The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

STUDY QUESTION: Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? SUMMARY ANSWER: The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. WHAT IS KNOWN ALREADY: The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. STUDY DESIGN, SIZE, DURATION: As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 µl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. LIMITATIONS, REASONS FOR CAUTION: The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03520933).

Noninvasive amino acid turnover predicts human embryo aneuploidy.

Assisted reproduction technology has two significant problems: low success rates and multiple pregnancies. Because of these problems, the priority in IVF clinics is to develop a potential diagnostic test that can be used to select the embryos with the ultimate developmental competence. Aneuploidy screening as embryo selection criteria will ensure that the transferred embryos are euploid and high implantation rate. We hypothesize that aneuploidy in human preimplantation embryos could be discriminated by their amino acid metabolism profile in the spent culture media. Preimplantation genetic testing for aneuploidy results and spent embryo culture medium amino acid content were analyzed for 58 couples. The next-generation sequencing technique was used and coupled with TE biopsy. Forty euploid and 71 aneuploid blastocysts were evaluated. Embryos were cultured individually until day 5 or 6 of embryo development. Spent culture medium was collected after finishing the culture. There was no statistical difference between D3 and D5 embryo morphology between euploid and aneuploid embryos (p > .05). Eight amino acids, including SER, GLY, HIS, ARG, THR, ALA, PRO, and TYR, were detected in the culture medium from the blank control group, euploid group, and aneuploid group. Only TYR amino acid concentration was found significantly higher in the aneuploid group compared to the euploid group (p < .003). Tyrosine amino acid levels equal to and above 76.38 µmol/L could be considered aneuploid. Aneuploid embryos demonstrate altered amino acid turnover in vitro relative to euploid counterparts. A noninvasive method of amino acid profiling will be of value as a tool for routine preimplantation embryo selection among all patient groups.

Birth of a Healthy Baby 9 Years After a Surgically Successful Deceased Donor Uterus Transplant.

OBJECTIVE: To describe surgical procedures, previous failed pregnancies, methods for overcoming pregnancy failure and, most importantly, birth of a healthy infant, in a uterus transplantation from a deceased donor. BACKGROUND: Majority of uterus transplants have involved live donors, but several advantages make deceased donor transplantation a practicable option, principally by eliminating surgical risks to the live donor. METHODS: Uterus transplantation from a deceased donor was performed in September 2011 in Turkey. After 5 miscarriages, perfusion computed tomography revealed an obstructed blood-outflow. To overcome this blood flow obstruction, a saphenous vein graft was anastomosed between utero-ovarian and left ovarian vein with laparotomy. Follow-up computed tomography confirmed resolution of venous congestion and a decrease in uterine volume. RESULTS: Following vascular augmentation surgery, fetal cardiac activity were observed 28 days after the first embryo transfer attempt. Preterm premature rupture of the membranes was diagnosed at 19 weeks' gestation. Cesarean section was planned at 28 weeks' gestation due to intrauterine growth restriction and suspected preeclampsia. A healthy 760 g male baby was delivered. The baby was discharged from the neonatal intensive care unit 79 days after delivery in good condition weighing 2475 g. CONCLUSIONS: Deceased donor uterus transplantation is a reasonable approach for treating uterine factor-related infertility. In case of recurrent miscarriages, regional vascular augmentation by arterial or venous supercharging may be required to overcome regional misperfused regions determined by imaging studies.

Being on the side of old findings: progesterone elevation on the day of oocyte maturation induction does not affect embryological parameters throughout the blastocyst culture period.

