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Gender dysphoria, a discrepancy between gender identity and genetically determined sex, is encountered in approximately 0.5% of people uniformly across the world. In the case of transgender men, formerly called female-to-male transsexuals, the available gender-affirming measures, hormone therapy and possible surgical procedures, are multiple and discussed in detail in this series of articles.

Objective To investigate the extent to which assisted oocyte activation (AOA) improves clinical outcomes in patients diagnosed with oocyte activation deficiencies (OADs). Design Retrospective cohort study comparing AOA cycles and previous intracytoplasmic sperm injection (ICSI) cycles in couples experiencing low or total failed fertilization after ICSI. Importantly, the sperm-related oocyte-activating capacity was examined in all patients before AOA with the use of the mouse oocyte activation test (MOAT). Setting Infertility center at a university hospital. Patient(s) A total of 122 couples with a history of low or total failed fertilization after ICSI. Intervention(s) ICSI, MOAT, AOA, and embryo transfer. Main Outcome Measure(s) Fertilization, pregnancy, and live birth rates. Result(s) MOAT revealed 19 patients with a sperm-related OAD (MOAT group 1), 56 patients with a diminished sperm-related oocyte-activating capacity (MOAT group 2), and 47 patients with a suspected oocyte-related OAD (MOAT group 3). AOA (191 cycles) significantly improved fertilization, pregnancy, and live birth rates in all MOAT groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among MOAT groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Between MOAT group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA. Conclusion(s) Patients undergoing diagnostic testing before AOA show a significant improvement in clinical outcomes compared with previous cycles. Our findings highlight that AOA should be reserved for patients with clear OADs.

Objective: To develop a random forest model (RFM) to predict implantation potential of a transferred embryo and compare it with a multivariate logistic regression model (MvLRM), based on data from a large cohort including in vitro fertilization (IVF) patients treated with the use of single-embryo transfer (SET) of blastocyst-stage embryos. Design: Retrospective study of a 2-year single-center cohort of women undergoing IVF or intracytoplasmatic sperm injection (ICSI). Setting: Academic hospital. Patient(s): Data from 1,052 women who underwent fresh SET in IVF or ICSI cycles were included. Intervention(s): None. Main Outcome Measure(s): The performance of both RFM and MvLRM to predict pregnancy was quantified in terms of the area under the receiver operating characteristic (ROC) curve (AUC), classification accuracy, specificity, and sensitivity. Result(s): ROC analysis resulted in an AUC of 0.74 ± 0.03 for the proposed RFM and 0.66 ± 0.05 for the MvLRM for the prediction of ongoing pregnancies of ≥11 weeks. This RFM approach and the MvLRM yielded, respectively, sensitivities of 0.84 ± 0.07 and 0.66 ± 0.08 and specificities of 0.48 ± 0.07 and 0.58 ± 0.08. Conclusion(s): The performance to predict ongoing implantation will significantly improve with the use of an RFM approach compared with MvLRM.

Objective To study the feasibility of in vitro maturation of ovarian tissue oocytes for fertility preservation in transgender men on testosterone treatment. Design Cross-sectional study Setting University hospital Patient(s) Eighty-three transgender men enrolled from November 2015 to January 2019 Intervention(s) In vitro maturation of cumulus-oocyte complexes (COCs) harvested at the time of gender confirmation surgery, and fertilization through intracytoplasmic sperm injection. Main Outcome Measure(s) In vitro maturation, fertilization, and blastulation rates; comparison of morphokinetics with vitrified-warmed oocytes; and analysis of the genetic profiles of embryos. Secondary outcomes: association between serum hormone levels; COCs’ morphologic characteristics, and vitrification rate. Result(s) All participants were on testosterone treatment for a median of 83 (64[Quartile 1]; 113.2[Quartile 2]) weeks. A total of 1,903 COCs (mean per participant, 23 ± 15.8) were collected. The in vitro maturation rate was 23.8%, vitrification rate was 21.5%, and survival rate after warming was 72.6% (n = 151). Intracytoplasmic sperm injection was performed in 139 oocytes. The rate of normal fertilized oocytes was 34.5%, and 25 (52.1%) embryos reached day 3. One blastocyst was achieved on day 5. Aberrant cleavage patterns and early embryo arrest were observed in 22 (45.8%) and 44 (91.7%) zygotes, respectively. Compared with vitrified-warmed donor oocytes, a delay was observed in pronuclei disappearance, t2 (time to reach 2 cell stage) timings, and CC1 (the duration of the 1st cell cycle) and SS3 (synchronization of cleavage pattern (calculated as t8-t5) time intervals. A normal genetic pattern was seen in 42% embryos. The proportion of vitrified oocytes was negatively associated with progesterone (odds ratio, 0.76) and positively associated with antimullerian hormone serum levels (odds ratio, 1.23). The highest vitrification rate was achieved by the morphologic characteristic 344 at day 0 and by 433 at day 2. Conclusion(s) Ovarian tissue oocytes matured in vitro show low developmental capacity in transgender men, when collected under testosterone treatment.

