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.

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.

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.

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.

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.

Importance of IGF-I levels in IVF: potential relevance for growth hormone (GH) supplementation.

PURPOSE: Growth hormone (GH) supplementation in association with in vitro fertilization (IVF) is worldwide again increasing, even though study outcomes have been discrepant. Since GH acts via insulin-like growth factor-1 (IGF-1), its utilization in IVF would only seem to make sense with low IGF-1. We, therefore, determined whether IGF-I levels affect IVF outcomes. METHODS: Retrospectively, 302 consecutive first fresh, non-donor IVF cycles were studied, excluding patients on GH supplementation. Patients were divided into 3 subgroups: IGF-1 in lower 25th percentile (group A, < 132 ng/mL, n = 64); 25th-75th percentile (B, 133-202 ng/mL, n = 164), and upper 25th percentile (C, > 202 ng/mL, n = 74). IGF-1 was tested immunochemiluminometric with normal range at 78-270 ng/mL. Because of the study patients' adverse selection and low pregnancy chances, the main outcome measure for the study was cycle cancellation. Secondary outcomes were oocyte numbers, embryos transferred, pregnancies, and live births. RESULTS: Group A was significantly older than B and C (P = 0.019). IGF-1 decreased with increasing age per year by 2.2 ± 0.65 ng/mL (P = 0.0007). FSH was best in group B and worst in A (trend, P = 0.085); AMH was best in B and worst in A (N.S.). Cycle cancellations were lowest in C (11.6%) and highest in A (25.0%; P = 0.042). This significance further improved with age adjustment (P = 0.021). Oocytes, embryo numbers, pregnancies, and live birth rates did not differ, though oocyte numbers trended highest in B. CONCLUSIONS: Here presented results support the hypothesis that IGF-1 levels affect IVF outcomes. GH treatments, therefore, may be effective only with low IGF-1.

Time associations between U.S. birth rates and add-Ons to IVF practice between 2005-2016.

Until 2010, the National Assisted Reproductive Technology Surveillance System (NASS) report, published annually by the Center for Disease Control and Prevention (CDC), demonstrated almost constantly improving live birth rates following fresh non-donor (fnd) in vitro fertilization (IVF) cycles. Almost unnoticed by profession and public, by 2016 they, however, reached lows not seen since 1996-1997. We here attempted to understand underlying causes for this decline. This study used publicly available IVF outcome data, reported by the CDC annually under Congressional mandate, involving over 90% of U.S. IVF centers and over 95% of U.S. IVF cycles. Years 2005, 2010, 2015 and 2016 served as index years, representing respectively, 27,047, 30,425, 21,771 and 19,137 live births in fnd IVF cycles. Concomitantly, the study associated timelines for introduction of new add-ons to IVF practice with changes in outcomes of fnd IVF cycles. Median female age remained at 36.0 years during the study period and center participation was surprisingly stable, thereby confirming reasonable phenotype stability. Main outcome measures were associations of specific IVF practice changes with declines in live IVF birth rates. Time associations were observed with increased utilization of "all-freeze" cycles (embryo banking), mild ovarian stimulation protocols, preimplantation genetic testing for aneuploidy (PGT-A) and increasing utilization of elective single embryo transfer (eSET). Among all add-ons, PGT-A, likely, affected fndIVF most profoundly. Though associations cannot denote causation, they can be hypothesis-generating. Here presented time-associations are compelling, though some of observed pregnancy and live birth loss may have been compensated by increases in frozen-thawed cycles and consequential pregnancies and live births not shown here. Pregnancies in frozen-thawed cycles, however, represent additional treatment cycles, time delays and additional costs. IVF live birth rates not seen since 1996-1997, and a likely continuous downward trend in U.S. IVF outcomes, therefore, mandate a reversal of current outcome trends, whatever ultimately the causes.

Predictive value of cytoplasmic granulation patterns during in vitro fertilization in metaphase II oocytes: part II, donor oocyte cycles.

