Impact of oxygen tension according to embryo stage of development: a prospective randomized study.

Human embryo culture under 2-8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional¼ conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B' (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B'(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B' and C. Furthermore, blastocyst quality was statistically equivalent between groups B' and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B' and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B' and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

[Prospective comparison of different techniques for cryopreservation of small numbers of human spermatozoa]. / Étude prospective comparant plusieurs techniques de congélation de faibles nombres de spermatozoïdes humains.

OBJECTIVES: This study aimed to determine if vitrification is more efficient than the slow freezing method when cryopreservation of small numbers of human spermatozoa is needed and, if so, which device is the most suitable. METHODS: This is a prospective experimental study conducted in a university-affiliated assisted reproductive center. Ejaculates were obtained from the same fertile man after written consent. Selected sperm were cryopreserved by slow freezing method or vitrified in two different devices: Cell Sleeper and Stripper tip. RESULTS: Vitrification in Cell Sleeper provided significantly higher recovery, motility and survival rates than slow freezing. Only recovery rate was higher in Cell Sleeper than when using Stripper tip. Moreover, recovery time per spermatozoon was faster in Cell Sleeper than in the two other groups. Furthermore, statistical significance was achieved when comparing the four above parameters between Stripper tip and slow freezing, concluding to the superiority of vitrification in Stripper tip as well. Finally, the theorical time needed per injected oocyte was significantly shorter after vitrification in the two devices than when using slow freezing, and shorter in Cell Sleeper in comparison with Stripper tip. CONCLUSIONS: Vitrification, in particular using Cell Sleeper, appears as the most suitable method to cryopreserve small numbers of spermatozoa.

[Dual trigger with gonadotropin-releasing hormone agonist and hCG to improve oocyte maturation rate]. / Intérêt du double déclenchement par agoniste de la GnRH et hCG en cas d'antécédent d'immaturité ovocytaire en FIV/ICSI.

OBJECTIVE: This study investigates dual trigger with GnRHa and hCG as a potential treatment in patients with a history of ≥25 % immature oocytes retrieved in IVF/ICSI cycles. METHODS: This is a retrospective case-control study performed between October 2008 and December 2017. Forty-seven patients who experienced high oocyte immaturity rate (≥25 %) during their first IVF/ICSI cycle (analyzed as control group) and received a dual trigger for their subsequent cycle, were involved. During dual trigger cycles, patients received antagonist protocol and ovulation triggering using triptorelin 0.2mg and hCG. Primary endpoint was maturation rate (MR). Secondary endpoints were fertilization, D2 top quality embryo (TQE) rates, clinical pregnancy rate per fresh embryo transfer and cumulative clinical pregnancy rate per couple. RESULTS: A significant increase in MR was achieved in case of dual trigger (71.0 %) when compared to control group (47.8 %; P<0.0001). Moreover, cumulative clinical pregnancy rate yielded 46.8 % in dual trigger group, which was statistically higher than 27.6 % obtained in control group (P=0.05). However, fertilization, D2 TQE rates and clinical pregnancy rates/transfer were statistically similar when compared between the two groups. CONCLUSION: Dual trigger seems efficient for managing patients with high oocyte immaturity rate.

Use of the endometriosis fertility index in daily practice: A prospective evaluation.

OBJECTIVE: To perform a prospective evaluation of postoperative fertility management using the endometriosis fertility index (EFI). STUDY: This prospective non-interventional observational study was performed from January 2013 to February 2016 in a tertiary care university hospital and an assisted reproductive technology (ART) centre. In total, 196 patients underwent laparoscopic surgery for endometriosis-related infertility. Indications for surgery included pelvic pain (dysmenorrhoea, and/or deep dyspareunia), abnormal hysterosalpingogram, and failure to conceive after three or more superovulation cycles with or without intra-uterine insemination. Multidisciplinary fertility management followed the surgical diagnosis and treatment of endometriosis. Three postoperative options were proposed to couples based on the EFI score: EFI score ≤4, ART (Option 1); EFI score 5-6, non-ART management for 4-6 months followed by ART (Option 2); or EFI score ≥7, non-ART management for 6-9 months followed by ART (Option 3). The main outcomes were non-ART pregnancy rates and cumulative pregnancy rates according to EFI score. Univariate and multivariate analyses with backward stepwise logistic regression were used to explain the occurrence of non-ART pregnancy after surgery for women with EFI scores ≥5. Adjustment was made for potential confounding variables that were significant (p<0.05) or tending towards significance (p<0.1) on univariate analysis. RESULTS: The cumulative pregnancy rate was 76%. The total number of women and pregnancy rates for Options 1, 2 and 3 were: 26 and 42.3%; 56 and 67.9%; and 114 and 87.7%, respectively. The non-ART pregnancy rates for Options 1, 2 and 3 were 0%, 30.5% and 48.2%, respectively. The ART pregnancy rates for Options 1, 2 and 3 were 50%, 60.6% and 80.3%, respectively. The mean time to conceive for non-ART pregnancies was 4.2 months. The benefit of ART was inversely correlated with the mean EFI score. On multivariate analysis, the EFI score was significantly associated with non-ART pregnancy (odds ratio 1.629, 95% confidence interval 1.235-2.150). CONCLUSION: In daily prospective practice, the EFI was useful for subsequent postoperative fertility management in infertile patients with endometriosis.