PURPOSE: To investigate whether there is any detrimental effect of progesterone elevation (PE) on the day of oocyte maturation induction on embryological development potentials. METHODS: This retrospective single-center cohort study included a total of 1485 individual intracytoplasmic sperm injection (ICSI) cycles between January 2014 and December 2018. Serum progesterone (P) levels were measured on the day of oocyte maturation induction following the GnRH antagonist suppression protocol. Embryological parameters such as maturation, fertilization rate (FR), top-quality embryo (TQE) formation rate per 2PN on day 3, and excellent-quality blastocyst (EQB) formation rate per 2PN on day 5/6 were recorded. The inclusion criteria for women were an age ≤ 37 years, a BMI ≤ 30 kg/m2, and access to a total sperm concentration ≥ 2 million. Groups were stratified according to the serum P levels using the cut-off levels of < 0.8 ng/ml; 0.8-1.49 ng/ml; and ≥ 1.5 ng/ml. RESULTS: Peak E2 level and total number of oocytes retrieved were significantly related to PE (p < 0.001). FR did not display a significance difference between groups (p = 0.108). The TQE and the blastulation rates were not affected by PE (p = 0.82 and p = 0.68, respectively). Chi square analysis revealed a significant relationship between PE and the EQB formation rate (p = 0.01). GEE analysis failed to present any statistical significance regarding the effect of PE on neither the TQE nor the EQB formation rates per 2PN [OR 1.07; 95% (0.98-1.16) p = 0.113 and OR 0.93; 95% (0.80-1.07) p = 0.32, respectively]. CONCLUSIONS: In accordance with previously published papers, our study could not find any detrimental effect of PE on embryological outcomes throughout the blastocyst culture period.

A 5-year multicentre randomized controlled trial comparing personalized, frozen and fresh blastocyst transfer in IVF.

RESEARCH QUESTION: Does clinical performance of personalized embryo transfer (PET) guided by endometrial receptivity analysis (ERA) differ from frozen embryo transfer (FET) or fresh embryo transfer in infertile patients undergoing IVF? DESIGN: Multicentre, open-label randomized controlled trial; 458 patients aged 37 years or younger undergoing IVF with blastocyst transfer at first appointment were randomized to PET guided by ERA, FET or fresh embryo transfer in 16 reproductive clinics. RESULTS: Clinical outcomes by intention-to-treat analysis were comparable, but cumulative pregnancy rate was significantly higher in the PET (93.6%) compared with FET (79.7%) (P = 0.0005) and fresh embryo transfer groups (80.7%) (P = 0.0013). Analysis per protocol demonstrates that live birth rates at first embryo transfer were 56.2% in PET versus 42.4% in FET (P = 0.09), and 45.7% in fresh embryo transfer groups (P = 0.17). Cumulative live birth rates after 12 months were 71.2% in PET versus 55.4% in FET (P = 0.04), and 48.9% in fresh embryo transfer (P = 0.003). Pregnancy rates at the first embryo transfer in PET, FET and fresh embryo transfer arms were 72.5% versus 54.3% (P = 0.01) and 58.5% (P = 0.05), respectively. Implantation rates at first embryo transfer were 57.3% versus 43.2% (P = 0.03), and 38.6% (P = 0.004), respectively. Obstetrical outcomes, type of delivery and neonatal outcomes were similar in all groups. CONCLUSIONS: Despite 50% of patients dropping out compared with 30% initially planned, per protocol analysis demonstrates statistically significant improvement in pregnancy, implantation and cumulative live birth rates in PET compared with FET and fresh embryo transfer arms, indicating the potential utility of PET guided by the ERA test at the first appointment.

The effect of the pooling method on the live birth rate in poor ovarian responders according to the Bologna criteria.

OBJECTIVE: Pooling is an alternative method to achieve in vitro fertilization outcomes. This study was to investigate the effect of pooling method on pregnancy outcomes in poor responder patients according to Bologna criteria. MATERIALS AND METHODS: Two hundred-fifty five poor responder patients were enrolled in this study. Pooling embryo transfer (ET) group had 110 and fresh ET group had 145 patients. RESULTS: Although, age was similar between both treatment groups (p=0.31), antral follicle count (p<0.001), total number of retrieved oocyte (p<0.001), total metaphase II oocyte count (p<0.001), number of stimulation cycles (p<0.001), were significantly different between the groups. The day of ET were similiar between two groups (p=0.72) but the number of ET procedure was significantly higher in pooling ET group compared to fresh ET (p<0.001). Positive pregnancy test [35/110 (32%) vs 53/145 (37%)] (p=0.43) and clinical pregnacy rates [31/110 (28%) vs 49/145 (34%)] (p=0.33) were similar between groups, whereas, implantation [31/191 (16%) vs 49/198 (25%)] (p=0.03) and live birth rates [15/110 (14%) vs 36/145 (25%)] (p=0.04) were significantly higher in fresh ET group. Despite that, abortion rates were significantly higher in pooling ET group [16/31 (52%) vs 13/49 (27%)] (p=0.04). Binary logistic regression analyese has revealed no effect of variables on live birth rates. CONCLUSION: Even though, pooling strategy seems to have a slight positive effect on pregnancy outcomes, there is no benefical effect on live birth rates. Furthermore, this strategy is increasing the abortion rates in parallel with clinical pregnancy rates.

