Is It Possible to Expand Oocyte Donors by Decreasing Number of Oocytes for Own Use? Insights From a Large Single-Center Study

BackgroundCurrently, in China, only women undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles can donate oocytes to others, but at least 15 oocytes must be kept for their own treatment. Thus, the aim of this study was to determine whether oocyte donation compromises the cumulative live birth rate (CLBR) of donors and whether it is possible to expand oocyte donors’ crowd.MethodsThis was a retrospective cohort study from August 2015 to July 2017 including a total of 2,144 patients, in which 830 IVF–embryo transfer (IVF-ET) patients were eligible for oocyte donation and 1,314 patients met all other oocyte donation criteria but had fewer oocytes retrieved (10–17 oocytes). All 830 patients were advised to donate approximately three to five oocytes to others and were eventually divided into two groups: the oocyte donation group (those who donated) and the control group (those who declined). The basic patient parameters and CLBR, as well as the number of supernumerary embryos after achieving live birth, were compared. These two factors were also compared in all patients (2,144) with oocyte ≥10.ResultsIn 830 IVF-ET patients who were eligible for oocyte donation, only the oocyte number was significantly different between two groups, and the donation group had more than the control group (25.49 ± 5.76 vs. 22.88 ± 5.11, respectively; p = 0.09). No significant differences were found between the two groups in other factors. The results indicate that the live birth rate in the donation group was higher than that in the control group (81.31% vs. 82.95%, p = 0.371), without significance. In addition, CLBR can still reach as high as 73% when the oocyte number for own use was 10. Supernumerary embryos also increased as the oocyte number increased in all patients (oocyte ≥10).ConclusionsCurrently, oocyte donation did not compromise CLBR, and oocyte donation can decrease the waste of embryos. In addition, in patients with 10 oocytes retrieved, the CLBR was still good (73%). Thus, it is possible to expand oocyte donors if the number of oocyte kept for own use was decreased from 15 to 10 after enough communication with patients.

P–624 Doxorubicin exposure detrimentally effects establishment of early ovarian reserve, ex-vivo mouse model

Study question Does exposure to chemotherapy agents used to treat cancer during pregnancy cause significant decrease in early establishment of the ovarian reserve? Summary answer Significant oocyte loss is noted following exposure of early mouse ovaries to doxorubicin. This apoptotic decline is not seen with cisplatin, docetaxel or paclitaxel exposure. What is known already Chemotherapy has been shown to adversely affect the ovarian reserve of pre-pubertal and reproductive age females. However, little is known regarding the effects in the next generation following treatment in pregnancy beyond observational population studies that examined rates of birth defects and developmental delays. Reproductive function in offspring develops over time and would be difficult to quantify in these short-term studies. An ex-vivo mouse ovary culture offers the ability to closely mimic maternal treatment level serum concentrations and directly evaluate the impact on oocyte number and cell death markers. Study design, size, duration In mice, the ovarian reserve is matured postnatally, mimicking the biologic second trimester activity of the human ovary. Wild-type C57bl/6 pups were collected on postnatal day 0. Ovaries were cultured in hanging well organ culture media with addition of DMSO or a chemotherapy agent. Immunofluorescence was used to quantify oocyte number and density. Power calculation showed a N of 11 per drug would be needed to demonstrate a decrease of ⅔ or more. Participants/materials, setting, methods: 83 ovaries were cultured, sectioned and analyzed in duplicate. Planned analysis at serum max and mid concentrations was performed at 48 hours and 5 days. Given clinically variation in concentrations of cisplatin, additional concentration samples were added. After noting the degradation following exposure to doxorubicin, additional samples at earlier time points of 12 and 24 hours of exposure were added for dynamic evaluation. Means were calculated and then compared using a 2-way ANOVA. Main results and the role of chance Doxorubicin exposure during establishment of the ovarian reserve resulted in a significant loss of oocyte number and density. At 12 hours a 22% decrease was noted; this increased to a loss of 91% of oocytes by 48 hours of exposure. The oocyte density fell from 693 oocytes/ mm² in the control to only 63 oocytes/ mm² in the serum max concentration (p = 0.003). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed early impact within the stroma by 12 hours with widespread apoptotic changes by 24 hours. Treatment with cisplatin resulted in a phenotypic change in the oocyte population with preservation of smaller, more peripheral cells. The average oocyte density remained similar to control even at the highest clinical concentrations, 536 oocytes/ mm² compared to 570 oocytes/ mm² (p = 0.772). Docetaxel and paclitaxel demonstrated an increase in oocyte number and density, though not enough to reach significance. The oocyte density 5 days following docetaxel exposure was 802 oocytes/ mm², a 41% increase (p = 0.12). The oocyte density 5 days following paclitaxel exposure was 780 oocytes/ mm², a 37% increase (p = 0.817). The drug exposure did impact stromal cells, as noted in TUNEL images. Limitations, reasons for caution This ex-vivo mouse model offers tight control of chemotherapy concentration, it does not account for filtration and modification by the placenta. Longer term cultures may also demonstrate that temporary arrest in small oocytes, such as those exposed to cisplatin, do not thrive and later progress to apoptosis. Wider implications of the findings: Doxorubicin is employed as the most common chemotherapy during pregnancy. Utilization may be to the serious detriment of the younger generation. If other alternatives are clinically effective they should be considered. No other models have explored the effect of fetal ovary exposure to chemotherapy on the establishment of ovarian reserve. Trial registration number Not applicable