Non-ART pregnancy predictive factors in infertile patients with peritoneal superficial endometriosis.

OBJECTIVE: To study the predictive factors for non-ART pregnancy in infertile women after laparoscopic diagnosis and surgery for isolated superficial peritoneal endometriosis (SUP). STUDY DESIGN: Retrospective observational study from January-2004 to December-2015 in a tertiary care university hospital and Assisted Reproductive Technology (ART) centre. Infertile women with laparoscopic surgery for SUP (with histologic diagnosis) were included. The surgical treatment was followed by spontaneous fertility or post-operative ovarian stimulation (pOS) using superovulation (gonadotrophins)±Intra Uterine Insemination (IUI). The main outcomes were the non-ART clinical pregnancy rates and its predictive factors. RESULT(S): Over the period study, 315 women were included. Of these, 133 (42.3%) women had non-ART pregnancy. The mean time to conceive was 6 months (±6days). Univariate analysis for non-ART pregnancy after surgery showed that: (i) no difference was observed according to age, length of infertility, Body Mass Index (BMI), the rate of previous pregnancy, and the pre-operative ovarian stimulation rate; (ii) diminished ovarian reserve and previous miscarriage were higher in the non-pregnant women group (8.3 versus 19.1%, p<0.05; 3.5% versus 9%, p=0.06, respectively); (iii) the mean EFI score and pOS were higher in pregnant women (7.7 versus 7.2, p=0.02; 49.2% versus 26.7%, p<0.01); and (iv) IUI did not show any benefit for pregnancy (22% after superovulation versus 27.2% after superovulation and IUI). In the multivariate analysis, only pOS (adjusted OR 2.504, 95% CI [1.537-4.077]) and DOR (aOR 0.420, 95% CI [0.198-0.891]) remained significantly associated with the incidence of pregnancy. CONCLUSION(S): After laparoscopic surgery for peritoneal superficial endometriosis related infertility, ovarian stimulation improved pregnancy rate, while diminished ovarian reserve had a worse prognosis for pregnancy.

[A prospective study to compare the efficiency of oocyte vitrification using closed or open devices]. / Étude prospective comparant l'efficacité de la vitrification ovocytaire en système fermé ou en système ouvert.

OBJECTIVES: Oocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). METHODS: Forty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). RESULTS: The global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). CONCLUSION: Taking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.

[Embryo development in two single-step media: Analysis of 2059 sibling oocytes]. / Développement embryonnaire dans 2 milieux de culture globale : étude prospective autocontrôlée sur 2059 ovocytes.

OBJECTIVE: The aim of this study was to compare embryo development cultured in two single-step media commercially available: Fert/Sage One Step® (Origio) and Continuous Single Culture® (CSC) (Irvine Scientific). METHODS: A prospective auto-controlled study of sibling oocytes from women undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed in our center from February to June 2015. After fertilization, for every patient, half of oocytes were cultured in the single-step Fert/Sage One Step® (serie SAGE) and the other half in the single-step CSC®(serie CSC). Fertilization and embryo morphology rates were assessed by day 1 to day 5-6 if needed. Embryo presenting<20% of fragmentation and 4 cells at day 2, 8 cells at day 3 were qualified as "top quality". Embryo with<20% of fragmentation and 3-5 cells at day 2, 6-10 cells at day 3 were qualified as "good quality". Blastocyst B3, B4, B5 with A or B inner cell mass and A or B trophectoderm were qualified as "good quality". Transferred or frozen embryos were qualified as useful embryos. RESULTS: Sixty-two attempts of IVF and 133 of ICSI were analyzed, corresponding to 2059 inseminated or micro-injected oocytes. Fertilization rate were not different between the 2 series, respectively SAGE vs CSC (IVF: 73.4% vs 68.3% [P=0.49]; ICSI: 58.9% vs 63.8% [P=0.12]). No difference was found for embryo morphology, respectively SAGE vs CSC, at day 2 (top quality embryo at day 2 IVF: 34.4% vs 33% [P=0.98]; ICSI: 42.4% vs 44.9% [P=0.37]; and good quality embryo at day 2 IVF: 44% vs 50.2% [P=0.07]; ICSI: 64% vs 71% [P=0.35]); no difference at day 3 (top quality embryo at day 3 IVF: 19.4% vs 21.3% [P=0.61]; ICSI: 28.7% vs 27.4% [P=0.54]; and good quality embryo at day 3 IVF: 40.4% vs 50.2% [P=0.91]; ICSI: 51% vs 47.6% [P=0.47]). Blastocyst development rate were not different, respectively SAGE vs CSC (IVF: 39.9% vs 41.5% [P=0.63] with 42.9% vs 42.2% of good quality blastocyst [P=0.70]; ICSI: 41.1% vs 37.8% [P=0.18] with 32.9% vs 40.8% of good quality blastocyst [P=0.13]). No difference was found in the useful embryo rate in the 2 series SAGE vs CSC (IVF: 52.8% vs 55.2% [P=0.83]; ICSI: 62.4% vs 61.7% [P=0.70]). CONCLUSION: Embryo development and rate of useful embryos, transferred or frozen, were not different according to the embryo culture in single-step media Fert/Sage One Step® vs single-step Continuous Single Culture®.

