The Impact of SARS-CoV-2 on Sperm Cryostorage, Theoretical or Real Risk?

Cryopreservation of human gametes and embryos as well as human reproductive tissues has been characterized as an essential process and aspect of assisted reproductive technology (ART). Notably, sperm cryopreservation is a fundamental aspect of cryopreservation in oncological patients or patients undergoing gonadotoxic treatment. Given that there is a risk of contamination or cross-contamination, either theoretical or real, during the procedures of cryopreservation and cryostorage, both the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) have provided updated guidelines for preventing or reducing the contamination risk of sexually transmitted viruses. Given the ongoing and worldwide COVID-19 pandemic, there is considerable interest in what measures should be taken to mitigate SARS-CoV-2 contamination during cryopreservation and cryostorage of semen samples. The SARS-CoV-2 virus is the virus that causes COVID-19, and whose transmission and infection is mainly aerosol-mediated. Several ART professional societies, including ESHRE and ASRM have proposed measures to mitigate the spread of the SARS-CoV-2 virus. Whether the proposed safety directives are enough to mitigate the possible SARS-CoV-2-contamination of sperm samples during cryopreservation or whether the policies should be re-evaluated will be discussed in this review. Additionally, insights regarding the possible impact of COVID-19 vaccination on the safety of sperm cryopreservation will be discussed.

Preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm.

The objective of this study is to compare aneuploidy rates between three distinct areas of the human trophectoderm: mural, polar and a region in between these two locations termed the 'mid' trophectoderm. This is a cohort study on in vitro fertilization (IVF) patients undergoing comprehensive chromosome screening at the blastocyst stage at a private IVF clinic. All embryos underwent assisted hatching on day 3 with blastocyst biopsy and comprehensive chromosome screening. Biopsied blastocysts were divided into three groups depending on which area (polar, mid, or mural) of the trophectoderm was protruding from the zona pellucida and biopsied. Aneuploidy rates were significantly higher with cells from the polar region of the trophectoderm (56.2%) compared with cells removed from the mural region of the trophectoderm (30.0%; P = 0.0243). A comparison of all three areas combined also showed a decreasing trend, but this did not reach clinical significance, polar (56.2%), mid (47.4%) and mural trophectoderm (30.0%; P = 0.1859). The non-concordance demonstrated between polar and mural trophectoderm can be attributed to biological occurrences including chromosomal mosaicism or procedural differences between embryologists.

Technique to 'Map' Chromosomal Mosaicism at the Blastocyst Stage.

The purpose of this study was to identify a technique that allows for comprehensive chromosome screening (CCS) of individual cells within human blastocysts along with the approximation of their location in the trophectoderm relative to the inner cell mass (ICM). This proof-of-concept study will allow for a greater understanding of chromosomal mosaicism at the blastocyst stage and the mechanisms by which mosaicism arises. One blastocyst was held by a holding pipette and the ICM was removed. While still being held, the blastocyst was further biopsied into quadrants. To separate the individual cells from the biopsied sections, the sections were placed in calcium/magnesium-free medium with serum for 20 min. A holding pipette was used to aspirate the sections until individual cells were isolated. Individual cells from each section were placed into PCR tubes and prepped for aCGH. A total of 18 cells were used for analysis, of which 15 (83.3%) amplified and provided a result and 3 (16.7%) did not. Fifteen cells were isolated from the trophectoderm; 13 (86.7%) provided an aCGH result, while 2 (13.3%) did not amplify. Twelve cells were euploid (46,XY), while 1 was complex abnormal (44,XY), presenting with monosomy 7, 10, 11, 13, and 19, and trisomy 14, 15, and 21. A total of 3 cells were isolated from the ICM; 2 were euploid (46,XY) and 1 did not amplify. Here, we expand on a previously published technique which disassociates biopsied sections of the blastocyst into individual cells. Since the blastocyst sections were biopsied in regard to the position of the ICM, it was possible to reconstruct a virtual image of the blastocyst while presenting each cell's individual CCS results.

Blastocyst euploidy and implantation rates in a young (<35 years) and old (≥35 years) presumed fertile and infertile patient population.