OBJECTIVE: To determine whether the ooplasm granulation patterns of donor oocytes, like those of oocytes from poor-prognosis patients, are predictive of in vitro fertilization (IVF) outcomes. DESIGN: Retrospective cohort study. SETTING: Academically affiliated private clinical infertility and research center. PATIENT(S): 770 fresh and 381 vitrified-thawed metaphase II oocytes from young donors (aged 21.0-34.6 years) used for IVF during 2017-2020. INTERVENTION(S): Determination of granulation patterns in every oocyte during intracytoplasmic sperm injection as fine, central, uneven, dispersed, and peripheral (thawed only). MAIN OUTCOME MEASURE(S): Fertilization, pregnancy, and live birth rates in fresh and thawed donor oocytes. Both overall and known-outcome analyses were performed for pregnancy and live birth. RESULT(S): In fresh donor oocytes, 2 pronuclei rates trended down from 96.1% to 90.2%, 88.9%, and 69.7% from fine to central, uneven, and dispersed granulations; overall pregnancy rates trended down from 50.4% to 29.0%, 17.7%, and 6.9%, as well as live birth rates (43.4%, 21.6%, 12.5%, and 6.4%), from fine to uneven, central, and dispersed granulations. Known pregnancy and known-live birth analyses showed similar findings. Thawed donor oocytes demonstrated similar trends in differences in fertilization, pregnancy, and live birth analyses with relatively worse outcomes. Peripheral granulation, unique to vitrification and thawing, always demonstrated the worst IVF outcomes. Moreover, granulation patterns were relatively disassociated from embryo morphological grades in fresh and largely disassociated in thawed donor oocytes. CONCLUSION(S): Predictive values of oocyte granulation patterns for fertilization, pregnancy, and live birth in IVF cycles are even more pronounced in young donors than results in older poor-prognosis patients, further supporting integration of oocyte granulation patterns into embryo selection.

Predictive value of cytoplasmic granulation patterns during in vitro fertilization in metaphase II oocytes: Part I, poor-prognosis patients.

OBJECTIVE: To determine whether 4 cytoplasmic granulation patterns of human metaphase II oocytes have a predictive value for in vitro fertilization outcomes. DESIGN: A retrospective cohort study. SETTING: An academically affiliated private clinical infertility and research center. PATIENT(S): A total of 2,690 consecutive fresh autologous oocytes collected from women aged 41.2 ± 5.0 years between 2017 and 2019. INTERVENTION(S): Determination of granulation pattern in every oocyte during intracytoplasmic sperm injection as fine, central, dispersed, and newly introduced uneven granulations. MAIN OUTCOME MEASURE(S): Fertilization outcomes (2 pronuclei [2PN], <2PN, and >2PN rates), pregnancy, and live birth rates for different granulation patterns at different ages. RESULT(S): Fine granulation produced the highest 2PN rate, followed by central, uneven, and dispersed granulation (91.8%, 83.9%, 77.9%, and 54.8%, respectively). Differences in fertilization were surprisingly relatively independent of age and other variables. Overall, compared with fine granulation, dispersed granulation resulted in lower pregnancy rates (4.6% vs. 10.7%) and known-outcome analysis (1.3% vs. 5.6%) as well as lower live birth rates (3.0% vs. 8.9%) and known-outcome analysis (0.6% vs. 5.6%). The known-outcome analysis demonstrated that uneven granulation had lower live birth rates than fine granulation (2.3% vs. 5.6%). Unexpectedly, the ooplasm granulation patterns were largely disassociated from embryo morphologic grades. CONCLUSION(S): We, for the first time, demonstrated that 4 distinct cytoplasmic granulation patterns in metaphase II oocytes had, largely independent of age and other variables, a predictive value for fertilization, pregnancy, and live birth outcomes in in vitro fertilization cycles of poor-prognosis patients. These data suggest that upstream ooplasm granulation patterns deserve closer attention in terms of embryo selection.

Depletion of aneuploid cells in human embryos and gastruloids.

Chromosomal instability leading to aneuploidy is pervasive in early human embryos1-3 and is considered as a major cause of infertility and pregnancy wastage4,5. Here we provide several lines of evidence that blastocysts containing aneuploid cells are worthy of in vitro fertilization transfer. First, we show clinically that aneuploid embryos can lead to healthy births, suggesting the presence of an in vivo mechanism to eliminate aneuploidy. Second, early development and cell specification modelled in micropatterned human 'gastruloids' grown in confined geometry show that aneuploid cells are depleted from embryonic germ layers, but not from extraembryonic tissue, by apoptosis in a bone morphogenetic protein 4 (BMP4)-dependent manner. Third, a small percentage of euploid cells rescues embryonic tissue in mosaic gastruloids when mixed with aneuploid cells. Finally, single-cell RNA-sequencing analysis of early human embryos revealed a decline of aneuploidy beginning on day 3. Our findings challenge two current dogmas: that a single trophectoderm biopsy at blastocyst stage to perform prenatal genetic testing can accurately determine the chromosomal make-up of a human embryo, and that aneuploid embryos should be withheld from embryo transfer in association with in vitro fertilization.

Commentary on two recently published formal guidelines on management of "mosaic" embryos after preimplantation genetic testing for aneuploidy (PGT-A).

