Conventional ICSI Research Articles

Parameters of poor prognosis in preimplantation genetic testing for monogenic disorders

Abstract STUDY QUESTION What is the likelihood of success of a single cycle of preimplantation genetic testing for monogenic disorders (PGT-M), measured as the cumulative live birth rate (CLBR) and based on various patient demographics? SUMMARY ANSWER For all women aged ≤40 years, the CLBR was at least 10% when the number of oocytes was ≥7 (range 10–30%) or was at least 5% when the number of oocytes was ≥3 (range 5–17%). WHAT IS KNOWN ALREADY The number of oocytes is significantly associated with the number of embryos for genetic testing and the clinical outcome in PGT-M. Embryos diagnosed as affected or embryos that remain without diagnosis cannot be used for embryo transfer. The size of the group of embryos non-suitable for transfer varies between 25% and 81%, depending on the indication. Thus, PGT-M is more likely to be more severely impacted by suboptimal ovarian response, poor fertilization and suboptimal embryo development than conventional IVF/ICSI schemes without PGT. STUDY DESIGN, SIZE, DURATION This was a single-centre retrospective comparative cohort study, of cycles between January 2011 and December 2015. A total number of 2265 PGT-M cycles were compared to 2833 conventional ICSI cycles. The principal aim of our study was the identification of the parameters of poor CLBR in couples undergoing PGT-M using multiplex short tandem repeat (STR) markers on blastomere biopsy DNA. The secondary aim was to compare the parameters of poor CLBR of the PGT-M population to those of couples undergoing ICSI without PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS The baseline characteristics of the PGT-M group were compared to the conventional ICSI group. A multiple regression analysis was applied to account for the following potential confounding factors: female age, number of previous ART cycles, number of oocytes/suitable embryos for transfer and dosage of gonadotrophins used for ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE The PGT-M group was younger (female age 32.0 vs 34.5 years), had a higher number of previous ART cycles (1.1 vs 0.9 cycles) and used more gonadotrophins (2367 vs 1984 IU). Per cycle, the PGT-M group had more retrieved oocytes (11.8 vs 8.3 oocytes), fewer suitable embryos for transfer (1.7 vs 2.8 embryos) and a lower CLBR (29.4% vs 35.0%). Multiple regression analysis showed that the CLBR in the PGT-M group was significantly influenced by female age, the number of previous ART cycles, the number of oocytes and the dose of ovarian stimulation. In both groups, the predicted CLBR increased with increasing numbers of oocytes and suitable embryos. At least two retrieved oocytes or one embryo per single PGT-M cycle could confer an estimated CLBR above 10%. By assessing female age and the number of retrieved oocytes together, it was shown that for all women aged ≤40 years, the predicted CLBR per single PGT-M cycle was ≥10% when the number of oocytes was ≥7 or was ≥5% when the number of oocytes was ≥3. LIMITATIONS, REASONS FOR CAUTION Despite the large sample size, the findings are confined by limited confounder adjustment and the lack of specific PGT-M comparators. WIDER IMPLICATIONS OF THE FINDINGS This study aimed to describe the likelihood of success of PGT-M treatment, measured as CLBR, based on various patient demographics. In a PGT-M program, couples need to be informed of the prognosis more specifically when it is futile. The table of predicted CLBRs presented in this study is a useful tool in counselling PGT-M couples for making reproductive choices. STUDY FUNDING/COMPETING INTEREST(S) No funding was required and there are no competing interests. TRIAL REGISTRATION NUMBER N/A.

Single-center thorough evaluation and targeted treatment of globozoospermic men

PurposeTo characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment.MethodsWe assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA).ResultsUltrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome.ConclusionsSperm bioassays and genomic studies can be used to characterize this gamete’s capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Relationship between follicular size and developmental capacity of oocytes under controlled ovarian hyperstimulation in assisted reproductive technologies.

PurposeWe investigate the relationships between oocyte developmental capacity and follicular size of its origin in Japanese women: those undergoing conventional IVF (cIVF) and ICSI, respectively.MethodsA total of 3377 follicles were punctured separately and were classified into three groups (large, medium, and small) by their diameters. A total of 1482 retrieved oocytes were individually cultured and received cIVF or ICSI. The oocytes receiving ICSI were denuded and the number of mature (MII) oocytes was counted.ResultsThe oocyte retrieval rates and the proportion of MII oocytes were significantly lower in small follicles than in large follicles. Under cIVF, the fertilization rate was significantly lower in oocytes from small follicles than large follicles. Under ICSI, the fertilization rate for MII oocytes was not significantly related to follicular size. Follicular size was not significantly related to the development potential to blastocyst and pregnancy rate for either the cIVF oocytes or the ICSI oocytes.ConclusionsAlthough the fertilization rate by cIVF is low in oocytes from small follicles due to the lower proportion of mature oocytes, their development potential is comparable to that of oocytes from larger follicles if they could be fertilized. Under ICSI using mature oocytes, their development potential is not related to follicular size.