In vitro fertilization for endometriosis-associated infertility.

Endometriosis is an enigmatic disease affecting 10-15% of reproductive aged women and is encountered in 25-35% of women suffering from infertility. IVF is an effective tool to overcome endometriosis-associated infertility when expectant management or surgery fails. Direct IVF should be envisioned if the female age is greater than 38 year and infertility is long lasting. Likewise, semen characteristics or tubal status that is incompatible with natural conception mandates going straight to IVF. IVF, not only bypasses the distortion of pelvic anatomy associated with advanced stage endometriosis, but also removes gametes from a hostile peritoneal environment. In this article, we address the impact, if any, of endometriosis and endometriomason IVF outcome, whether surgical treatment of early-stage disease, endometriomas or deep infiltrating endometriosis would enhance pregnancy rates in IVF, which protocol to employ for controlled ovarian hyperstimulation for IVF and finally the impact, if any, of controlled ovarian hyperstimulation for IVF on progression of endometriosis.

The effects of fresh embryo transfers and elective frozen/thawed embryo transfers on pregancy outcomes in poor ovarian responders as defined by the Bologna criteria.

OBJECTIVE: To compare the effects of fresh embryo transfers (ET) and elective frozen/thawed embryo transfers (eFET) on implantation, clinical pregnancy, and live birth rates in poor ovarian responders, as defined by the Bologna criteria. MATERIALS AND METHODS: All electronic databases of embryo transfers between January 2011 and January 2014 were retrospectively reviewed. Two hundred fifty-nine of all the fresh ET and 96 of all eFET were included into the study. An antagonist protocol with letrozole was used for the controlled ovarian hyperstimulation (COH) in all participants. RESULTS: The mean age was 36.9 years (range, 21-43 years) in the fresh ET arm and 37.2 years (range, 21-43 years) in the eFET arm (p=0.45). The clinical pregnancy rate was 35% (90/259) versus 29% (28/96); the abortion rate was 27% (20/75) versus 36% (9/25); and the live birth rate was 21% (55/259) versus 17% (16/99). There were no significant differences between groups and p values were 0.32, 0.52, and 0.42, respectively. The mean E2 level was 389 (range, 50-2055 pg/mL) in the fresh ET group (on hCG day) and 418 pg/mL (range, 121-3073 pg/mL) in the eFET group (on day 14 of cycle) (p=0.122). No differences were found between the two groups with respect to the total number of retrieved oocytes (p=0.55) and number of metaphase II (MII) oocytes (p=0.81). The number of embryo transfers was statistically different (p=0.005). The effects of age, total number of retrieved oocytes, number of MII oocytes, type of treatment, number of ET, and the day of ET and E2 level to live birth outcomes were investigated using binary logistic regresion analyses, and no stastical effect was determined by any of the parameters. P values were p=0.50, 0.66, 0.45, 0.30, 0.30, 0.08, and 0.90, respectively. CONCLUSION: E2 levels tend to be lower in poor responders, thus the receptivity of the endometrium may be damaged less than normal, which may explain why pregnancy results are the same between eFET and ET groups.

Comparison of WHO and endometrial intraepithelial neoplasia classifications in predicting the presence of coexistent malignancy in endometrial hyperplasia.