STUDY QUESTION: What progress has been made in fertility preservation (FP) over the last decade?SUMMARY ANSWER: FP techniques have been widely adopted over the last decade and therefore the establishment of international registries on their short- and long-term outcomes is strongly recommended.WHAT IS KNOWN ALREADY: FP is a fundamental issue for both males and females whose future fertility may be compromised. Reproductive capacity may be seriously affected by age, different medical conditions and also by treatments, especially those with gonadal toxicity. There is general consensus on the need to provide counselling about currently available FP options to all individuals wishing to preserve their fertility.STUDY DESIGN, SIZE, DURATION: An international meeting with representatives from expert scientific societies involved in FP was held in Barcelona, Spain, in June 2015.PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty international FP experts belonging to the American Society of Reproductive Medicine, ESHRE and the International Society of Fertility Preservation reviewed the literature up to June 2015 to be discussed at the meeting, and approved the final manuscript. At the time this manuscript was being written, new evidence considered relevant for the debated topics was published, and was consequently included.MAIN RESULTS AND THE ROLE OF CHANCE: Several oncological and non-oncological diseases may affect current or future fertility, either caused by the disease itself or the gonadotoxic treatment, and need an adequate FP approach. Women wishing to postpone maternity and transgender individuals before starting hormone therapy or undergoing surgery to remove/alter their reproductive organs should also be counselled accordingly. Embryo and oocyte cryopreservation are first-line FP methods in postpubertal women. Metaphase II oocyte cryopreservation (vitrification) is the preferred option. Cumulative evidence of restoration of ovarian function and spontaneous pregnancies after ART following orthotopic transplantation of cryopreserved ovarian tissue supports its future consideration as an open clinical application. Semen cryopreservation is the only established method for FP in men. Testicular tissue cryopreservation should be recommended in pre-pubertal boys even though fertility restoration strategies by autotransplantation of cryopreserved testicular tissue have not yet been tested for safe clinical use in humans. The establishment of international registries on the short- and long-term outcomes of FP techniques is strongly recommended.LIMITATIONS, REASONS FOR CAUTION: Given the lack of studies in large cohorts or with a randomized design, the level of evidence for most of the evidence reviewed was 3 or below.WIDER IMPLICATIONS OF THE FINDINGS: Further high quality studies are needed to study the long-term outcomes of FP techniques.STUDY FUNDING/COMPETING INTEREST(S): None.TRIAL REGISTRATION NUMBER: N/A.

Objective To create a pool of frozen donated human oocytes and find the optimal stage for slow controlled-rate freezing of human in vitro matured oocytes. Design Oocytes at different developmental stages of maturation (germinal vesicle, metaphase I, or metaphase II) and oocytes that failed to fertilize after IVF or intracytoplasmic sperm injection (ICSI) were frozen using a slow controlled-rate freezing protocol. Frozen/thawed oocytes were artificially activated to verify activation potential and compared with oocytes that were not frozen. Setting University hospital–based fertility center. Patient(s) Stimulated patients undergoing IVF/ICSI treatment donated oocytes left over during their infertility treatment. Intervention(s) Human oocytes were frozen at different stages of maturation. Fresh and frozen/thawed oocytes were activated by electrical pulses followed by incubation in 6-dimethylaminopurine. Main Outcome Measure(s) Survival rate, maturation rate and kinetics, and activation potential. Result(s) Human oocytes at all developmental stages have high survival rates after slow controlled-rate freezing. Frozen/thawed germinal vesicle oocytes showed decreased and delayed maturation after thawing. Activation potential was not affected. Conclusion(s) A pool of donated human oocytes could be established using slow controlled-rate freezing. Immature oocytes should be frozen after in vitro maturation.