Do in vitro fertilization, intrauterine insemination or female infertility impact the risk of congenital anomalies in singletons? A longitudinal national French study.

Do IVF, IUI or female infertility (i.e. endometriosis, polycystic ovary syndrome [PCOS] and primary ovarian insufficiency [POI]) lead to an increased risk of congenital anomalies in singletons? After multivariable adjustments, the increased risks of congenital defects associated with IUI were no longer significant, but the underlying maternal infertility presented a potential emental risk, in addition to the risk associated with IVF. Most epidemiological studies suggest that singletons born from ART have a higher risk of birth defects, specifically musculoskeletal, cardiovascular and urogenital disorders. However, most of these studies were established on data obtained at birth or in the neonatal period and from relatively small populations or several registries. Moreover, to our knowledge, female infertility, which is a potential confounder, has never been included in the risk assessment. Using data from the French National Health System database, we conducted a comparative analysis of all singleton births (deliveries ≥22 weeks of gestation and/or >500 g of birthweight) in France over a 5-year period (2013-2017) resulting from fresh embryo or frozen embryo transfer (fresh-ET or FET from IVF/ICSI cycles), IUI and natural conception (NC). Data were available for this cohort of children at least up to early childhood (2.5 years old). A total of 3 501 495 singleton births were included (3 417 089 from NC, 20 218 from IUI, 45 303 from fresh-ET and 18 885 from FET). Data were extracted from national health databases and used to identify major birth defects. Malformations were classified according to the 10th revision of the International Classification of Disease. To analyse the effect of mode of conception, multivariable analyses were performed with multiple logistic regression models adjusted for maternal age, primiparity, obesity, smoking, history of high blood pressure or diabetes and female infertility. In our cohort of children, the overall prevalence of congenital malformations was 3.78% after NC, 4.53% after fresh-ET, 4.39% after FET and 3.91% after IUI (132 646 children with major malformations). Compared with infants conceived naturally, children born after fresh-ET and after FET had a significantly higher prevalence of malformations, with an adjusted odds ratio (aOR) of 1.15 [95% CI 1.10-1.20, P < 0.0001] and aOR of 1.13 [95% CI 1.05-1.21, P = 0.001], respectively. Among the 15 relevant subgroups of malformations studied, we observed a significantly increased risk of eight malformations in the fresh-ET group compared with the NC group (i.e. musculoskeletal, cardiac, urinary, digestive, neurological, cleft lip and/or palate and respiratory). In the FET group, this increased risk was observed for digestive and facial malformations. The overall risk of congenital malformations, and the risk by subtype, was similar in the IUI group and the NC group (overall risk: aOR of 1.01 [95% CI 0.94-1.08, P = 0.81]). In addition, there was an overall independent increase in the risk of congenital defects when the mothers were diagnosed with endometriosis (1.16 aOR [95% CI 1.10-1.22], P < 0.0001), PCOS (1.20 aOR [95% CI 1.08-1.34], P = 0.001) or POI (1.52 aOR [95% CI 1.23-1.88], P = 0.0001). Chromosomal, cardiac and neurological anomalies were more common in the three maternal infertility groups. Male infertility, the in vitro fertilization method (i.e. in vitro fertilization without or with sperm injection: conventional IVF vs ICSI) and embryo stage at transfer could not be taken into account. Furthermore, residual confounding cannot be excluded as well as uncertainties regarding the diagnostic criteria used for the three female infertilities. Findings for specific malformations should be interpreted with caution because the number of cases was small in some sub-groups (potentially due to the Type I error or multiple testing). In this large study, after multivariable maternal adjustments, a moderately increased risk of defects subsisted after IVF, while those associated with IUI were no longer significant. In addition, our results showed that underlying maternal infertility could contribute to the increased risk of defects associated with IVF. These novel findings highlight the importance of taking into account the ART treatment methods and the type of infertility. This work was supported by the National Agency of Biomedicine. The authors have no competing interests to disclose. NA.

