From oocytes to a live birth: Are we improving the biological efficiency?

OBJECTIVE(S): The objectives of our study were to investigate the live birth rate (LBR) per oocyte retrieved during in vitro fertilization, in patients who had used all their embryos and to extrapolate the LBR in patients with remaining frozen embryos by calculating the expected LBR from these embryos. DESIGN: A retrospective cohort study. SETTING: A single academically affiliated fertility clinic. PATIENT(S): Autologous in vitro fertilization cycles from January 2014 to December 2020. Data on the number of oocytes retrieved, number of embryos obtained and transferred (at cleavage or blastocyst-stage), use of preimplantation genetic testing for aneuploidy (PGT-A), and number of live births were obtained. The expected LBR was estimated in patients with remaining frozen embryos according to nationally reported Society for Assisted Reproductive Technology LBR data. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live birth rate per oocyte retrieved. RESULT(S): A total of 12,717 patients met the inclusion criteria and underwent a total of 20,677 oocyte retrievals which yielded a total of 248,004 oocytes and 57,268 embryos (fresh and frozen). In patients who had fully utilized all their embryos the LBR per oocyte was 2.82% (ranging from 11.3% aged <35 years to 1.2% aged >42 years). Stratification of the population based on PGT-A utilization yielded similar results (with PGT-A: 2.88% and without PGT-A: 2.79%). When stratified by the Society for Assisted Reproductive Technology age groups, the addition of PGT-A in patients aged 35-37 and 38-40 years yielded higher LBR per oocyte compared with patients who did not add PGT-A (P<.05). In patients with remaining frozen embryos who had added PGT-A, the projected LBR per oocyte was 8.34%. Use of PGT-A in patients aged <35 and 35-37 years decreased LBR per oocyte (P<.001 and P=.03, respectively) but improved LBR per oocyte in patients aged 38-40 and 41-42 years (P=.006 and P=.005, respectively). Poisson regression analysis demonstrated an age threshold of 38.5, below which PGT-A lowers LBR per oocyte compared with no PGT-A. CONCLUSION(S): Despite clinical and scientific advances in Assisted Reproductive Technology, with the current protocols of ovarian stimulation, the LBR per oocyte remains low reflecting a biological barrier that has yet to be overcome. Overall, the addition of PGT-A did not demonstrate improved outcomes.

The utility of all-freeze IVF cycles depends on the composition of study populations.

BACKGROUND: Because often introduced without proper validation studies, so-called "add-ons" to IVF have adversely affected in vitro fertilization (IVF) outcomes worldwide. All-freeze cycles (embryo banking, EB) with subsequently deferred thaw cycles are such an "add-on" and, because of greatly diverging reported outcomes, have become increasingly controversial. Based on "modeling" with selected patient populations, we in this study investigated whether reported outcome discrepancies may be the consequence of biased patient selection. RESULTS: In four distinct retrospective case control studies, we modeled in four cohort pairings how cryopreservation with subsequent thaw cycles affects outcomes differently in good-, average- and poor-prognosis patients: (i) 127 fresh vs. 193 frozen donor-recipient cycles to model best-prognosis patients; (ii) 741 autologous fresh non-donor IVF cycles vs. 217 autologous frozen non-donor IVF cycles to model average prognosis patients; (iii) 143 favorably selected autologous non-donor IVF cycles vs. the same 217 frozen autologous cycles non-donor to monitor good- vs. average-prognosis patients; and (iv) 598 average and poor-prognosis autologous non-donor cycles vs. the same 217 frozen autologous non-donor cycles to model poor vs. average prognosis patients. In best-prognosis patients, EB marginally improved IVF outcomes. In unselected patients, EB had no effects. In poor-prognosis patients, EB adversely affected IVF outcomes. Unexpectedly, the study also discovered independent-of-age-associated chromosomal abnormalities, a previously unreported effect of recipient age on miscarriage risk in donor-egg recipients. CONCLUSIONS: In poor-prognosis patients, EB cycles should be considered contraindicated. In intermediate-prognosis patients EB does not appear to change outcomes, not warranting additional cost and time delays. Therefore, only good-prognosis patients are candidates for EB, though they will experience only marginal benefits that may not be cost-effective.