Electroacupuncture Reduces Oocyte Number and Maintains Vascular Barrier Against Ovarian Hyperstimulation Syndrome by Regulating CD200

Ovarian hyperstimulation syndrome (OHSS) is a common complication caused by ovulatory stimulation therapy, which manifests as an increase in ovarian volume, an increase in the number of oocytes retrieved, and increased vascular permeability throughout the body and especially in ovarian tissue. In our previous study, we found that electroacupuncture (EA) could prevent the progression of OHSS, by mainly affecting ovary. However, the specific molecules and the mechanism of this process were still unknown. In order to explore the underlying mechanism, OHSS rat model was established and EA treatment was performed, which was followed by proteomic analysis of ovaries. Results showed a significant increase in the expression level of CD200 in the ovaries of OHSS group treated with EA than those of OHSS group. Clinical data showed that the level of CD200 in follicular fluid was negatively correlated with the number of oocytes retrieved and serum E2 level. Further in vitro experiments showed a concentration-dependent role of human chorionic gonadotropin (hCG) in reducing CD200 and CD200R levels, and increasing inflammatory cytokine levels in cultured KGN cells. In human umbilical vein endothelial cells (HUVECs), the vascular barrier function was improved by CM (cultural medium from KGN cell) which treated with CD200Fc (CD200R agonist). Meanwhile, the results of in vivo experiments indicated that EA reduced the number of ovarian corpora lutea, decreased inflammatory response, and improved the vascular barrier function by increasing the expression of CD200 and CD200R in rat ovaries. These findings suggest that EA treatment may reduce oocyte number and maintain vascular barrier against OHSS through ovarian anti-inflammatory response mediated by CD200. Therefore, this study is the first to identify CD200 as a main of EA in the ovary and elucidate the possible mechanism of EA on preventing and treating OHSS, which provide a scientific basis for CD200 as an effector and indicator in EA treatment.

C57BL/6J mouse superovulation: schedule and age optimization to increase oocyte yield and reduce animal use

Superovulation protocols have been described for different mouse strains, however the numbers of animals used are still high and still little information is known about hormone administration schedules and estrous cycle phases. In this study, we aimed to optimize a superovulation protocol by injecting 5 IU of pregnant mare serum gonadotropin followed by 5 IU of hCG 48 h later, using three different schedules related to the beginning of the dark cycle (3, 5 and 7 pm) in a light cycle of 7 am to 7 pm, with light on at 7 am. C57BL/6J mice at 3, 4 and 5 weeks of age were used and the estrous cycle phase for times of PMSG and hCG injections was also analyzed. Total oocyte number was counted in the morning after hCG injection. Hormones given at 3 weeks of age at 3 pm (59 ± 15 oocytes) and 7 pm (61 ± 10 oocytes) produced a significantly higher oocyte number compared with oocytes numbers collected from females at the same age at 5 pm (P = 0.0004 and <0.0001 respectively). Females at 4 and 5 weeks of age produced higher numbers of oocytes when superovulated at 7 pm. No statistical differences between females at different phases of the estrous cycle were found. These results showed that in C57BL/6J mice, hormones should be given at 3 or 7 pm for females at 3 weeks of age, however older females should be superovulated closer to the beginning of the dark cycle to reduce female mouse use and increase the numbers of oocytes produced per female.

Effect of Female Age on Infertility Diagnostic Factors and In-vitro Fertilization Treatment Outcomes: A Single-center Retrospective Cohort Study