A randomized controlled trial of laser-assisted ICSI

Does the use of a laser to open the zona pellucida during ICSI (laser assisted or LA-ICSI) improve oocyte survival, embryo development and clinical outcomes? Compared to conventional ICSI, LA-ICSI increased rates of oocyte survival and some aspects of embryo development but it did not alter the ongoing pregnancy rate; after adjusting for oocyte survival, there was no beneficial effect of LA-ICSI on embryo development and utilization. Oocyte degeneration occurs in a 10th of mature oocytes after ICSI. Pilot studies suggest that LA-ICSI may improve oocyte survivability. In a randomized controlled trial, 966 couples (16122 metaphase II oocytes) were allocated to receive LA-ICSI (intervention) or conventional ICSI (control) between 17 September 2018 and 5 August 2019. Oocyte survival (primary endpoint), embryo development and ongoing pregnancy rates were compared. Couples included in this study were recommended for ICSI due to female or male factor, unexplained infertility or a combination of factors. Patients were ineligible to participate in the study if they had uterine abnormality including thin endometrium, recurrent pregnancy loss, endometriosis or a severe medical condition. Concealed randomization to LA-ICSI or conventional ICSI, allocated in a 1:1 ratio, took place on stimulation Day 1 with replacement of blastocysts on only Day 5. The primary endpoint was oocyte survival with membrane integrity 24 h after the ICSI procedure. The sample size was estimated to detect a 3% increase in oocyte survival after LA-ICSI with 99% power at a 1% significance level. This also permitted the detection of 10% increase in ongoing pregnancy rate after LA-ICSI with 85% power at 5% alpha level. We used Poisson regression with zero-inflation for count data to estimate relative risk (RR) with 95% CI and logistic regression for clinical outcomes to estimate odds ratio (OR) with 95% CI. Both models adjusted for age as a covariate. Compared with conventional ICSI, LA-ICSI resulted in a higher number of surviving oocytes (RR 1.08, 95% CI 1.05-1.12, P < 0.001), as well as a higher number of fertilized oocytes (RR 1.08, 95% CI 1.04-1.13, P < 0.001) and utilizable blastocysts (RR 1.09, 95% CI 1.04-1.15, P < 0.001). Sensitivity analyses adjusted for oocyte survival showed no between-group difference in utilizable blastocysts (OR 1.01, 95% CI 0.95-1.08, P = 0.73) and by calculating the mean rate, a reduction in utilizable blastocysts was shown (RR 0.95, 95% CI 0.94-0.97, P < 0.001). Ongoing pregnancy showed no between-group difference (LA-ICSI 179/489 (37%) vs ICSI 201/477 (42%), OR 0.79, 95% CI 0.61-1.03, P = 0.09). It was not possible to blind the embryologists involved in the ICSI procedure. However, there was concealment of randomization and blinding of outcome assessments reducing the risk of selection and measurement bias. A beneficial effect of LA-ICSI on oocyte survival should be shown to improve clinical outcomes, before its use in clinical practice is justified. The study received no funding, and the authors declare that there are no conflicts of interest. NCT03665103. 11 September 2018. 17 September 2018.

Análisis de los indicadores de calidad entre los diferentes centros públicos españoles en técnicas de reproducción asistida; benchmarking

IntroductionPublic centers with IVF/ICSI laboratory (In Vitro Fertilization/Intracytoplasmic Sperm Injection) must guarantee both the efficacy and safety of treatment in patients. Benchmarking offers us a vision of where we should direct our efforts in this regard. The objective of this work is to identify if the procedures carried out by national public centers are effective and safe. Material and methodsBased on the National Activity Registry - Spanish Fertility Society Registry and an online survey, data on results, clinical practice and physical and human resources were obtained from 42 public centers that are included in its IVF/ICSI service portfolio. The results were compared with the specification at the desirable level of the quality indicators of UNE 179007:2013 (Quality Management Systems for Assisted Reproduction Laboratories) established by the Association for the Study of Reproduction Biology (ASEBIR). Those of greater efficiency and safety were defined and identified as ‘excellent’ centers, in order to carry out a benchmarking study. ResultsThe average of the quality indicators analyzed in public centers, reached and/or exceeded the ASEBIR quality specifications at the desirable level. Two centers were considered ‘excellent’ for presenting a higher gestation rate with a low percentage of multiple gestations. These centers had a percentage of contraceptive cycle programming, embryonic transfer in blastocyst, deferred transfer, elective transfer of single embryo and percentage of cycles with conventional IVF versus ICSI higher than the average of public centers. Evaluating the human, physical resources and the weekly activity, no relation was obtained with better results. ConclusionsExcellence in Public Centers is related more to a set of procedures than to a single one.

The impact of male factor infertility on early and late morphokinetic parameters: a retrospective analysis of 4126 time-lapse monitored embryos.

