The effect of laser-assisted hatching on vitrified/warmed blastocysts: the ALADDIN randomized controlled trial.

OBJECTIVE: To evaluate whether laser-mediated assisted hatching (AH) performed on vitrified/warmed blastocysts before embryo transfer can improve live birth rate. DESIGN: The "pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN)" is a 2-center comparative study with a parallel randomized controlled design. SETTING: University hospital. PATIENTS: Participants were recruited between September 2018 and November 2021. They were aged 18-39 years, underwent nondonor in vitro fertilization cycles, and were scheduled for elective single embryo transfer with vitrified/warmed blastocysts. Those with uterine abnormalities, body mass index of >35 kg/m2, severe male factor infertility, or performing preimplantation genetic testing were excluded. INTERVENTION: Assisted hatching was performed using a 1,480 nm diode laser, removing approximately one-third of the zona pellucida with continuous 0.2 ms pulses applied from the 1-5 o'clock positions. MAIN OUTCOME MEASURES: The primary outcome was the live birth rate. Secondary end points included clinical pregnancy, miscarriage, multiple pregnancies, preterm births, obstetric and neonatal complications, and congenital anomalies. RESULTS: Overall, 698 participants met the inclusion criteria and were randomized: 352 patients were assigned to the AH arm and 346 to the control arm. Of the participants, 105 (29.8%) and 101 (29.2%), respectively, achieved a live birth after treatment. The relative risk of live birth in patients with vitrified/warmed blastocysts treated with AH was 1.02 (95% confidence interval, 0.86-1.19). Exploratory subgroup analyses for women's age, recruiting centers, indications for in vitro fertilization, method of insemination, blastocyst quality, and days of blastocyst development failed to highlight any clinical situation that could benefit from AH in thawed blastocysts. CONCLUSION: In patients undergoing frozen embryo transfer with vitrified/warmed blastocysts, laser AH does not improve the live birth rate. Further studies are required to rule out milder but potentially interesting benefits in specific subgroups of patients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03623659.

The Task Matters: A Comprehensive Review and Proposed Literature Score of the Effects of Chemical and Physical Parameters on Embryo Developmental Competence.

To explore the effects of chemical and physical parameters on embryo developmental competence, we conducted a systematic search on PubMed for peer-reviewed original papers using specific keywords and medical subject heading terms. Studies of interest were selected from an initial cohort of 4141 potentially relevant records retrieved. The most relevant publications were critically evaluated to identify the effect of these parameters on embryo development. Moreover, we generated a literature score (LS) using the following procedure: (i) the number of studies favoring a reference group was expressed as a fraction of all analyzed papers; (ii) the obtained fraction was multiplied by 10 and converted into a decimal number. We identified and discussed six parameters (oxygen, temperature, humidity, oil overlay, light, pH). Moreover, we generated a LS according to five different comparisons (37 °C vs. <37 °C; 5% vs. 20% oxygen; 5-2% vs. 5% oxygen; humidity conditions vs. dry conditions; light exposure vs. reduced/protected light exposure). Only two comparisons (37 °C vs. <37 °C and 5% vs. 20% oxygen) yielded a medium-high LS (8.3 and 7, respectively), suggesting a prevalence of studies in favor of the reference group (37 °C and 5% oxygen). In summary, this review and LS methodology offer semi-quantitative information on studies investigating the effects of chemical and physical parameters on embryo developmental competence.

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps. When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 °C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified. In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

Phthalate Exposure and Biomarkers of Oxidation of Nucleic Acids: Results on Couples Attending a Fertility Center.

Phthalates are substances used as plasticizing agents and solvents that can increase the risk of infertility and that appear to induce oxidative stress. The aim of the study was to show the possible relationship between urinary concentrations of phthalates metabolites, namely MEP, MBzP, MnBP, MEHP, MEHHP, and MnOP and biomarkers of nucleic acids oxidation, methylation, or protein nitroxidation. The oxidative stress biomarkers measured in human urine were 8-oxo-7,8-dihydroguanine, 8-oxo-7,8-dihydroguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 3-nitrotyrosine, and 5-methylcytidine. Two hundred and seventy-four couples were enrolled, undergoing an assisted reproduction technology (ART) treatment, urine samples were analyzed in HPLC/MS-MS, and then two sub-groups with urinary concentration > 90th or <10th percentile were identified, reducing the sample size to 112 subjects. The levels of oxidative stress biomarkers were measured in both groups, reduced to 52 men and 60 women. A statistically significantly difference for 8-oxoGuo and 3-NO2Tyr between men and women, with higher levels in men, was found. The levels of oxidative stress biomarkers were directly correlated with some phthalate concentrations in both sexes.