Delivery rates after elective single cryopreserved embryo transfer related to embryo survival.

OBJECTIVE: The objective of this study was to assess if eSCET (elective Single Cryopreserved Embryo Transfer) outcome is related to blastomere survival rate. The final objective was to avoid multiple pregnancies and offer the best chances to women to achieve pregnancy even during their frozen-thawed embryo transfer (FET) cycles. STUDY DESIGN: Patients were included in this prospective observational study if they met the following criteria: (i) women age <37 years old; (ii) IVF of ICSI cycle rank ≤2, (iii) eSET proposed during fresh embryo transfer cycle and (iv) ≥1 good quality cryopreserved embryos available (<20% fragmentation and 4-5 blastomeres at day-2 or 7-9 blastomeres at day-3). Live birth rates (LBR) were compared into eSCET groups according to embryo survival (partially damaged or intact transferred embryo). RESULTS: We observed among selected patients, that partial loss of blastomeres (1 blastomere for day-2 embryos, 1 or 2 blastomeres for day-3 embryos) following FET cycles did not affect LBR compared with intact embryo. CONCLUSION: These results underline the relevance of eSCET as a strategy to reduce multiple pregnancies frequency without reducing LBR.

How to select the spermatozoon for intracytoplasmic sperm injection in 2015?

The selection of the individual spermatozoon in intracytoplasmic sperm injection (ICSI) is routinely performed by the observation of its motility and morphology. However, in case of severe oligoasthenozoospermia or non-obstructive azoospermia requiring the use of testicular sperm, other methods are necessary to help the embryologist making this choice. According to some authors, sperm processing before ICSI seems to limit the DNA fragmentation index, and in this way improves ICSI outcomes. Moreover, intracytoplasmic morphologically selected sperm injection is potentially a good option in some specific indications such as severe teratozoospermia, or repeated ICSI failures. Other methods based on sperm structure, as sperm head birefringence observation, or based on its function, like the hyaluronic acid or zona pellucida binding capacity, could be of interest, but still need to be evaluated. Finally, in case of akinetozoospermia, the use of functional tests, such as pentoxifylline test, HOS-test, or to a lesser extent laser touch, makes the selection of viable spermatozoa easier. Nevertheless, studies on larger series have to be conducted to evaluate and precise the interest of each of these methods and their indications, before considering an application on larger scale.

[How to select the spermatozoon in ICSI?]. / Comment choisir le spermatozoïde en ICSI ?

The selection of the individual spermatozoon in ICSI is routinely performed by the observation of its motility and morphology. However, in case of severe oligoasthenozoospermia or non-obstructive azoospermia needing the use of testicular sperm, other methods are necessary to help the embryologist making this choice. According to some authors, sperm processing before ICSI seems to limit the DNA fragmentation index, and in this way improve ICSI outcomes. Moreover, IMSI is potentially a good option in some specific indications such as severe teratozoospermia, or repeated ICSI failures. Other methods based on sperm structure, as sperm head birefringence observation, or based on its function, like the hyaluronic acid or zona pellucida binding capacity, could be of interest, but still need to be confirmed. Finally, in case of akinetozoospermia, the use of functional tests, such as pentoxifylline test, HOS-test, or to a lesser extent laser touch, makes the selection of viable spermatozoa easier. Nevertheless, studies on larger series have to be conducted to evaluate and precise the interest of each of these methods and their indications, before considering an application on larger scale.