Is there an effect of male factor infertility (MFI) on either early or late morphokinetic parameters obtained during embryonic culture to blastocyst stage in a time-lapse imaging (TLI) incubator? Neither mild nor severe MFI had an impact on overall time to blastocyst or duration of individual cleavage stages in the total embryo population. Prior studies have suggested that paternal DNA and sperm quality affect embryo morphokinetic parameters, but the impact of MFI is not fully understood. This retrospective cohort study, at a major academic fertility centre, included 536 couples (women, ≤44years of age) undergoing IVF between September 2013 and September 2016. Data from 4126 embryos cultured to the blastocyst stage in a TLI-monitored incubator were retrospectively reviewed. Embryos derived from the sperm of men with MFI were compared with those derived from patients with other infertility diagnoses. Generalized fixed and random effects models, t-test and χ2 were used as appropriate. Couples with MFI had a higher rate of ICSI utilization and fewer usable embryos on average, and the men were older compared with couples with other diagnoses. Additionally, the women in MFI couples were younger and had higher antral follicle counts (AFCs) and higher anti-Müllerian hormone (AMH) levels compared with the other women undergoing IVF. When controlling for maternal and paternal ages, AMH and fertilization method (conventional IVF versus ICSI), neither mild nor severe MFI affected duration of individual cleavage stages or overall time to the blastocyst stage, when all or only usable embryos were examined (coefficient 0.44hours in all embryos, P = 0.57; coefficient 0.39hours in usable embryos, P = 0.60). Whether the sperm was surgically extracted similarly had no significant effect on embryo morphokinetic parameters. When the fertilization method was assessed independently, ICSI lengthened the overall time to blastocyst stage by 1.66hours (P = 0.03) on average, primarily due to an increase in duration of the time from 5-cell embryo stage to early blastulation (P5SB). This large cohort study avoided embryo selection bias due to random assignment of embryos to the TLI incubators. However, our findings may not be generalizable to groups under-represented in our clinic population. Future studies should also evaluate the impact of male hormonal status and detailed sperm morphology, such as head versus flagellum defects, on embryo morphokinetic development. Our findings suggest that the fertilization method rather than MFI per se impacts time to early blastulation. The clinical implications of this effect on embryo development warrant further investigation. There were no sources of funding for this study. There are no competing interests. N/A.

The clinical results of piezo-ICSI compared to conventional-ICSI: a sibling-oocyte study

Clinically, conventional ICSI (CI) is a common, widely-used method, while there are few reports with respect to Piezo ICSI (PI). PI is effective in degeneration rate and fertilization rate (Kimura, Y & Yanagimachi, R, 1995). It is known that the survival rates of mice oocytes are low after CI; however, degeneration rate improved markedly using PI. PI is an effective technique for cases with fragile oocytes. It has been reported that the survival rate is similarly improved in human oocytes (Hiraoka, K & Kitamura, S, 2015). However, most of the studies reporting on PI are retrospective studies. Here, we prospectively compared the degeneration rates, fertilization rates and embryo development of PI and CI in a sibling study. This is a prospective randomized controlled single-center study, using sibling oocytes, conducted from August 2018 to March 2019. Written informed consent was obtained from all patients involved in this study. This sibling oocyte study comprised 26 cycles in 26 cases. CI was performed in 149 mature oocytes. CI consists of mechanical penetration of the zona pellucida, breaking the oocyte membrane by aspiration of cytoplasm. PI was performed in 162 mature oocytes. PI consists of breaking the oocyte membrane and zona pellucida by Piezo pulse. P-value of 0.05 or less was considered to be statistically significant.　Limitation: The clinical results using vitrified oocytes, artificial oocyte activation, cryptozoospermia, azoospermia cases, and females aged over 40 years old were not included in this study. The pregnancy outcome has not been confirmed in our study. There were no statistically significant differences in the fertilization rates, degeneration rates, cleavage rates, blastocyst formation rates or good quality blastocyst rates (according to the Gardner criteria) between CI and PI (75.8% vs. 78.4% (P=0.592), 7.4% vs. 3.7% (P=0.146), 100% vs. 96.8% (P=0.160), 61.9% vs. 64.0% (P=0.743) and 41.6% vs. 36.8% (P=0.450), respectively). In conclusion, the present study has demonstrated there was no significant difference in the clinical results of piezo-ICSI and conventional-ICSI. However, this may be attributable to the limited number of cases with fragile oocytes, etc. In our experience, PI is safer and easier to learn and perform in clinical work in a shorter period, especially for beginners. Further studies are necessary.Tabled 1GroupConventional ICSIPiezo ICSIPPatients (N)26-No. of ICSI cycles26-Female age, years (mean ± SD)34.8 ± 4.2-Male age, years (mean ± SD)36.9 ± 5.4-No. of oocytes149162-N(%) of zygotes (2PN)113 (75.8)127 (78.4)0.592N(%) of degenerated oocytes11 (7.4)6 (3.7)0.146N(%) of cleaved oocytes113 (100)122 (96.8)0.167N(%) of blastocysts70 (61.9)80 (64.0)0.477N(%) of good quality blastocysts47 (41.6)46 (36.8)0.498 Open table in a new tab

Blastocyst formation and pregnancy rate comparison between standard large box (Sanyo) incubators and a novel Geri® time-lapse incubator