Objective To assess the effect of two assisted oocyte activation (AOA) protocols with the use of two calcium (Ca 2+ ) ionophores, ionomycin and A23187 (calcimycin), on the intracellular Ca 2+ level in mouse and human oocytes and the fertilization rates. Design Comparison of two Ca 2+ ionophores, ionomycin and A23187, regarding their capacity to increase the intracellular Ca 2+ level and to support subsequent oocyte activation and development. Setting University hospital research laboratory. Patient(s)/Animal(s) Patients undergoing intracytoplasmic sperm injection (ICSI) treatment and B6D2F1 mice. Intervention(s) Assisted oocyte activation and microinjection of mouse and human oocytes with sperm. Main Outcome Measure(s) Measurement of the fertilizing and Ca 2+ -releasing ability of human sperm. Result(s) Ionomycin was more potent than A23187 in provoking Ca 2+ increases in both mouse and human oocytes with significantly higher amplitude and area under the receiver operating characteristic curve. The oocyte activation rate was significantly higher when mouse oocytes were activated with the use of the ionomycin- rather than the A23187-based AOA protocol. Furthermore, oocyte activation rate was higher when human in vitro matured oocytes were activated with the ionomycin-based AOA protocol, but the difference did not reach statistical significance. Conclusion(s) In both mouse and human oocytes, the AOA protocol that used ionomycin was more efficient than the one that used A23187. Bearing in mind that mammalian fertilization is successful when the total dose of Ca 2+ released reaches a minimal threshold, the use of ionomycin for human AOA might be justified instead of the use of A23187.

Objective: To assess the Ca2þ-releasing ability of sperm involved in partial hydatidiform moles. Design: Analysis of the activating and Ca2þ-releasing ability of human sperm. Setting: University hospital research laboratory. Patient(s): Patients undergoing intracytoplasmic sperm injection (ICSI) treatment. Intervention(s): Microinjection of mouse and human oocytes with sperm. Main Outcome Measure(s): Measurement of the fertilizing and Ca2þ-releasing ability of human sperm. Result(s): The mouse oocyte Ca2þ analysis showed that only 19.0% (4/21) of the mouse oocytes injected with sperm involved in molar pregnancies exhibited a normal pattern of Ca2þ oscillations versus 63.2% (36/57) of those injected with control sperm. Further, 83.3% (15/18) of donated in vitromatured human oocytes injected with deficient sperm did not exhibit any Ca2þ release, while 76.9% (10/13) failed to show normal pronuclear development. Yet the sperm oocyte activation factor phospholipase C zeta (PLCz) was present in the majority (96.6%, n ¼ 113) of the analyzed sperm at a normal expression level. Eventually, fertilization failure was overcome with assisted oocyte activation in subsequent therapeutic ICSI cycles, which led to normal deliveries. Conclusion(s): Sperm that previously provoked recurrent partial hydatidiform mole pregnancies due to dispermic fertilization is not able to activate human oocytes or trigger the normal pattern of Ca2þ oscillations in mouse and human oocytes in vitro. (Fertil Steril 2014;102: 5818. 2014 by American Society for Reproductive Medicine.)

Objective: To investigate a method of assisted activation of human oocytes for the treatment of infertility resulting from globozoospermia associated with deficient oocyte activation capacity. Design: The mouse oocyte activation test was used to analyze the oocyte activation capacity of the sperm cells of a patient with globozoospermia. Fresh donor human oocytes were used for determining the most appropriate procedure for oocyte activation. Setting: Infertility Center, University Hospital of Ghent. Patient(s): A couple with infertility resulting from globozoospermia. Intervention(s): Intracytoplasmic sperm injection, assisted oocyte activation, and embryo transfer. Main Outcome Measure(s): Oocyte activation and fertilization rates, implantation, and pregnancy. Result(s): Deficiency in oocyte activation capacity was found in the sperm of a patient with globozoospermia. This deficiency was proven by the mouse oocyte activation test and was confirmed further by lack of activation of human oocytes after simple sperm injection. Only human oocytes that were injected with sperm and subjected to calcium chloride and ionophore treatment underwent activation. Transfer of embryos obtained by this procedure of assisted oocyte activation resulted in an ongoing pregnancy. Conclusion(s): Assisted oocyte activation of human oocytes is useful when globozoospermia is associated with absence of oocyte activation capacity in the sperm. These cases can be identified by the mouse oocyte activation test.