Does Endometriosis Influence the Embryo Quality and/or Development? Insights from a Large Retrospective Matched Cohort Study.

In vitro fertilization can be an effective tool to manage the endometriosis-associated infertility, which accounts for 10% of the strategy indications. Nevertheless, a negative effect of endometriosis on IVF outcomes has been suggested. The aim of this study was to evaluate the potential effect of endometriosis in the development of embryos at cleavege stage in assisted reproduction treatment cycles. A total of 429 cycles from women previously operated for moderate/severe endometriosis were compared with 851 cycles from non-affected women. Patients were matched by age, number of oocyte retrieved and study period. A total of 3818 embryos in cleavage stage have been analyzed retrospectively. Overall, no difference was found between women with and without endometriosis regarding the number of cleavage stage embryos obtained as well as the percentage of good/fair quality embryos. Excluding cycles in which no transfers were performed or where embryos were frozen in day three, no difference was observed for blastulation rate or the percentage of good/fair blastocysts obtained. Despite similar fertilization rate and number/quality of embryos, a reduction in ongoing pregnancy rate was observed in patients affected, possibly due to an altered endometrial receptivity or to the limited value of the conventional morphological evaluation of the embryo.

Assessment of pre-implantation genetic testing for embryo aneuploidies: A SWOT analysis.

The recently re-named pre-implantation genetic testing for determining embryo aneuploidies (PGT-A) is presently very popular although its acceptance by the scientific community is controversial. This approach still encounters drawbacks. This paper uses a SWOT (strengths, weaknesses, opportunities and threats) analysis to discuss salient points to be considered when examining the pre-implantation genetic testing (PGT-A) strategy to gather information from a range of perspectives. One of the strengths associated with the procedure is represented by an increase in implantation rate although data from the highest level of evidence do not support an increase in cumulative pregnancy rates. The current difficulty in the management of mosaicisms represents a weakness of PGT-A. The application of the strategy represents an opportunity to favor the single embryo transfer while other advantages, such as reduction of time to pregnancy and emotional distress are controversial. Potential important threats, at present still undefined, are represented by the biopsy-related damage to the blastocyst and the impact on neonatal and long-term outcomes.

Vitamin D and Endometrium: A Systematic Review of a Neglected Area of Research.

Growing evidence supports a role of vitamin D (VD) in reproductive health. Vitamin D receptor (VDR) is expressed in the ovary, endometrium, and myometrium. The biological actions of VD in fertility and reproductive tissues have been investigated but mainly using animal models. Conversely, the molecular data addressing the mechanisms underlying VD action in the physiologic endometrium and in endometrial pathologies are still scant. Levels of VDR expression according to the menstrual cycle are yet to be definitively clarified, possibly being lower in the proliferative compared to the secretory phase and in mid-secretory compared to early secretory phase. Endometrial tissue also expresses the enzymes involved in the metabolism of VD. The potential anti-proliferative and anti-inflammatory effects of VD for the treatment of endometriosis have been investigated in recent years. Treatment of ectopic endometrial cells with 1,25(OH)2D3 could significantly reduce cytokine-mediated inflammatory responses. An alteration of VD metabolism in terms of increased 24-hydroxylase mRNA and protein expression has been demonstrated in endometrial cancer, albeit not consistently. The effect of the active form of the vitamin as an anti-proliferative, pro-apoptotic, anti-inflammatory, and differentiation-inducing agent has been demonstrated in various endometrial cancer cell lines.

Application of failure mode and effect analysis in an assisted reproduction technology laboratory.

Assisted reproduction technology laboratories have a very high degree of complexity. Mismatches of gametes or embryos can occur, with catastrophic consequences for patients. To minimize the risk of error, a multi-institutional working group applied failure mode and effects analysis (FMEA) to each critical activity/step as a method of risk assessment. This analysis led to the identification of the potential failure modes, together with their causes and effects, using the risk priority number (RPN) scoring system. In total, 11 individual steps and 68 different potential failure modes were identified. The highest ranked failure modes, with an RPN score of 25, encompassed 17 failures and pertained to "patient mismatch" and "biological sample mismatch". The maximum reduction in risk, with RPN reduced from 25 to 5, was mostly related to the introduction of witnessing. The critical failure modes in sample processing were improved by 50% in the RPN by focusing on staff training. Three indicators of FMEA success, based on technical skill, competence and traceability, have been evaluated after FMEA implementation. Witnessing by a second human operator should be introduced in the laboratory to avoid sample mix-ups. These findings confirm that FMEA can effectively reduce errors in assisted reproduction technology laboratories.