Changing clinical significance of oocyte maturity grades with advancing female age advances precision medicine in IVF.

In current IVF practice, metaphase-2 (M2) oocytes are considered most efficient in producing good quality embryos. Maximizing their number at all ages is standard clinical practice, while immature germinal vesicle (GV) oocytes are mostly automatically discarded. We present preliminary evidence that oocyte maturity grades with advancing age significantly change in their abilities to produce good quality embryos, with M2 oocytes significantly declining, GV oocytes improving, and M1 oocytes staying the same. These data contradict the over-40-year-old dogma that oocyte grades functionally do not change with advancing age, supporting potential changes to current IVF practice: (1) Stimulation protocols and timing of oocyte retrieval can be adjusted to a patient's age and ovarian function. (2) In older and younger women with prematurely aging ovaries, GV oocytes may no longer be automatically discarded. (3) In some infertile women, rescue in vitro maturation of immature oocytes may delay the need for third-party egg donation.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

A Monocentric Randomized Controlled Clinical Trial to Compare Single- and Double-Lumen Needles in Oocyte Retrieval Procedure in Assisted Reproductive Technologies.

The purpose was to determine any difference in outcomes, primarily in terms of number of retrieved oocytes per procedure, between two different needles used for oocytes retrieval procedure in Assisted Reproductive Technologies: the single-lumen needle (SLN) versus the double-lumen needle (DLN) with follicle flushing after aspiration. This randomized controlled trial included oocyte retrieval (OR) cycles for IVF and ICSI performed in 18 to 42-year-old women between March 2019 and January 2021 at a tertiary-care Fertility Center. A total of 200 ORs were randomized, 100 in each group. The mean number of retrieved oocytes was not different between groups (10.2 ± 6.5 for DLNs vs. 10.7 ± 7.0 for SLNs, p = 0.810). No significant differences were observed also in terms of number of retrieved oocytes/punctured follicles (83.0% ± 27.0% vs. 81.0% ± 22.0%, p = 0.916), number of retrieved oocytes/follicles at trigger (78.0% ± 29.0% vs. 78.0% ± 27.0%, p = 0.881), number of mature oocytes (7.6 ± 5.3 vs. 8.0 ± 5.1, p = 0.519), and pregnancy rate (27% vs. 23%, p = 0.514). However, the time required to retrieve each oocyte was longer using the DLN (1.5 ± 1.3 vs. 1.1 ± 0.9 minutes, p = 0.002). The present study confirmed the new perspectives on the sole use of SLNs in terms of saving time, without affecting the number of retrieved oocytes. Trial registration number and date of registration NCT03611907; July 26, 2018.

A review of the 2021/2022 PGDIS Position Statement on the transfer of mosaic embryos.

The practice of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF) since 2016 has been mostly directed by three highly controversial guidance documents issued by the Preimplantation Genetic Diagnosis International Society (PGDIS). Because these documents are so influential on worldwide IVF practice, the most recent one is here the subject of a detailed review, again revealing important misrepresentations and internal contradictions. Most importantly, however, this most recent guidance document still does not prevent the non-use and/or disposal of large numbers of embryos with substantial pregnancy and live-birth potential and, therefore, continues to propagate an IVF practice harmful to many infertile women.

Previously reported and here added cases demonstrate euploid pregnancies followed by PGT-A as "mosaic" as well as "aneuploid" designated embryos.