Objective To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. Design Retrospective data analysis study. Setting Academic centers for reproductive medicine and genetics. Patient(s) Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. Intervention(s) Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. Main Outcome Measure(s) This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. Result(s) We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). Conclusion(s) The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer.

Objective: To examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLCζ H398P, histidine > proline) and PLCζ H233L (histidine > leucine) mutations were previously identified. Design: Laboratory-based study. Setting: University laboratory. Patient(s): An infertile 38-year-old man with significantly impaired oocyte activation ability. Intervention(s): Minisequencing of individual sperm for PLCζ H398P and PLCζ H233L, and investigation of localization patterns arising from the expression of fluorescently tagged PLCζ isoforms in HEK293T cells. Main Outcome Measure(s): The presence/absence of PLCζ H398P and PLCζ H233L determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCζ isoforms quantified in HEK293T cells. Result(s): Sperm possessed either PLCζ H233L or PLCζ H398P, but never both at the same time. Fluorescent PLCζ H233L and PLCζ H233L+H398P (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLCζ WT > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCζ H233L+H398P exhibited a statistically significantly reduced level of fluorescence compared with PLCζ H398P at 48 hours but not 56 hours. Conclusion(s): Both H398P and H233L mutations are present on different alleles and do not alter PLCζ localization in HEK293T cells. Loss-of-activity mutations in PLCζ may contribute not only toward male infertility but also male subfertility in cases where PLCζ is mutated on a single allele. Copyright © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.

Objective To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Design Laboratory study. Setting University laboratory. Patient(s) Control subjects (men with proven oocyte activation capacity; n=16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n=5). Intervention(s) Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Main Outcome Measure(s) Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Result(s) Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. Conclusion(s) The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis.

Objective Is there any correlation between follicular fluid (FF) steroid levels and the occurrence of cytogenetic abnormalities in unfertilized human oocytes? Design Cytogenetic analysis was carried out on 397 oocytes, and the steroid content of 104 corresponding FF was analyzed using high-pressure liquid chromatography. Ovarian stimulation was performed by clomiphene citrate and human menopausal gonadotropin (hMG) or by hMG combined with a gonadotropin-releasing hormone agonist (GnRH-a) pretreatment. Results Oocyte maturity was correlated with an increasing FF progestin content and a significant decrease of androstenedione (A) levels. Chromosomal analysis revealed 84 of all oocytes to be abnormal (polyploid or aneuploid and/or prematurely condensed chromosomes present). In this group, A levels and A to estradiol ratios were significantly higher. Although progestin levels were higher in GnRH-a/hMG cycles, the incidence of oocyte normality was not different between the two stimulation schemes. More abnormal oocytes were found in patients with good sperm morphology. Conclusions Oocyte abnormality correlates with higher A levels in the corresponding FF. Oocyte fertilization is also determined by intrinsic oocytic factors other than maturity.

Objective To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. Design Study of diagnostic accuracy. Setting University hospital. Patient(s) Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. Intervention(s) Trophectoderm biopsy was performed on 47 blastocysts. Main Outcome Measure(s) In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. Result(s) In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼4.5 Mb deletion/duplication. Conclusion(s) This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.

Objective: To investigate whether cumulative pregnancy rates using life table analysis but without considering dropouts are representative of the whole population of patients entering an in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) program. Design: Retrospective study. Setting: University hospital-based infertility center. Patient(s): One thousand one hundred sixty-nine patients entering our IVF/ICSI program from January 1993 to December 1996. Intervention(s): Comparison of prognostic factors between pregnant and nonpregnant patients, and between patients continuing IVF/ICSI treatment and dropouts. Main Outcome Measure(s): Prognostic factors, such as patient age, cancellation of oocyte retrieval because of poor response to ovarian stimulation, number of oocytes retrieved, fertilization rate, number and quality of embryos transferred. Result(s): No statistical differences in prognostic factors were found between patients continuing IVF/ICSI treatment and dropouts. Conclusion(s): Cumulative pregnancy rates using life table analysis can be considered representative of the whole population of patients for at least the first three treatment cycles.