OBJECTIVE: The most commonly used classification system for endometrial hyperplasia is the World Health Organization system which is based on subjective criteria. Another classification system is endometrial intraepithelial neoplasia (EIN) system which uses diagnostic criteria including cytological demarcation, crowded gland architecture, minimum size of 1 mm, and careful exclusion of mimics, and aims to identify a precancer or cancer. The objective of this study was to compare the two classification systems in terms of predicting the presence of a coexistent cancer in surgically treated patients. METHODS: Biopsy and hysterectomy specimens of 49 women who were subjected to surgery with a preoperative diagnosis of endometrial hyperplasia (EH) according to the WHO system were re-evaluated retrospectively by using EIN system. RESULTS: Among the 49 patients, 69.4% had complex atypical EH and 75.5% had EIN at biopsy specimens. EIN was detected in 94.1% of complex atypical EH, and 41.7% of non-atypical EH. Nine women (18.4%) had endometrial cancer. Among women with cancer, all had complex atypical EH or EIN. The rate of coexistent endometrial cancer was 26.5% in women with complex atypical EH and 24.3% in women with EIN. CONCLUSION: Diagnoses of atypical or complex atypical EH and EIN had similar sensitivities and negative predictive values in predicting the coexistent endometrial cancer. Either of these two classification systems may be used safely when an experienced pathologist is available. However, use of the objective EIN system may be preferred whenever possible to prevent diagnostic errors in centers where an experienced pathologist is not available.

Obesity does not affect the number of retrieved lymph nodes and the rate of intraoperative complications in gynecologic cancers.

OBJECTIVE: Lymphadenectomy, in general, is a safe and well-tolerated procedure in gynecologic oncology. However, some technical difficulties may be experienced in obese women which may result in inadequate lymphadenectomy and increased complications. The purpose of this study is to retrospectively evaluate the effect of obesity on lymph node counts retrieved and complication rates observed during lymphadenectomy in gynecologic cancers. METHODS: Patients with ovarian, endometrial or cervical cancers treated with initial surgery including bilateral pelvic and paraaortic lymph node dissection were grouped as non-obese and obese. These two groups were compared in terms of the number of retrieved lymph nodes and the rate of intraoperative complications directly related to lymph node dissection. RESULTS: One hundred twenty-three patients were eligible with a mean age of 55.1 years and mean body mass index of 29.2 kg/m(2). Fifty-nine patients were obese while 64 were non-obese. Lymph node counts obtained in different stations and in total were similar among non-obese and obese patients. Rates of lymphadenectomy-related intraoperative complications including vascular, neural, intestinal, and bladder injury were also similar in non-obese and obese patients. CONCLUSION: The obesity does not affect the lymph node counts and intraoperative complication rates adversely in women with gynecologic cancers. Therefore, adequate lymph node dissection should not be omitted based solely upon obesity in gynecologic oncology patients.

Visfatin and retinol-binding protein 4 concentrations in lean, glucose-tolerant women with PCOS.

Since insulin resistance is accepted to be a common feature of polycystic ovary syndrome (PCOS), the exact molecular mechanism(s) involved in glucose and lipid metabolism have been under investigation in the syndrome. Recently, two novel adipokines, namely visfatin and retinol-binding protein 4 (RBP4), have been suggested to play a role in insulin resistance and diabetes. This study sought to determine whether plasma concentrations of visfatin and RBP4 are altered in PCOS by comparing a total of 27 lean, normal glucose-tolerant PCOS patients with 19 age- and body mass index-matched healthy controls. The mean plasma visfatin concentrations were higher in PCOS patients than those in healthy subjects (37.9+/-18.2 versus 19.8+/-17.5, P<0.01), while RBP4 concentrations were similar between the two. Both adipokines were correlated with each other in the whole (r=0.50, P<0.01) and in PCOS (r=0.52, P<0.01) groups but not in controls. The results suggest that lean, glucose-tolerant women with PCOS have increased circulating visfatin and unaltered RBP4 concentrations compared with healthy lean women. In order to clarify overlapping effects and their potential contribution to the pathophysiology of PCOS, further studies are needed.