OBJECTIVE: To examine the relationship between blastocyst euploidy and implantation rates in a presumed fertile patient population. DESIGN: Retrospective analysis. SETTING: Private IVF clinic. PATIENT(S): IVF patients undergoing comprehensive chromosome screening (CCS). INTERVENTION(S): Embryo biopsy at the blastocyst stage with preimplantation genetic screening using CCS. MAIN OUTCOME MEASURE(S): Euploidy, chemical pregnancy, and implantation rates. RESULT(S): There was no significant difference in the number of euploid blastocysts between presumed fertile (68/118, 57.6%) and infertile (75/132, 56.8%) patients<35 years old. Likewise, there was no significant difference in the number of euploid blastocysts between presumed fertile (42/86, 48.8%) and infertile (97/206, 47.1%) patients≥35 years old. When those same patients underwent a corresponding frozen embryo transfer cycle, presumed fertile patients demonstrated a significantly higher chemical pregnancy rate when compared with infertile patients, 28/33 (84.8%) and 50/81 (61.7%), respectively. Moreover, presumed fertile patients exhibited significantly higher implantation rates compared with infertile patients, 36/42 (85.7%) and 54/109 (66.7%), respectively. CONCLUSION(S): When subdivided by maternal age, no significant difference was seen in blastocyst euploidy rates between presumed fertile and infertile patients; however, chemical pregnancy and implantation rates were significantly higher in a presumed fertile patient population even when transferring only euploid blastocysts. This would indicate that infertility, as a disease, may encompass other aspects such as uterine or other unknown embryological factors that can influence outcomes.

Comparison of aneuploidy, pregnancy and live birth rates between day 5 and day 6 blastocysts.

Comprehensive chromosome screening is typically used for aneuploidy analysis of blastocysts. It is believed that either day of blastocyst development is acceptable. Euploidy rates and outcomes were examined between day 5 and day 6 blastocysts in two studies. First, euploidy rates of day 5 and day 6 blastocysts were examined on a per-embryo and per-patient basis. Second, outcomes were compared when only euploid day 5 or day 6 blastocysts were transferred in a cryopreserved embryo transfer cycle. In cycles (n = 70) that had blastocysts biopsied on both day 5 and day 6, day 5 blastocysts had a higher chance of being euploid than day 6 blastocysts (125/229 [54.6%]) and (77/180 [42.8%]), respectively (P = 0.0231). Similarly, euploid rates in blastocysts from patients (n = 193) with day 5 biopsy, day 6 biopsy, or both, were significantly higher in day 5 (235/421 [55.8%]) compared with day 6 (184/413 [44.6%]) blastocysts (P = 0.0014). In the second study, 50 women (36.1 ± 4.3 years) and 39 women (35.1 ± 3.8 years) with only euploid day 5 or euploid day 6 blastocysts transferred during a cryopreserved embryo transfer had similar cycle outcomes. Although underpowered, these data suggest that euploid day 6 blastocysts are as capable of positive outcomes as their euploid day 5 counterparts.

Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer.

Trophectoderm biopsy with comprehensive chromosome screening (CCS) has been shown to increase implantation and pregnancy rates. Some patients desire CCS on previously cryopreserved blastocysts, resulting in blastocysts that are thawed/warmed, biopsied, vitrified and then warmed again. The effect of two cryopreservation procedures and two thawing/warming procedures on outcomes has not been effectively studied. Cycles were divided into two groups: group 1 patients underwent a cryopreserved embryo transfer with euploid blastocysts that were vitrified and warmed once; group 2 patients had a cryopreserved embryo transfer of a euploid blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified and warmed. Groups 1 and 2 included 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years, respectively (not significantly different). Blastocyst survival in group 1 (114/116, 98.3%) and survival of second warming in group 2 (21/24, 87.5%) was significantly different (P = 0.0354). There was no difference between biochemical (68.2% and 62.5%) and clinical (61.2% and 56.3%) pregnancy rates, implantation rate (58.4% and 52.4%) and live birth/ongoing pregnancy rate (54.0% and 47.6%) between groups 1 and 2, respectively. Although it is unconventional to thaw/warm, biopsy, revitrify and rewarm blastocysts for cryopreserved embryo transfer, the results indicate that outcomes are not compromised. Trophectoderm biopsy and screening the embryos for chromosomal abnormalities has been reported to increase implantation and pregnancy rates. There is a category of patients requesting chromosomal screening on previously cryopreserved blastocysts. This scenario requires blastocysts to be thawed/warmed, biopsied, cryopreserved, and thawed/warmed again. The effect of double cryopreservation procedures and double thawing/warming procedures on pregnancy is unknown. Patients were divided into two groups, group 1 underwent a cryopreserved embryo transfer with a chromosomally normal blastocyst that was vitrified and warmed once and group 2 included patients that had a cryopreserved embryo transfer of a chromosomally normal blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified, and rewarmed. A total of 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years were included in groups 1 and 2, respectively. The survival rate for group 1 (114 of 116, 98.3%) compared with the second warming for group 2 (21 of 24, 87.5%) was significantly higher. There was no difference between biochemical (68.2% and 62.5%), and clinical pregnancies (61.2% and 56.3%), implantation (58.4% and 52.4%), and live birth/ongoing rates (54.0% and 47.6%) between groups 1 and 2. Although it is unconventional to twice cryopreserve and twice thaw/warm a blastocyst, our results indicate that outcomes are not compromised.