Two professional societies recently published opinions on the clinical management of "mosaic" results from preimplantation genetic testing for aneuploidy (PGT-A) in human blastocyst-stage embryos in associations with in vitro fertilization (IVF). We here point out three principal shortcomings: (i) Though a most recent societal opinion states that it should not be understood as an endorsement of the use of PGT-A, any discussion of how PGT-A should be clinically interpreted for all practical purposes does offer such an endorsement. (ii) The same guideline derived much of its opinion from a preceding guidance in favor of utilization of PGT-A that did not follow even minimal professional requirements for establishment of practice guidelines. (iii) Published guidelines on so-called "mosaic" embryos from both societies contradict basic biological characteristics of human preimplantation-stage embryos. They, furthermore, are clinically unvalidated and interpret results of a test, increasingly seen as harmful to IVF outcomes for many infertile women. Qualified professional organizations, therefore, should finally offer transparent guidelines about the utilization of PGT-A in association with IVF in general.

Rate of rebound in follicle growth after cessation of ovarian stimulation in initial non-responders: a prospective cohort study.

Previously anecdotally observed rebounds in follicle growth after interruption of exogenous gonadotropins in absolute non-responders were the impetus for here reported study. In a prospective cohort study, we investigated 49 consecutive patients, absolutely unresponsive to maximal exogenous gonadotropin stimulation, for a so-called rebound response to ovarian stimulation. A rebound response was defined as follicle growth following complete withdrawal of exogenous gonadotropin stimulation after complete failure to respond to maximal gonadotropin stimulation over up to 5-7 days. Median age of study patients was 40.5 ± 5.1 years (range 23-52). Women with and without rebound did not differ significantly (40.0 ± 6.0 vs. 41.0 ± 7.0 years, P = 0.41), with 24 (49.0%) recording a rebound and 25 (51.0%) not. Among the former, 21 (87.5%) reached retrieval of 1-3 oocytes and 15 (30.6%) reached embryo transfer. A successful rebound in almost half of prior non-responders was an unsuspected response rate, as was retrieval of 1-3 oocytes in over half of rebounding patients. Attempting rebounds may, thus, represent another incremental step in very poor prognosis patients before giving up on utilization of autologous oocytes. Here presented findings support further investigations into the underlying physiology leading to such an unexpectedly high rebound rate.

A form of secondary ovarian insufficiency (SOI) due to adrenal hypoandrogenism as new infertility diagnosis.

BACKGROUND: Mediated via the androgen receptor on granulosa cells, models of small growing follicle stages demonstrate dependence on testosterone. Androgen deficiency reduces ovarian response to follicle stimulation hormone (FSH), granulosa cell mass and estradiol (E2) production falls and FSH, therefore, rises. Though potentially of adrenal and/or ovarian origin, androgen deficiency in association with female infertility is almost universally primarily of adrenal origin, raising the possibility that women with presumptive diagnosis of primary ovarian insufficiency (POI), also called primary ovarian failure (POF) may actually suffer from secondary ovarian insufficiency (SOI) due to adrenal hypoandrogenism that leads to follicular arrest at small-growing follicle stages. METHODS: This retrospective cohort study was performed in a private, academically affiliated infertility center in New York City. We searched the center's anonymized electronic research data bank for consecutive patients who presented with a diagnosis of POI, defined by age <41 year, FSH > 40.0 mIU/mL, amenorrhea for at least 6 month, and low testosterone (T), defined as total T (TT) in the lowest age-specific quartile of normal range. This study did not include patients with oophoritis. Since dehydroepiandrosterone sulfate (DHEAS) is the only androgen almost exclusively produce by adrenals, adrenal hypoandrogenism was defined by DHEAS < 100ug/dL. Thirteen of 78 presumed POI women (16.67%) qualified and represented the original study population. POI patients are usually treated with third-party egg donation; 6/13, however, rejected egg donation for personal or religious reasons and insisted on undergoing at least one last IVF cycle attempt (final study population). In preparation, they were supplemented with DHEA 25 mg TID and CoQ10 333 mg TID for at least 6 weeks prior to ovarian stimulation for IVF with FSH and human menopausal gonadotropins (hMG). Since POI patients are expected to be resistant to ovarian stimulation, primary outcome for the study was ovarian response, while secondary outcome was pregnancy/delivery. RESULTS: Though POI/POF patients usually are completely unresponsive to ovarian stimulation, to our surprise, 5/6 (83.3%) patients demonstrated an objective follicle response. In addition, 2/6 (33.3%) conceived spontaneously between IVF cycles, while on DHEA and CoQ10 supplementation and delivered healthy offspring. One of those is currently in treatment for a second child. CONCLUSIONS: This preliminary report suggests that a surprising portion of young women below age 41, tagged with a diagnosis of POI/POF, due to adrenal hypoandrogenism actually suffer from a form of SOI, at least in some cases amenable to treatment by androgen supplementation. Since true POI/POF usually requires third-party egg donation, correct differentiation between POI and SOI in such women appears of great importance and may warrant a trial stimulation after androgen pre-supplementation for at least 6 weeks.