BACKGROUND: After the longest time opposing all transfers of embryos by preimplantation genetic testing for aneuploidy (PGT-A) diagnosed as "chromosomal-abnormal," the field has over recent years slowly been moving toward selective transfers of by PGT-A as "mosaic" diagnosed embryos, but is still rejecting transfers of embryos by PGT-A defined as "aneuploid." METHODS: Upon review of the literature, we report published cases of euploid pregnancies following transfers of PGT-A as "aneuploid" diagnosed embryos and add several additional, ongoing cases at our center. RESULTS: Among the published cases from our center, we identified seven euploid pregnancies from "aneuploid" embryos, four of which preceded the PGT-A industry's 2016 switch from binary "euploid" - "aneuploid" reporting to "euploid," "mosaic," and "aneuploid" reporting. That those four cases post 2016 PGT-A definition involving "mosaic" embryos, therefore, cannot be ruled out. Since then, we recently established three additional ongoing pregnancies from transfers of "aneuploid" embryos which still await confirmation of euploidy after delivery. A recent fourth pregnancy from the transfer of a trisomy 9 embryo miscarried before a fetal heart. Outside our own center's experience, the literature revealed only one additional such transfer, involving PGT-A as a "chaotic-aneuploid" diagnosed embryo with six abnormalities, leading to normal euploid delivery. In reviewing the literature, we furthermore demonstrate why current PGT-A reporting that differentiates between "mosaic" and "aneuploid" embryos based on relative percentages of euploid and aneuploid DNA in a single trophectoderm biopsy of on average 5-6 cells, is biologically non-sensical. CONCLUSION: Basic biological evidence and a clinically still very limited experience with transfers of PGT-A as "aneuploid" labeled embryos demonstrate beyond reasonable doubt that at least some "aneuploid" embryos can lead to healthy euploid births. Therefore, this observation establishes beyond reasonable doubt that the rejection of all "aneuploid" embryos from transfer reduces pregnancy and live birth chances for IVF patients. Whether (and to what possible degree) pregnancy and live birth chances differ between "mosaic" and "aneuploid" embryos, remains to be determined. The answer will likely depend on the aneuploidy(ies) of an embryo and to what degree percentages of "mosaicism" in a single, on average 5/6-cell trophectoderm biopsy can reflect the ploidy-status of a complete embryo.

Endometrial Cancer in Reproductive Age: Fertility-Sparing Approach and Reproductive Outcomes.

Endometrial cancer is the most common gynecologic malignancy in developed countries and approximately 7% of the women with endometrial cancer are below the age of 45. Management of endometrial cancer in young women who desire to maintain fertility presents a unique set of challenges since the standard surgical treatment based on hysterectomy and salpingo-oophorectomy is often not compatible with the patient's goals. A fertility-preserving approach can be considered in selected patients with early stage and low-grade endometrial cancer. An increasing amount of data suggest that oncologic outcomes are not compromised if a conservative approach is utilized with close monitoring until childbearing is completed. If a fertility-preserving approach is not possible, assisted reproductive technologies can assist patients in achieving their fertility goals.

Ectopic pregnancy risk factors in infertile patients: a 10-year single center experience.

The present retrospective study included both intrauterine insemination and in vitro assisted reproductive technologies (ART) procedures performed from January 2009 to December 2018 at a tertiary-care Fertility Centre. The purpose was to assess the incidence of ectopic pregnancy (EP) in infertile population who undergoes ART and to identify any risk factor impacting the occurrence of EP after ART. Among 27,376 cycles, 7352 pregnancies were achieved, of which 132 were EPs, the 1.80% (95% CI 1.5-2.1) of all pregnancies. In fresh embryo transfer cycles, a history of prior pelvic adhesions showed the greatest impact on the incidence of EP (aOR 2.49 95% CI 1.53-4.07 p < 0.001). Other factors associated with EP incidence were also identified, such as female age, basal FSH, the transfer of blastocyst embryos and difficulties during the embryo transfer procedure. In frozen embryo transfer cycles, the only factor influencing the incidence of EP was anti Müllerian hormone (AMH) serum concentration (aOR 0.81 95% CI 0.65-1.00, p = 0.048). To conclude, the incidence of EP observed was comparable to that reported after natural conception. On the other hand, pre-existing risk factors, traditionally more common in infertile population, appeared to influence the incidence of EP and should thus be modified if possible.