The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans.

BACKGROUND: Chromosomal mosaicism, the presence of two or more distinct cell lines, is prevalent throughout human pre- and post-implantation development and can lead to genetic abnormalities, miscarriages, stillbirths or live births. Due to the prevalence and significance of mosaicism in the human species, it is important to understand the origins, mechanisms and incidence of mosaicism throughout development. METHODS: Literature searches were conducted utilizing Pubmed, with emphasis on human pre- and post-implantation mosaicism. RESULTS: Mosaicism persists in two separate forms: general and confined. General mosaicism is routine during human embryonic growth as detected by preimplantation genetic screening at either the cleavage or blastocyst stage, leading to mosaicism within both the placenta and fetus proper. Confined mosaicism has been reported in the brain, gonads and placenta, amongst other places. Mosaicism is derived from a variety of mechanisms including chromosome non-disjunction, anaphase lagging or endoreplication. Anaphase lagging has been implicated as the main process by which mosaicism arises in the preimplantation embryo. Furthermore, mosaicism can be caused by any one of numerous factors from paternal, maternal or exogenous factors such as culture media or possibly controlled ovarian hyperstimulation during in vitro fertilization (IVF). Mosaicism has been reported in as high as 70 and 90% of cleavage- and blastocyst-stage embryos derived from IVF, respectively. CONCLUSIONS: The clinical consequences of mosaicism depend on which chromosome is involved, and when and where an error occurs. Mitotic rescue of a meiotic error or a very early mitotic error will typically lead to general mosaicism while a mitotic error at a specific cell lineage point typically leads to confined mosaicism. The clinical consequences of mosaicism are dependent on numerous aspects, with the consequences being unique for each event.

Pregnancy after rebiopsy and vitrification of blastocysts following allele dropout after day 3 biopsy.

OBJECTIVE: To report a clinical pregnancy after rebiopsy and vitrification of blastocysts following allele dropout (ADO) of biopsied day 3 embryos. DESIGN: Case report. SETTING: Private center. PATIENT(S): Thirty-year-old woman and her 33-year-old husband who carries the single-gene condition paraganglioma. INTERVENTION(S): In vitro fertilization with day 3 embryo biopsy-ET-blastocyst biopsy and vitrification-subsequent frozen ET cycle. MAIN OUTCOME MEASURE(S): Results from preimplantation genetic diagnosis and pregnancy results after fresh and frozen ETs. RESULT(S): Nineteen oocytes were retrieved of which 13 were mature and 12 fertilized. Eleven embryos were biopsied on day 3: two were normal, five were affected, and four exhibited ADO. The two normal blastocysts were transferred, and three of the ADO blastocysts were biopsied and sent for reanalysis. The biopsied blastocysts were vitrified. No pregnancy resulted from the fresh ET. One of the biopsied blastocysts was normal, one received no result, and one exhibited ADO. A singleton clinical pregnancy resulted from a subsequent frozen ET of the thawed biopsied normal blastocyst. CONCLUSION(S): Rebiopsy and vitrification of blastocysts could be used in cases of ADO or lack of results after day 3 embryo biopsy.

The utility of embryo banking in order to increase the number of embryos available for preimplantation genetic screening in advanced maternal age patients.

PURPOSE: to determine if embryo banking with PGS is more optimal than proceeding with PGS regardless of embryo number. METHODS: patients were divided into 2 groups, group 1 were those that banked embryos and proceeded through another round of IVF prior to PGS, and group 2 underwent PGS regardless of embryo number. Group 2 was divided into group 2A (patients with >10 embryos) and group 2B (patients who had <10 embryos). RESULTS: there was no difference in embryos biopsied, normal embryos, number transferred, and pregnancy rate between group 1 and 2. A significant number of patients did not have a transfer in group 2B (6/11) compared to group 1 (3/19) (P = 0.0419). There was no significance between pregnancy rates per transfer between group 1 (6/16) and group 2B (2/5). CONCLUSION: our data suggests that banking will increase the odds of going to transfer but there was no increase in pregnancy rates.