Objective We compared the blastulation rates and pregnancy success from embryos cultured in standard “large box” Sanyo incubators and the Geri® time-lapse incubator. Design A retrospective study in a private IVF laboratory. Materials and Methods Mature oocytes were fertilized by either conventional insemination or ICSI and cultured to the blastocyst stage undisturbed in Continuous Single Culture®-NX (Irvine Scientific) at 37°C in 6% CO2. Patients could self-elect to be included in the Geri incubator prior to retrieval. Availability for the Geri was limited to six culture chambers. Patients with a minimum of one mature oocyte with normal fertilization were eligible to have their embryos cultured in the Geri. All embryos were either biopsied and/or frozen on Day 5, 6 or 7 of development. Results The evaluation was performed over a six-month period (August 2018-February 2019). A total of 67 patients had their embryos cultured in the Geri. A total of 611 zygotes were cultured to the blastocyst stage, resulting in 247 blastocysts – a 40.4% blastulation rate. In comparison, 288 patients yielded 2,387 zygotes that were cultured in the Sanyo incubators, resulting in 1,055 blastocysts a 44.2% blastulation rate. Chi-square analysis showed no significant difference in blastulation rates (p=0.0932) between incubator types. In addition, 279 frozen embryo transfers (16 Geri and 264 control) were performed with no significant difference (p=0.9556) in pregnancy success, 33% and 34%, respectively. Pregnancy success was identified by positive fetal cardiac activity at seven weeks gestation. Time-lapse morphokinetic assessment was not applied during this evaluation period. Conclusions In the IVF laboratory, several types of incubators are available, and each requires careful consideration. The Geri, an independent chamber/microscope incubator, offers a safe environment for embryo culture with minimal disturbance. Blastulation rates did not differ when compared to the large box incubators, allowing for another option for embryo culture. The Geri has six chambers, which can accommodate a maximum of 16 embryos per Geri Dish, which may be limiting factor to some laboratories. However, the Geri Dish is unique in that it allows for potential benefits of group culture while maintaining individual embryo growth tracking. The ability to assess the embryos in real time does provide opportunities for clinical applications for embryo selection. We are continuing to monitor pregnancies and embryo culture outcomes accordingly. Disclosures EMD Serono provided the incubator at no cost during the embryo evaluation period. Funding None

Artificial oocyte activation (AOA) treatment offers a new option in previous unsuccessful cases due to poor fertilization history; a paired cohort study

To improve of treatment outcome in terms of fertilization, implantation and pregnancy rates as well as cancelation rate after applying AOA in the following treatments for those patients with previous very low or failed fertilization attempts. Additionally were tested both delivery rate and obstetric outcomes in children born after AOA use. Retrospective Cohort Study. Period included between April 2013 and December 2018. The total number of oocytes included in the study was 1125 distributed in two groups: the control group formed by 509 oocytes from 66 patients who were injected without AOA, acquiring either a failure or unusually low fertilization values (<30%) and the study group consisted of 616 oocytes from the same patients (66) in a second attempt but using AOA. Injection technique with AOA is comparable to conventional injection (ICSI) but in this case each oocyte was injected by spermatozoon together with a previous phase of buffered medium mixed with ionophore calcium (ICa) aspirating it up to filling one third of the overall length of the pipette under 40X microscope magnification. Just after, they were kept for ten minutes with Ica into benchtop incubator and finally were incubated under the same culture conditions (37ºC, 6% CO2, 5% O2 atmosphere) Fertilization, pregnancy, implantation and abortion rates were analysed and compared in both groups by Χ2, t-Student and ANOVA tests. Both live birth and neonatal outcome was also reported. After applying AOA, we observed a significant increase in the fertilization rate (51% vs 13.1%), ongoing pregnancy rate (47% vs. 21.7%) as well as implantation rate (31.1% vs 13.1%) and lower chances of cancellation (22.7% vs 69.3%). Additionally, no adverse obstetric and perinatal outcomes effects were found after the use of AOA compare to conventional ICSI. Our findings support the use of AOA for a specific population of patients with previous very low fertilization or fertilization failure and in consequence, the outcome was impaired. After applying AOA, the reproductive success was significantly enhanced, mainly by the significant increase in the fertilization rate, the number of embryos available for selection, reducing dramatically the cancellation rate. The safety of AOA is reflected in the current practice after confirming non-detrimental effects in the offspring.

Piezo-assisted ICSI improves fertilization and blastocyst development rates compared with conventional ICSI in women aged more than 35years.

PurposePiezo‐assisted intracytoplasmic sperm injection (Piezo‐ICSI) is reported to be an effective method for inseminating fragile oocytes compared with conventional ICSI (c‐ICSI). However, infertile patient groups suitable for Piezo‐ICSI have not been elucidated. This study was conducted to determine age groups suitable for Piezo‐ICSI using sibling egg controls inseminated by a well‐trained embryologist to reduce technical inequalities.MethodsA total of 947 matured oocytes were inseminated either by Piezo‐ICSI or by c‐ICSI in sibling oocytes as controls. Fertilization (2 pronuclei, PN), survival, and blastocyst development rates on day (D) 5 and D6 after insemination were compared between the Piezo‐ICSI and c‐ICSI groups. Further analyses were applied to groups of women >35 or ≤35 years of age.ResultsThere were no significant differences in fertilization, survival, or blastocyst development rates between the two insemination treatment groups. However, for women >35 years of age, the fertilization (P = .008) and blastocyst development (P = .016) rates with Piezo‐ICSI on D5 and D6 were significantly higher than in those subjected to c‐ICSI.ConclusionsPiezo‐ICSI was useful for inseminating oocytes from women >35 years of age.

