysts survived significantly better (90.4%) than Day 6 blastocysts (70.8%, P < 0.01). No difference in survival rate was observed between early cavitating (89.2%) and full/expanded blastocysts (93.3%). In nine cycles, no blastocyst was available for transfer. The clinical pregnancy rate was 19.2% (15/78) after single-embryo transfer (SET) and 38.5% (5/13) after double-embryo transfer (DET). In SET, the implantation rate for blastocysts frozen on Day 5 was 13.7% (7/51), which was not different from the implantation rate of Day 6 blastocysts (18.5%, 5/27). The overall implantation rate of vitrified PGD biopsied blastocysts (14.4%) was comparable with that of vitrified blastocysts without biopsy (20.4%), but lower than the implantation rate obtained in the fresh PGD cycles (24.4%). CONCLUSION: Blastocysts on Day 5 and Day 6 of development derived from biopsied embryos can be successfully vitrified using a closed system.

Outcome of closed blastocyst vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single-embryo transfer policy.

BACKGROUND: In order to optimize blastocyst cryopreservation, vitrification was introduced as the routine procedure instead of slow freezing. The outcome of a closed blastocyst vitrification system was evaluated in relation to the blastocyst score before cryopreservation in single embryo transfers (SETs). METHODS: Supernumerary blastocysts of IVF/ICSI patients with a fresh Day 5 transfer were vitrified using CBS-VIT High Security (HS) straws. In 759 warming cycles, morphological survival and transfer rates were assessed in relation to the blastocyst score and the day of vitrification. Pregnancy rates were assessed in 530 SET and 156 double embryo transfer (DET) cycles. Implantation rates per embryo transferred in SET cycles were analysed according to blastocyst quality and day of cryopreservation. RESULTS: Immediate morphological survival was 77.8% (921/1185) and the transfer rate per warmed blastocyst was 70.7% (838/1185). Survival rates were higher for Day 5 early blastocysts (86.7%) compared with full (78.7%) or expanded blastocysts (72.7%). A reduced survival rate of 70.1% was found for Day 6 blastocysts compared with Day 5 blastocysts (80.6%, P < 0.001). The overall clinical/ongoing pregnancy rate after SET was 16.4/14.2% and 24.4/20.5% after DET with an ongoing multiple pregnancy rate of 21.8% (7/32) after DET. Significantly lower implantation rates were found for Day 5 early blastocysts (10.6%) compared with advanced blastocysts (17.5%, P < 0.05). Similar implantation potentials for Day 5 and 6 blastocysts (14.3 versus 13.7%) were found. CONCLUSIONS: Successful cryopreservation of blastocysts from the early cavitating up to expanded blastocyst stages is possible using a closed HS device. The choice between single or double frozen blastocyst transfer should depend on blastocyst expansion after vitrification.

Offering excess oocyte aspiration and vitrification to patients undergoing stimulated artificial insemination cycles can reduce the multiple pregnancy risk and accumulate oocytes for later use.

BACKGROUND: The prevention of multiple pregnancies remains a major challenge in patients treated with ovarian stimulation prior to intrauterine insemination (IUI). The pilot study presented here investigates whether multiple pregnancies can be minimized by a microscopically confirmed aspiration of oocytes from supernumerary follicles immediately before intrauterine insemination and evaluates the benefit of concomitant excess oocyte cryopreservation for future use. METHODS: Thirty-four aspirations of supernumerary follicles were performed immediately prior to IUI in 31 patients undergoing ovarian stimulation. sIUI was only performed if cumulus-oocyte complexes were microscopically observed in the aspirated follicular fluid. All collected mature excess oocytes were cryopreserved using the vitrification technique. RESULTS: Only four sIUI procedures had to be cancelled due to failed oocyte retrieval or premature ovulation. IUI treatment resulted in a clinical pregnancy rate of 23.5% per cycle. All were singleton pregnancies. A total of 111 oocytes were cryopreserved. Patients with polycystic ovary syndrome (PCOS) had an average of 6.07 oocytes vitrified, whereas patients without PCOS had 1.3 oocytes vitrified per cycle. CONCLUSION: Microscopically confirmed collection of excess oocytes prior to stimulated IUI reduced cancellation rates, further reduced the risk for multiple pregnancy and may lead to future additional pregnancies because, based on current information, approximately 5% of the vitrified oocytes could potentially establish a pregnancy.

The incidence of monozygotic twinning following PGD is not increased.

BACKGROUND: Monozygotic (MZ) twin pregnancies are associated with increased perinatal mortality and morbidity, and risk of congenital anomalies. The causes of MZ twinning in humans are unclear but the incidence may increase after PGD, for example, as a result of holes created in the zona pellucida. We compared the incidence of MZ twin pregnancies in ICSI cycles with PGD, versus ICSI cycles without PGD. METHODS: In this retrospective comparative cohort study, we analysed incidence of twin pregnancies in unselected patients undergoing ICSI and PGD (group A; 1992 cycles) with blastocyst transfer at Day 5, versus a period-matched control population of unselected patients undergoing ICSI and blastocyst transfer at Day 5 without PGD (group B; 2429 cycles) from January 2001 to December 2006. RESULTS: Clinical pregnancy per embryo transfer was established in 618/1992 (31.0%) and 947/2429 (39.0%) in group A versus B, respectively (P < 0.01). Overall MZ twin rate was 29/4421 (0.7%) per embryo transfer and 29/1565 (1.9%) per established clinical pregnancy. The incidence of MZ twinning per established clinical pregnancy did not differ between groups (1.5 versus 2.1%, group A and B, respectively). In group A, seven MZ twins were born versus 19 MZ twins in group B. In group B, one MZ twin pregnancy resulted in two stillbirths. In group A, two MZ twins had severe congenital malformations versus none in group B. CONCLUSIONS: The incidence of MZ twinning was not increased in PGD compared with regular ICSI with blastocyst transfer. This information is useful in counselling patients about potential risks of PGD.

Does the estradiol level on the day of human chorionic gonadotrophin administration have an impact on pregnancy rates in patients treated with rec-FSH/GnRH antagonist?

BACKGROUND: The purpose of this prospective observational study was to evaluate the association between estradiol (E(2)) levels on the day of human chorionic gonadotrophin (hCG) administration and pregnancy rates in a recombinant FSH (rec-FSH) antagonist fixed protocol. METHODS: A group of 207 patients (or=3 follicles of >or=17 mm diameter. One to two embryos were transferred on Day 3 after oocyte retrieval. RESULTS: The area under the curve (AUC) for E(2) on the day of hCG could not distinguish between pregnant and non-pregnant women (AUC:0.5; 95% confidence interval (CI): 0.42-0.59). No significant difference was observed between the three percentile groups of E(2) values on the day of hCG administration [group 1, lower 25th percentile (<1142 pg/ml); group 2, medium 50th percentile (1142-2446 pg/ml) and group 3, higher 75th percentile (>2446 pg/ml)] for the ongoing pregnancy rates (P = 0.52). On the contrary, the linear regression model showed that higher E(2) values on the day of hCG administration significantly improved the scores of transferred embryos (P = 0.01) as well as the total embryo score (P = 0.02). Yet subgroup analysis only in this high responders group revealed lower E(2) and progesterone levels on the day of hCG in pregnant women compared with the non-pregnant (P = 0.01). CONCLUSIONS: E(2) concentrations on the day of hCG administration in GnRH antagonist cycles are not associated with pregnancy rates. A potential deleterious impact of estradiol on endometrial receptivity is shown for the high responders who have high E(2) levels and improved embryo quality without a concomitant rise in pregnancy rate.