The strategy of group embryo culture based on pronuclear pattern on blastocyst development: a two center analysis.

PURPOSE: To compare two embryo grouping strategies. METHODS: Retrospective time-course analysis in two different centres. Two culture protocols were used at the zygote stage: "Random Group" in which zygotes were randomly grouped and "Definite Group" in which zygotes were grouped based on pronuclear pattern. Embryo culture was extended to blastocyst stage. Primary and secondary outcomes were respectively the blastulation rate and the cumulative clinical pregnancy and implantation rates. RESULT(S): A similar blastulation rate [42 and 41% day (5 + 6) blastocysts] was obtained in the two groups. Conversely, after adjusting for baseline and cycle variables, cumulative pregnancy [adjusted Odds Ratio = 2.10 (95%CI: 1.08-4.07)] and implantation [adjusted Odds Ratio = 1.78 (95%CI: 1.06-2.97)] rates were significantly higher in the "Random Group" compared to the "Definite Group". CONCLUSION(S): Two strategies of group culture gave similar results in terms of blastulation rate but the random grouping of zygotes improves pregnancy and implantation rates in IVF-cycles.

Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law.

OBJECTIVE: To retrospectively evaluate whether fresh day-5 embryo transfer could overcomes the detrimental effect of subtle progesterone elevations at hCG administration on pregnancy outcomes in women undergoing ovarian hyperstimulation for IVF/ICSI cycles. STUDY DESIGN: Retrospective study of 204 infertile patients aged 23-44 years who underwent IVF/ICSI treatment and fresh blastocyst transfer under the Italian law (embryos cryopreservation cannot be planned in advance). Women were divided into those with a progesterone level <1.5 ng/ml and those with a progesterone concentration ≥ 1.5 ng/ml at hCG triggering. The clinical pregnancy rate (CPR) after blastocyst transfer was the primary outcome. RESULTS: Age, body mass index (BMI), antral follicle count, anti-Mullerian hormone (AMH) and FSH values, mean number of stimulation days, ratio of GnRH agonist and antagonist cycles and total dose of gonadotrophins administered did not differ between the two groups. Serum estradiol and number of retrieved oocytes were significantly increased in the group with elevated progesterone and a significantly higher number of oocytes was used in this group. Fertilization rate, percentage of top quality embryos, and number of transferred blastocysts were similar in the two groups. The CPR was significantly higher in women with progesterone levels <1.5 ng/ml at hCG (50%) compared with women with progesterone concentration ≥ 1.5 ng/ml (33.3%) (odds ratio = 2.00, 95% confidence interval 1.07-3.75). CONCLUSIONS: A fresh blastocyst transfer does not completely overcome the detrimental effect of progesterone rise at hCG on IVF/ICSI pregnancy outcomes.

Exposure of embryos to oxygen at low concentration in a cleavage stage transfer program: reproductive outcomes in a time-series analysis.

BACKGROUND: Embryos are exposed to oxygen at a concentration of 2% to 8% under in vivo conditions. Laboratory culture of embryos with oxygen at atmospheric tension impairs embryo metabolism and blastocyst development in several species. Indeed, a high rate of live births after a day 5 transfer has been obtained by lowering oxygen concentration in the incubator atmosphere, thus definitively proving the damaging effect of oxygen at atmospheric tension on late stage development of human embryos. Conversely, the possible beneficial effect of low oxygen tension on assisted reproductive technology (ART) outcomes in a selective cleavage stage transfer program remains controversial. Therefore, the present study validated the hypothesis that oxygen at reduced concentration may improve ART outcomes when cleavage stage embryos are transferred. METHODS: The effect of oxygen at atmospheric versus reduced concentration on laboratory and clinical outcomes of both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles were compared. Evaluation of outcomes included fertilization, cleavage, and implantation rates. Clinical outcomes were also assessed in our analysis. RESULTS: From May 2010 to Mid-October 2010 357 ART cycles (ICSI n = 273 and IVF n = 84) were performed using atmospheric oxygen laboratory incubators while from Mid-October 2010 until March 2011 306 cycles (ICSI n = 224 and IVF n = 82) were carried out under low oxygen concentration. The multi-ovulation protocols were not significantly different between the two study periods. The medical and laboratory staff, the laboratory, and its procedures and the operating room did not differ between the two groups, neither did the culture media. For ICSI procedures, no significant difference in ART outcomes was found between the two culture conditions. For conventional IVF cycles, both fertilization rate (59 +/- 36 vs. 71 +/- 32, respectively) and proportion of embryos obtained (38% vs. 50%, respectively) were significantly improved under low oxygen condition. CONCLUSIONS: Culture of embryos in oxygen at low tension improved ART outcomes during a selective cleavage stage transfer program.