Increased circulating soluble P-selectin in polycystic ovary syndrome.

OBJECTIVE: To determine whether the P-selectin-von Willebrand factor (vWF) pathway is altered in patients with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING(S): Tertiary care academic medical center. PATIENT(S): Thirty-two normal glucose-tolerant patients with PCOS and 21 age- and body mass index-matched healthy women were prospectively enrolled. All the patients with PCOS had clinical and/or biochemical hyperandrogenism and chronic oligoanovulation, and 89% had polycystic ovaries on ultrasound. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Soluble P-selectin (sP-selectin), vWF, total T, sex hormone-binding globulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, fasting glucose and insulin, 2-hour glucose, and homeostatic model assessment-insulin resistance. RESULT(S): Soluble P-selectin levels were significantly higher in patients with PCOS compared with controls (58.7 +/- 19.0 vs. 45.3 +/- 15.0 ng/mL), whereas PCOS and control groups had similar vWF levels (46.7 +/- 24.2 vs. 39.5 +/- 22.3, respectively). There was no correlation between sP-selectin and anthropometric measurements or any of the androgen, lipid, or insulin resistance parameters. CONCLUSION(S): Our results suggest increments in the circulating sP-selectin concentrations associated with unaltered vWF levels in PCOS. Increased sP-selectin might potentially contribute to the future risk of cardiovascular disease in patients with PCOS.

Single intramural leiomyoma with normal hysteroscopic findings does not affect ICSI-embryo transfer outcome.

Where there is no distortion of the endo-myometrial junction, the effect of an intramural leiomyoma on reproductive performance is controversial. The current study compared the performance of patients having a single leiomyoma and intact endometrium confirmed by hysteroscopy (study group) with that of controls having intact endometrium alone in intracytoplasmic sperm injection (ICSI) cycles. A total of 61 consecutive infertile patients were retrospectively enrolled into the study group from a computerized IVF database. The control group consisted of 444 age-matched patients undergoing ICSI-embryo transfer without any endocervical or intrauterine pathology confirmed by both transvaginal ultrasonography and office hysteroscopy. The baseline characteristics, performance of ovarian stimulation and embryological data were similar between the two groups. The clinical pregnancy per embryo transfer (36 versus 38%) and implantation rate (20 versus 19%) were also comparable. Although the miscarriage rate tended to be higher in the leiomyoma group (27 versus 19%), the difference did not reach statistical significance. In conclusion, in the presence of intact endometrium, a single intramural leiomyoma does not seem to have a deleterious effect on ICSI cycles. Before ICSI is attempted, hysteroscopy may be useful for ruling out distortion of the endometrium due to leiomyoma in selected cases.

The role of reoperation in the management of endometrial carcinoma found in simple hysterectomy.

OBJECTIVE: To evaluate our experience in patients with endometrial cancer found in simple hysterectomy. METHODS: Forty patients treated for endometrial cancer after simple hysterectomy were evaluated, retrospectively. Twenty-one patients (Group 1) underwent surgical staging procedure while 19 patients were not subjected to complementary surgical staging procedure (Group 2). RESULTS: The mean age was 53.80 years. Residual disease following reoperation was found in only four patients (19.0%). The three of them who were considered Stage IC (two Grade 1, one Grade 2) were found to be Stage IIIC and one patient who was considered Stage IB (Grade 2) was found to be Stage IIIC. The overall recurrence rates in Groups 1 and 2 were 4.8% versus 10.5% (P > 0.05). The overall disease-free survival rates were 95.24% in Group 1 and 87.50% in Group 2 (P > 0.05). Considering the patients who received postoperative adjuvant radiotherapy (RT), the overall disease-free survival rates were 88.89% in Group 1 and 84.62% in Group 2 (P > 0.05). CONCLUSION: We believe that complementary surgical staging may have benefit in patients with incompletely staged endometrial carcinoma excluding Stage IA G1 cases, since the recurrence rate has rising and disease-free survival estimate has decreasing tendencies in incompletely staged patients than that in complementary surgically staged ones.