The effects of different laser pulse lengths on the embryo biopsy procedure and embryo development to the blastocyst stage.

PURPOSE: a laser is commonly used to remove a blastomere from an embryo for genetic testing. The laser uses intense heat which could possibly disrupt embryo development. It is the goal of this study to test the effects of different laser pulse lengths (and consequently heat) on the embryo biopsy procedure and embryo development. METHODS: each embryo biopsy was performed randomly utilizing laser pulse lengths of 0.604mS (group I), 0.708mS (group II), and 1.010mS (group III). RESULTS: for groups I, II, and III, 83, 86, and 71 embryos were biopsied, respectively. There was no difference in day 5 embryo quality or lysed blastomeres between groups. Average number of blastomeres biopsied between group I (1.0 ± 0.0), II (1.0 ± 0.2), and III (1.1 ± 0.2) was significant (0.0001). CONCLUSION: our data demonstrates that laser pulse length does not influence the embryo biopsy procedure or embryo development.

Comparison of ICSI and conventional IVF in patients with increased oocyte immaturity.

Using sibling oocytes, the objective of this study was to compare the intracytoplasmic sperm injection (ICSI) fertilization rates to those achieved with conventional IVF in patients with high rates of oocyte immaturity. This study was observational in nature, and included 91 patients who were treated using split insemination techniques. The fertilization rates for the ICSI group and the IVF group were 41.1 +/- 15.0% and 53.2 +/- 19.8%, respectively (P <: 0.0001). There was no significant difference in day-3 embryo quality between the two groups. There was a significantly higher number of embryos frozen in the IVF group than in the ICSI group: 357 (84.8%) and 297 (76.7%), respectively (P = 0.037). Furthermore, the number of embryos either transferred or frozen was significantly higher in the IVF group than the ICSI group: 459 of 1173 (39.1%) and 385 of 1268 (30.4%), respectively (P < 0.0001). These data indicate that conventional IVF results in a higher fertilization rate than ICSI. Furthermore, IVF provided more embryos available for transfer or cryopreservation when compared with ICSI, thereby optimizing the patient's cycle.

Lysed cell removal promotes frozen-thawed embryo development.

OBJECTIVE: To develop a mouse model to investigate the possible causes for increased success rates when lysed cells are removed from thawed embryos. DESIGN: Experimental study. SETTING: Clinical IVF laboratory. INTERVENTION(S): Assisted hatching, cell lysis, and removal of lysed cells. MAIN OUTCOME MEASURE(S): Embryonic growth rate and morphology. RESULT(S): The mouse embryos were divided into three groups; control (no cell lysis), group 1 (cell lysis and removal), and group 2 (cell lysis only). There was no significant difference in the initial number of blastomeres in each group or the number of cells lysed artificially in groups 1 and 2. The rate of embryonic development showed a significant delay in group 2 (7.97 +/- 4.92; control, 10.42 +/- 8.18; group 1, 5.74 +/-4.42; group 2). The embryo morphology on day 4 was significantly improved in group 1 and the control group when compared with group 2. CONCLUSION(S): Mouse embryos with artificially lysed cells after thawing had poorer developmental quality and growth rates compared with control embryos. However, removal of lysed cells restored the embryo's developmental potential to that of the control. Cell number and morphology was also significantly improved compared with embryos without lysed cell removal. These findings are consistent with human embryo development after thawing when lysed cells are present and thus mechanical lysis seems to be an appropriate method by which to further study frozen-thawed lysed cell removal.

Comparison of bovine- and recombinant human-derived hyaluronidase with regard to fertilization rates and embryo morphology in a sibling oocyte model: a prospective, blinded, randomized study.

The objective of the present study was to compare a traditionally used bovine-derived hyaluronidase (Hyase) with the newly developed recombinant human-derived enzyme product (Cumulase) in intracytoplasmic sperm injection (ICSI) procedures using a sibling oocyte model in a prospective randomized design. The results of the study demonstrate that Cumulase is safe and effective in an ICSI treatment program and can provide comparable if not improved parameters, including fertilization and embryo developmental rates.