Embryos Arising from Apronuclear (0PN) and Unipronuclear (1PN) Have Similar Euploidy Rates with Those from 2PN and Should be Considered for Transfer

Background: Fertilisation assessment is routinely made at 16–18 hours post-ICSI and 18–20 hours post-insemination. However, the absence of pronuclei (PN) during standard fertilisation assessment does not necessarily indicate fertilisation failure. The aim of this study is to assess the chromosomal status of blastocysts derived from 0PN and 1PN zygotes as well as to assess the clinical outcome after transfer of such embryos. Methods: In this study, we use microarray comparative genomic hybridisation (MaCGH) or next generation sequencing (NGS) to analyse the chromosomal status of 271 blastocysts (204 from 2PN, 41 from 0PN, 26 from 1PN) obtained from 42 patients who underwent conventional IVF (cIVF) and ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Results: Euploidy was confirmed in 126 (126/204; 61.8%), 31 (31/41; 75.6%) and 18 (18/26; 69.2%) 2PN-, 0PN- and 1PN-derived blastocysts respectively while the remaining 96 blastocysts displayed various chromosomal abnormalities. A Y-chromosome was observed in 0PN-derived blastocysts (19/41; 46.3%) and 1PN-derived blastocysts (13/26; 50%), indicating that sperm had penetrated the oocyte and not due to parthenogenetic activation. Four euploid 0PN-derived blastocysts were transferred to 4 patients and 3 healthy live births were achieved. Four euploid 1PN-derived blastocysts were transferred to 4 patients and 1 on-going pregnancy was achieved. Conclusion(s): 0PN- and 1PN-derived zygotes can be chromosomally normal and result in healthy live births. Such zygotes should not be discarded but instead be subjected to extended culture with PGT-A to ascertain the chromosomal and ploidy status and be considered for transfer.

Follicular fluid progesterone concentration is associated with fertilization outcome after IVF: a systematic review and meta-analysis.

Follicular fluid is a key biochemical environment for oocyte development. The potential effect of follicular progesterone level on successful fertilization is a subject of debate, and so the aim of this study was to provide a summary of the currently available evidence on the association between follicular fluid progesterone level and fertilization outcome. To do so, a systematic review and a meta-analysis were performed, with the literature searches being conducted in three databases (PubMed, Embase and the Cochrane Library) to identify all relevant studies published up to 19 August 2017. Data were available from 13 studies (four intracytoplasmic sperm injection [ICSI] and nine conventional IVF) and 1009 individually aspirated follicular fluid samples were included in the analysis. The progesterone levels in follicular fluid were significantly higher in normal fertilization than in failed fertilization, both in conventional IVF (33% difference, P<0.001) and ICSI (34% difference, P=0.004). Although these data show that fertilized oocytes are derived from follicles with higher levels of progesterone, the results must be interpreted with caution, because of various progesterone measurement methods and different treatment protocols and it is too early to state that follicular fluid progesterone level could be considered as a marker for oocyte quality.

In vitro maturation in subfertile women with polycystic ovarian syndrome undergoing assisted reproduction.

Polycystic ovarian syndrome (PCOS) occurs in 4% to 7% of all women of reproductive age and 50% of women presenting with subfertility. Subfertility affects 15% to 20% of couples trying to conceive. A significant proportion of these women ultimately need assisted reproductive technology (ART). In vitro fertilisation (IVF) is one of the assisted reproduction techniques employed to raise the chances of achieving a pregnancy. For the standard IVF technique, stimulating follicle development and growth before oocyte retrieval is essential, for which a large number of different methods combining gonadotrophins with a gonadotrophin-releasing hormone (GnRH) agonist or antagonist are used. In women with PCOS, the supra-physiological doses of gonadotrophins used for controlled ovarian hyperstimulation (COH) often result in an exaggerated ovarian response, characterised by the development of a large cohort of follicles of uneven quality, retrieval of immature oocytes, and increased risk of ovarian hyperstimulation syndrome (OHSS). A potentially effective intervention for women with PCOS-related subfertility involves earlier retrieval of immature oocytes at the germinal-vesicle stage followed by in vitro maturation (IVM). So far, the only data available have derived from observational studies and non-randomised clinical trials. To assess the effectiveness and safety of IVM followed by IVF or ICSI versus conventional IVF or ICSI among women with PCOS undergoing assisted reproduction. This is the second update of this review. We performed the search on 17 April 2018.The search was designed with the help of the Cochrane Gynaecology and Fertility Group Information Specialist, for all published and unpublished randomised controlled trials (RCTs).We searched the the Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials, CENTRAL via the Cochrane Central Register of Studies Online, MEDLINE, Embase, CINAHL, and the trial registers for ongoing and registered trials and the Open Grey database for grey literature from Europe. We made further searches in the National Institute for Health and Care Excellence (NICE) fertility assessment and treatment guidelines. We handsearched reference lists of relevant systematic reviews and RCTs, together with PubMed and Google for any recent trials that have not yet been indexed in the major databases. All RCTs on the intention to perform IVM before IVF or ICSI compared with conventional IVF or ICSI for subfertile women with PCOS, irrespective of language and country of origin. Two review authors independently selected studies, assessed risk of bias, extracted data from studies, and attempted to contact the authors of studies for which data were missing. Our primary outcomes were live birth per woman randomised and miscarriage. We performed statistical analysis using Review Manager 5. We assessed evidence quality using GRADE methods. We found two RCTs suitable for inclusion in the review and six ongoing trials that have not yet reported results. Both included studies were published as abstracts in international conferences.Both studies were at unclear or high risk of bias for most of the seven domains assessed. Common problems were unclear reporting of study methods and lack of blinding. The main limitations in the overall quality of the evidence were high risk of bias and serious imprecision.There were no data on the primary outcomes of this review, namely live birth per woman randomised and miscarriage.Both studies reported clinical pregnancy rate: there was evidence of an effect between IVM and IVF, favouring the former (odds ratio 3.10, 95% confidence interval 1.06 to 9.00; 71 participants; 2 studies; I2 = 0%; very low-quality evidence). The incidence of OHSS was zero in both studies in both groups.There were no data for the other outcomes specified in this review. Though promising data on the in vitro maturation (IVM) technique have been published, unfortunately there is still no evidence from properly conducted randomised controlled trials upon which to base any practice recommendations regarding IVM before in vitro fertilisation (IVF) or intracytoplasmic sperm injection for women with polycystic ovarian syndrome. Regarding our secondary outcomes, very low-quality evidence showed that clinical pregnancy was higher with IVM when compared to IVF, whereas the incidence of ovarian hyperstimulation syndrome was zero in both studies in both groups. We are awaiting the results of six ongoing trials and eagerly anticipate further evidence from good-quality trials in the field.