The case for mild stimulation for IVF: recommendations from The International Society for Mild Approaches in Assisted Reproduction.

The practice of ovarian stimulation for IVF is undergoing a fundamental re-evaluation as recent data begin to successfully challenge the traditional paradigm that ovarian stimulation should be aimed at the retrieval of as many oocytes as possible, in the belief that this will increase pregnancy rates. An opposing view is that live birth rate should not be the only end-point in evaluating the success of IVF treatment and that equal emphasis should be placed on safety and affordability. The International Society for Mild Approaches in Assisted Reproduction (ISMAAR) committee has carried out an up-to-date literature search, with the evidence being graded according to the University of Oxford's Centre for Evidence-Based Medicine. The recommendations were formulated taking into account the quality of evidence on the efficacy, risk and cost of each intervention. ISMAAR recommends adopting a mild approach to ovarian stimulation in all clinical settings as an increasing body of evidence suggests that mild stimulation is as effective as conventional stimulation, while being safer and less expensive. Mild ovarian stimulation could replace conventional stimulation, thus making IVF safer and more accessible worldwide.

Biochemical Hazards during Three Phases of Assisted Reproductive Technology: Repercussions Associated with Epigenesis and Imprinting.

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques involved may have an impact on crucial stages of early embryo and fetal development highly vulnerable to epigenetic influence. In this paper, the key role of methylation processes in epigenesis, namely the essential biochemical/metabolic pathways involving folates and one-carbon cycles necessary for correct DNA/histone methylation, is discussed. Furthermore, potential contributors to epigenetics dysregulation during the three phases of assisted reproduction: preparation for and controlled ovarian hyperstimulation (COH); methylation processes during the preimplantation embryo culture stages; the effects of unmetabolized folic acid (UMFA) during embryogenesis on imprinting methyl "tags", are described. Advances in technology have opened a window into developmental processes that were previously inaccessible to research: it is now clear that ART procedures have the potential to influence DNA methylation in embryonic and fetal life, with an impact on health and disease risk in future generations. Critical re-evaluation of protocols and procedures is now an urgent priority, with a focus on interventions targeted toward improving ART procedures, with special attention to in vitro culture protocols and the effects of excessive folic acid intake.

Different actors for the same play: the impact of the embryologist performing the embryo transfer.

RESEARCH QUESTION: Does the embryologist performing the embryo transfer impact the cycle outcome, in terms of ongoing pregnancy rate (OPR)? DESIGN: This single-centre retrospective study analysed the results, corrected for main confounders, from 28 embryologists and 32 physicians who performed respectively 24,992 and 24,669 fresh embryo transfers (either at cleavage or blastocyst stage) during a 20-year period from January 2000 to December 2019, in a university-affiliated tertiary care assisted reproductive technology (ART) centre. Primary outcome was OPR, defined as the number of viable pregnancies that had completed at least 12 weeks of gestation on the total number of embryo transfers performed. The study also assessed whether the embryologist's experience, measured in terms of number of embryo transfers performed prior to the day of the procedure, had an impact on their performance. The secondary aim was to assess which variable, between the embryologist and physician, more significantly impacted OPR. RESULTS: The overall unadjusted OPR was 22.54%. The embryologist performing the embryo transfer was found to significantly affect the OPR (P < 0.0001), corrected for potential confounders. However, the physician factor made a slightly greater contribution to the model (likelihood ratio 21.86, P < 0.001 versus likelihood ratio 17.20, P < 0.0001). No significant association was found between the experience of the embryologist and OPR (P = 0.067). CONCLUSIONS: These results show how the 'human factor' influences the chances of a positive outcome in the final step of a high-tech procedure and underline the importance of implementing an operator quality performance programme (both for physicians and embryologists) to ensure the maintenance of benchmark results and eventually retrain underperforming operators.