Pathologic findings in hysteroscopy before in vitro fertilization-embryo transfer (IVF-ET).

BACKGROUND: The aim of this study was to evaluate hysteroscopy routinely performed prior to in vitro fertilization-embryo transfer (IVF-ET). METHODS: We analyzed in a prospective study 300 patients who underwent hysteroscopy before the first IVF-ET cycle. We analyzed then in a retrospective manner 300 patients who did not perform hysteroscopy. RESULTS: One-hundred-and-eighty (60%) hysteroscopies were normal but 120 (40%) revealed an unsuspected intrauterine abnormality. We did not find statistically significant differences between patients with normal or abnormal hysteroscopy in any characteristic. We found a statistically significant difference in pregnancy rate between women who performed hysteroscopy before IVF-ET cycle and in women who did not perform it. CONCLUSIONS: Hysteroscopy, as a routine examination, should be performed before the first IVF-ET cycle in all patients.

Polar body morphology and spindle imaging as predictors of oocyte quality.

It has been suggested that first polar body (PBI) morphology reflects oocyte competence. Oocytes with an intact normal-sized PBI have been described as generating better day 2 embryos, higher blastocyst yield, and increased pregnancy and implantation rates. In other studies, PBI morphology was found to be unrelated to fertilization rate, embryo quality, and blastocyst formation. In a prospective analysis, the predictive value of the PBI was investigated by comparing the development of oocytes retrieved from intracytoplasmic sperm injection patients and displaying different PBI morphology, classified according to the following characteristics: normal size and smooth surface (I), fragmented (II), rough surface (III), or large size (IV). Fertilization rates were 59, 57, 64 and 60% respectively. No significant differences were found between the various groups. The proportions of high quality (grade A) day 2 embryos were also comparable among groups I-III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV (two embryos) did not allow comparison with the other classes. These data do not suggest that PBI selection can contribute to identification of embryos with high developmental ability. In order to establish alternative criteria for oocyte selection, a metaphase II (MII) spindle analysis was also conducted via Polscope. In oocytes of patients of different age, spindle retardance (which reflects the high order and density of microtubules) was compared with parameters of embryo development. In aged patients, a trend was observed between low retardance and poor embryo quality, although in general the association between retardance and oocyte developmental performance did not reach statistical significance.

Consecutive cycles in in vitro fertilization--embryo transfer.

BACKGROUND: The decline of female fertility with advancing age is well documented. The aim of this study was to compare the ovarian performance after repeated ovarian stimulation cycles in women of different ages. METHODS: Four hundred patients who started at least three in vitro fertilization (IVF) cycles during the 5-year period between 1998 and 2002 were identified. The patients were divided into four groups: the 25-30 age group (n = 90), the 31-35 age group (n = 150), the 36-40 age group (n = 110) and the 41-45 age group (n = 50). RESULTS: Comparing subsequent cycles versus the first treatment cycle we found a statistically significantly increased number of ampules of recombinant follicle stimulating hormone (rFSH) needed to reach follicles maturation (p < 0.001). The number of ampules of gonadotropin required was significantly higher (p < 0.001) in the groups of advanced age compared with the groups of young women. For women in the 36-40 group and in the 41-45 group we found the number of follicles, the number of oocytes and the proportion of grade A embryos, in every cycle, were significantly lower than in the groups of young women. We compared the characteristics of ovarian stimulation and response of a single age group in different consecutive cycles. We found significant differences (p < 0.05) only in the number of ampules required. CONCLUSIONS: Maternal age adversely affected ovarian performance. During repeated IVF cycles we also noted an age-independent decline of ovarian response.