Blastocyst Rates and Kinetics of Sperm Processing After Conventional vs. Piezo-driven ICSI

Blastocyst Rates and Kinetics of Sperm Processing After Conventional vs. Piezo-driven ICSI

Morphokinetic analysis of embryos in conventional IVF and ICSI cycles according to the level of sperm DNA fragmentation

Morphokinetic analysis of embryos in conventional IVF and ICSI cycles according to the level of sperm DNA fragmentation

Day 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles

ObjectiveEmbryo transfers during cleavage stage (day 2 or day 3) and blastocyst stages (day 5 or day 6) are common in current daily practice in fresh IVF/ET cycles. Data regarding transferring day 4 embryos, morula/compact stage, is still restricted and the grading system is also inconsistent, as between IVF clinics. This study provided a new detailed classification system for morula/compact stage embryos and compared successes rates between day 4 and day 5 ET. Materials and methodsThis was a retrospective study. A review of medical records from January 1st, 2013, to December 31st 2015, performed for all conventional insemination and ICSI cycles with a GnRH-antagonist protocol at the Infertility Division of MacKay Memorial Hospital in Taipei City, Taiwan. ResultsThere were 427 cycles included in our study, 107 in study group (day 4 MET) and 320 in control group (day 5 BET). Pregnancy rates and live birth rate were compatible, as between morula embryo transfer (MET) and blastocyst embryo transfer (BET). The implantation rate (36.3% vs. 39.6%, respectively, p = 0.500), clinical pregnancy rate (49.5% vs. 51.9%, respectively, p = 0.737), and live birth rate (42.1% vs. 45.6%, respectively, p = 0.574) were statistically insignificant between groups. The term birth rate was statistically higher in the MET group than in the BET group (95.7% vs. 79.5%, respectively, p = 0.006). When the clinical outcomes between day 4 good MET and day 5 good BET were compared, the results were compatible. The implantation rate (48.8% vs. 41.1%, respectively, p = 0.335), clinical pregnancy rate (55.0% vs. 53.2%, respectively, p = 0.867), and live birth rate (47.5% vs. 47.1%, respectively, p = 1.000) showed no significant difference. The term birth rate was also higher in day 4 good MET group than in day 5 good BET group (100% vs. 78.3%, respectively, p = 0.025). ConclusionIn this study, we performed day 4 MET avoid BET on Sunday. The grading system we provided was more detailed for embryo selection and it was easier to remember. Our data showed that morula embryo transfer might be a flexible, easier and applicable method for embryo transfer in daily routine.

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI.

Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare.