Reconsidering the Polycystic Ovary Syndrome (PCOS).

Though likely the most common clinical diagnosis in reproductive medicine, the Polycystic Ovary Syndrome (PCOS) is still only poorly understood. Based on previously published research, and here newly presented supportive evidence, we propose to replace the four current phenotypes of PCOS with only two entities-a hyperandrogenic phenotype (H-PCOS) including current phenotypes A, B, and C, and a hyper-/hypoandrogenic phenotype (HH-PCOS), representing the current phenotype D under the Rotterdam criteria. Reclassifying PCOS in this way likely establishes two distinct genomic entities, H-PCOS, primarily characterized by metabolic abnormalities (i.e., metabolic syndrome) and a hyperandrogenic with advancing age becoming a hypoandrogenic phenotype (HH-PCOS), in approximately 85% characterized by a hyperactive immune system mostly due to autoimmunity and inflammation. We furthermore suggest that because of hypoandrogenism usually developing after age 35, HH-PCOS at that age becomes relatively treatment resistant to in vitro fertilization (IVF) and offer in a case-controlled study evidence that androgen supplementation overcomes this resistance. In view of highly distinct clinical presentations of H-PCOS and HH-PCOS, polygenic risk scores should be able to differentiate between these 2 PCOS phenotypes. At least one clustering analysis in the literature is supportive of this concept.

A New Methodology to Assess Fallopian Tubes Microbiota and Its Impact on Female Fertility.

Tubal factor is an important contributor to female infertility, and the current diagnostic approaches cannot correctly identify many subtle causes of tubal dysfunction. While it is known that the most common cause of tubal factor infertility is pelvic inflammatory disease (PID), creating critical alterations of the tubal epithelium, little attention has been devoted to understanding the tubal modifications caused by the resident microbial population and their interaction with the surrounding tubal epithelium. Furthermore, most of these samples are obtained by traumatic procedures such as direct sampling during laparoscopy using a cytobrush. However, as in any other organ of the female genital tract, the microbiota environment of the fallopian tube plays an essential role in maintaining tubal functioning, counteracting the pathogenic effect of acquired microbes. Consequentially, to better analyze the tubal microbiota without causing anatomical and/or functional alteration of the fallopian tube and preserving fertility, the hysteroscopic approach might be the method of choice, guarantying maximal integrity of the uterine cavity and tubal lumen. Here we describe our plan for using atraumatic hysteroscopic sampling methods to investigate the correlation between tubal microbiota and female infertility.

We have reached a dead end for preimplantation genetic testing for aneuploidy.

The hypothesis of preimplantation genetic testing for aneuploidy (PGT-A) was first proposed 20 years ago, suggesting that during IVF elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos and, therefore, increase pregnancy and live birth rates, while also reducing miscarriages. Subsequently, unvalidated and increasingly unrestricted clinical utilization of PGT-A called for at least one properly randomized controlled trial (RCT) to assess cumulative live birth rates following a single oocyte retrieval, utilizing all fresh and frozen embryos of an IVF cycle. Only recently two such RCTs were published, however both, when properly analysed, not only failed to demonstrate significant advantages from utilization of PGT-A, but actually demonstrated outcome deficits in comparison to non-use of PGT-A, when patient selection biases in favour of PGT-A were reversed. Moreover, because of high embryo mosaicism at the blastocyst stage and, therefore, high false-positive rates from trophectoderm biopsies, large numbers of chromosomal-normal embryos with normal pregnancy potential are unnecessarily left unused or discarded, indisputably causing harm to affected couples. We, therefore, strongly call for restricting PGT-A to only research protocols and, as of this point in time, encourage professional societies in the field to follow suit with appropriate practice guidelines.