Faculty Bibliography

The ovaries have a fixed pool of immature (primordial) follicles at birth known as the ovarian reserve. Activation or loss of follicles in this reserve causes an irreversible decline in reproductive function that culminates in menopause. Premenopausal women with cancer who are treated with conventional genotoxic chemotherapy have accelerated loss of the ovarian reserve, leading to subfertility and infertility. Cyclophosphamide (CY), a highly gonadotoxic drug, is widely used as part of combination cancer chemotherapy. This drug induces ovarian damage in large part by activating the mammalian/mechanistic target of rapamycin (mTOR) pathway, leading to activation of primordial follicles, follicular burnout, and premature menopause. As a result, the probability of pregnancy in premenopausal female cancer survivors is significantly diminished. There has been little progress in preserving ovarian function during cancer chemotherapy. As part of multiagent chemotherapeutic regimens, inhibitors of themTORC1 pathway have a growing role in cancer treatment and are being studied as treatment for a growing number of malignant and nonmalignant conditions. Mammalian/mechanistic target of rapamycin inhibitors block the primordial-to-primary follicle transition. The investigators used a clinically relevant mouse model of chemotherapy-induced gonadotoxicity to investigate whether inhibitors of mTOR could block CY-induced premature activation of primordial follicles and also preserve fertility during chemotherapy. Two inhibitors of the mTOR pathway were used: everolimus (RAD001), a drug clinically approved for treatment of tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer), and INK128, an experimental drug. Female mice were treated with CY weekly and then randomized to also receive either everolimus, INK128, or nothing.

Study question: What happens to telomere length during human meiotic maturation and pre-implantation development? What is the impact of DNA damage and telomere attrition on human development? Summary answer: DNA damage and telomere attrition limit oocyte maturation in vitro. Telomeres are short in oocytes, increase markedly, and develop increased heterogeneity during pre-implantation development. What is known already: Telomere length reflects aging in many cell types. Sperm, which emerge from spermatogonia throughout the life of the man, have long telomeres. Oocytes from mouse and women maintain short telomeres. Telomerase, the enzyme that lengthens telomeres, is minimally active in mouse and human oocytes and pre-implantation embryos until blastocyst stage. Lacking appreciable telomerase activity, early mouse embryos elongate telomeres via Alternative Lengthening of Telomeres (ALT), which provides robust telomere elongation, but increased genomic instability. We do not know whether ALT occurs during early human development, and if so, when it takes place. Study design, size, duration: 60 immature germinal vesicle (GV) and metaphase I (M1) human oocytes donated to research from women ages 18-45 were collected and in vitro matured (IVM) for up to 48 hours following the subjects' retrieval. Additionally, 28 cryopreserved human embryos donated to research from 7 couples (age 27-42 years old), at the New York University Langone Fertility Center. Participants/materials, setting, methods: Immature oocytes were in vitro matured to metaphase II (M2). Frozen embryos between the 2 pronuclear (2PN) and day 3 (8-10-cell) stage were thawed and dissociated into single blastomere, intact blastocysts were processed whole. Telomere length was evaluated using Single Cell Amplification of Telomere Repeats PCR (SCATRPCR), expressed as a telomere to reference gene ratio (T/R ratio). DNA damage was assessed by immunoflorescent staining. Statistical analysis was performed using one-way ANOVA or T-test where appropriate. Main results and the role of chance: During oocyte maturation, immunostaining revealed that oocytes which arrested at the GV stage and failed to mature, contained robust and abundant DNA damage signaling on their chromosomes compared with successfully matured M2s (mean total florescent units = 35073.4 +/- 10051.8 vs. 843.7 +/- 74.9). Telomere length however did not differ significantly between arrested GV and M2 oocytes (mean T/R ratio = 0.074 +/- 0.040 vs 0.105 +/- 0.067). Telomere length increased significantly (p < 0.05) between M2 oocytes and both 2PN embryos (mean T/R ratio = 0.837 +/- 0.546) and blastocysts (mean T/R ratio = 0.634 +/- 0.260). The most significant elongation (p < 0.0002) occurred by day 2 (2-4 cell) (mean T/ R ratio = 0.957 +/- 767). Between day 2 and day 3 (mean T/R ratio = 0.385 +/- 0.418) telomere length decreased. These data suggest early activation of a telomere lengthening mechanism, prior to zygotic genome activation. Additionally, Intra-embryo telomere length increased until its peak day 3 (Coefficient of Variation = 108.51%) and was at its lowest at the blastocyst stage (Coefficient of Variation = 40.97%) when telomerase becomes active. Together these data suggest that an Alternative Lengthening of Telomeres (ALT) mechanism may be responsible for both increases in telomere length and increased heterogeneity among blastomeres within early embryos. Limitations, reasons for caution: The limited sample size for some of the earliest embryonic stages in addition to the freezing method for the embryos may have affected quality, and an inability to infer developmental ability as the embryos were not cultured to blastocyst. Wider implications of the findings: Our study is the first to perform a molecular characterization of telomere dynamics and the role of DNA damage in human oocytes and embryos at the single cell level and provides the first evidence that ALT is part of normal embryonic development in humans

BACKGROUND: Transgender individuals, individuals whose gender identity does not align with their sex assigned at birth, undergoing gender-affirming hormonal or surgical therapies may experience loss of fertility. Assisted reproductive technologies have expanded family-building options for transgender men who were assigned female at birth. CASES: Three transgender men underwent oocyte cryopreservation before gender-affirming hormonal therapy. One patient underwent fertility preservation as an adolescent. Two adult patients had children using their cryopreserved oocytes, with the pregnancies carried by their sexually intimate partners. CONCLUSION: Transgender men with cryopreserved gametes can build families in a way that affirms their gender identity. Obstetrician-gynecologists should be familiar with the fertility needs of transgender patients so appropriate discussions and referrals can be made.

Ovarian aging occurs earlier than somatic aging. We tested the hypothesis that ovarian functions could be artificially reconstructed by transplantation of primordial germ cells (PGCs). We compared various methods for transplantation of PGCs aggregated with gonadal somatic cells and showed that reconstituted ovaries exhibited folliculogenesis after transplantation of PGCs-aggregates into either kidney capsule or ovarian bursa. Neo-oogenesis occurred early after transplantation, as evidenced by the presence of prophase I meiocytes displaying homologous pairing. Moreover, endocrine function was recovered in ovariectomized recipients, including elevated levels of AMH and estradiol. Interestingly, folliculogenesis in the reconstituted ovaries failed to sustain past four weeks. Regardless of transplantation method, follicles diminished after 45 days, accompanied by increased apoptosis, and were undetectable after two months. Meanwhile, no replicative PGCs or prophase I meiocytes could be found. Together, transplantation of PGCs can effectively reconstitute ovarian functions but for limited time. These data suggest that PGCs do not undergo self-renewal but rapidly enter meiosis following transplantation. Global activation of primordial follicles in artificial ovaries can result in further rapid loss of germ cells. Methods for maintaining self-renewal and expansion in vivo of PGCs and controlling follicle activation will be essential for continuing maintenance of the functional reconstructed ovaries.