Evaluation of a new sperm density gradient and sperm wash for assisted reproductive technology

Sperm preparation is an important factor that affects the results of conventional IVF and ICSI. Typically, separation methods that yield a higher number of motile spermatozoa combine density-gradient centrifugation with a subsequent swim-up procedure. However, fertilization in vivo is not only based on absolute sperm numbers, but also on their functional capability. A sperm preparation solution that mimics the physiology of the sperm environment in vivo was developed as the ORIGIO® GradientTM (OG) system. The effect of this new sperm preparation system in ART is still unknown. The aim of this study was to evaluate and compare sperm quality and IVF/ICSI outcomes following density gradient sperm preparation with two different media: Percoll and the OG. A prospective quasi-randomized controlled study was performed in a single IVF center between January 2016 and October 2016. We examined 87 conventional IVF cycles and 167 ICSI cycles. Patients were allocated to two groups randomly: for the control group, sperm preparation was performed using 80% Percoll solution (Sigma) with Sperm Washing Medium (Irvine Scientific); for the test group, sperm preparation was performed using 80% OG with ORIGIO® Sperm Wash (Origio). Only cycles using fresh autologous gametes were included. We excluded testicular sperm extraction-ICSI cycles and cycles combining IVF/ICSI. Sperm preparation was performed using density gradient centrifugation in the control or test solutions (25 min at 500 g) with a subsequent swim-up (30 min). We analyzed 668 inseminated (IVF) and 966 microinjected (ICSI) oocytes. All embryos were cultured using the same protocol. Sperm quality and IVF/ICSI outcomes were compared between the two groups. In both the IVF and ICSI cycles, there were no significant differences in patient characteristics, semen parameters before preparation (ejaculate volume, total and motile sperm concentrations, and percentage motility) or sperm quality after preparation (motile sperm concentration, and percentage motility), when comparing the two groups. Among the 87 conventional IVF cycles, 44 were in the control group and 43 were in the test group. The fertilization rate in the test group was significantly higher (P < 0.05) than in the control group (77.6% vs. 68.2%, respectively). The high-quality embryo formation rate on day 2 in the test group was significantly higher (P < 0.05) than in the control group (48.5% vs. 37.3%, respectively). The cleavage and blastocyst formation rates were similar between the test and control group (97.4% vs. 99.5%, and 58.3% vs. 57.6%, respectively). Among the 167 ICSI cycles, 87 were in the control group and 80 were in the test group. The fertilization, cleavage, high quality embryo rate on day 2 and blastocyst formation rates were similar between the test and control groups (83.8% vs. 82.3%, 98.1% vs. 99.3%, 47.6% vs. 50.9%, and 56.1% vs. 54.6%, respectively). This new sperm preparation system improved the fertilization and early embryonic development rates in conventional IVF cycles.

Use of ICSI in IVF cycles in women with tubal ligation does not improve pregnancy or live birth rates.

Does ICSI improve outcomes in ART cycles without male factor, specifically in couples with a history of tubal ligation as their infertility diagnosis? The use of ICSI showed no significant improvement in fertilization rate and resulted in lower pregnancy and live birth (LB) rates for women with the diagnosis of tubal ligation and no male factor. Prior studies have suggested that ICSI use does not improve fertilization, pregnancy orLB rates in couples with non-male factor infertility. However, it is unknown whether couples with tubal ligation only diagnosis and therefore iatrogenic infertility could benefit from the use of ICSI during their ART cycles. Longitudinal cohort of nationally reported cycles in the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS) of ART cycles performed in the USA between 2004 and 2012. There was a total of 8102 first autologous fresh ART cycles from women with the diagnosis of tubal ligation only and no reported male factor in the SART database. Of these, 957 were canceled cycles and were excluded from the final analysis. The remaining cycles were categorized by the use of conventional IVF (IVF, n=3956 cycles) or ICSI (n=3189 cycles). The odds of fertilization, clinical intrauterine gestation (CIG) and LB were calculated by logistic regression modeling, and the adjusted odds ratios (AORs) with 95% confidence intervals were calculated by adjusting for the confounders of year of treatment, maternal age, race and ethnicity, gravidity, number of oocytes retrieved, day of embryo transfer and number of embryos transferred. The main outcome measures of the study were odds of fertilization (2PN/total oocytes), clinical intrauterine gestation (CIG/cycle) and live birth (LB/cycle). The fertilization rate was higher in the ICSI versus IVF group (57.5% vs 49.1%); however, after adjustment this trend was no longer significant (AOR 1.14, 0.97-1.35). Interestingly, both odds of CIG (AOR 0.78, 0.70-0.86), and odds of LB were lower (AOR 0.77, 0.69-0.85) in the ICSI group. Plurality at birth, mean length of gestation and birth weight did not differ between the two groups. This was a retrospective study, therefore only the available parameters could be included, with parameters of interest such as smoking status not available for inclusion. Smoking status may have led practitioners to use ICSI to improve pregnancy and LB outcomes. Studies have shown that in the USA there is an increasing usage of ICSI for non-male factor infertility despite a lack of evidence-based benefit. Our study corroborates this increasing use over the last 8 years, specifically in the tubal ligation only patient population. Even after adjusting for multiple confounders, the patients who underwent ICSI had no statistically significant improvement in fertilization rate and actually had a lower likelihood of achieving a clinical pregnancy and LB.Therefore, our data suggest that the use of ICSI in tubal ligation patients has no overall benefit. This study contributes to the body of evidence that the use of ICSI for non-male factor diagnosis does not improve ART outcomes over conventional IVF. None.