BackgroundInfertility has become an important issue in modern world because of the increasing number of infertile couples around the world. Advanced maternal age was considered to be a main factor of infertile problem. With second child policy published in China and more women in China intend to seek help for infertility problem, this study provided information of fertility diagnostic factors and IVF treatment outcomes of female IVF patients in different age groups, which can be a guidance for infertility diagnostic and treatment.MethodsA retrospective cohort study was conducted to IVF patient data collected by Jinjiang District Maternal and Child Health Hospital, Chengdu, China. Clinical and laboratory data of 45,743 fresh, autologous IVF cycles from January 2008 to September 2017 were included in the analysis. The diagnostic factors and treatment outcomes were analyzed for different age groups (age<35, n=30,708; age 35-41, n=11,921 and age≥42, n=3,114) as well as further stratified advanced age groups (age 42, n=933; age 43, n=744; age 44, n=556; age≥45, n=881). The characteristics including number of previous cycles, duration of infertility, BMI, basic FSH, basic AFC, AMH, retrieved oocyte number, fertilized oocyte number, transferred embryo number, baby number and economic cost were stratified according to patient age.ResultsThe basic characteristics of obesity rate, basic FSH and cancellation rate of IVF cycles in ≥42 years old group were significantly higher than other groups (p<0.01). Basic AFC, AMH, retrieved oocyte number, fertilized oocyte number and transferred embryo number in ≥42 years old group were significantly lower than other groups (p<0.01). Diagnostics characteristics and IVF-ET outcome declined significantly when maternal age increased (p<0.05). In the meanwhile, a preliminary analysis of cost per cycle showed that cost per child increased with patient’s age increase.ConclusionAlthough with increasing number of advanced maternal age IVF cycles, the age group of ≥42 years would intend to get unsatisfied outcome and higher cost per child. More guidance and considerations should be focused on encouraging earlier age treatment of infertility.Plain English summaryWith more and more women in the global range choose to get late pregnancy because of changes in society and economy, age has become an unavoidable factor in infertility diagnostic and treatment. Advanced age women may experience more infertility problems and negative IVF outcomes. A better understanding of the effect of maternal age on infertility would offer help to both diagnostic and treatment of IVF patient. This study conducted a single-center retrospective cohort analysis to female patients of different age groups and found that women with more advanced age (≥42) would be more easily to experience unsatisfied IVF outcome and higher economic cost to obtain one child. After the publication of second child policy in China in 2013, the number of advanced age patients increased, the necessity of special guidance for AMA patient may need to be taken into consideration.

The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

ERK phosphorylates chromosomal axis component HORMA domain protein HTP-1 to regulate oocyte numbers

Oocyte numbers, a critical determinant of female reproductive fitness, are highly regulated, yet the mechanisms underlying this regulation remain largely undefined. In the Caenorhabditis elegans gonad, RAS/extracellular signal–regulated kinase (ERK) signaling regulates oocyte numbers; mechanisms are unknown. We show that the RAS/ERK pathway phosphorylates meiotic chromosome axis protein HTP-1 at serine-325 to control chromosome dynamics and regulate oocyte number. Phosphorylated HTP-1(S325) accumulates in vivo in an ERK-dependent manner in early-mid pachytene stage germ cells and is necessary for synaptonemal complex extension and/or maintenance. Lack of HTP-1 phosphorylation leads to asynapsis and persistence of meiotic double-strand breaks, causing delayed meiotic progression and reduced oocyte number. In contrast, early onset of ERK activation causes precocious meiotic progression, resulting in increased oocyte number, which is reversed by removal of HTP-1 phosphorylation. The RAS/ERK/HTP-1 signaling cascade thus functions to monitor formation and maintenance of synapsis for timely resolution of double-strand breaks, oocyte production, and reproductive fitness.

The DNA damage response regulates the oocyte pool in mammals

SummaryMammalian oogonia proliferate without completing cytokinesis producing germ cell cysts. Within these cysts, oocytes differentiate and enter meiosis, promote genome-wide double-strand break (DSBs) formation which repair by homologous recombination leads to synapsis of the homologous chromosomes. Errors in homologous recombination or synapsis trigger the activation of surveillance mechanisms, traditionally called ‘pachytene checkpoint’, to either repair them or send the cells to programmed death. Contrary to what is found in spermatocytes, most oocytes present a remarkable persistence of unrepaired DSBs at pachynema. Simultaneously, there is a massive oocyte death accompanying the oocyte cyst breakdown. This oocyte elimination is thought to be required to properly form the follicles, which constitute the pool of germ cells females will use during their adult life. Based on all the above mentioned, we hypothesized that the apparently inefficient meiotic recombination occurring in mouse oocytes may be required to eliminate most of the oocytes in order to regulate the oocyte number, promote cyst breakdown and follicle formation in mammalian females. To test this idea, we analyzed perinatal ovaries to evaluate the oocyte population, cyst breakdown and follicle formation in control and mutant mice for the effector kinase of the DNA damage response, CHK2. Our results confirm the involvement of CHK2 in the elimination of oocytes that accumulate unrepaired DSBs and show that CHK2 regulates the number of oocytes in fetal ovaries. We also show that CHK2 is required to eliminate oocytes as a result of LINE-1 activation, which was previously shown to be responsible for fetal oocyte loss. Nonetheless, the number of oocytes found in Chk2 mutant ovaries three days after birth was similar to that of control ovaries, suggesting the existence of CHK2-independent mechanisms capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2 mutant ovaries suggesting that CHK1 regulates postnatal oocyte death. Moreover, both CHK1 and CHK2 functions are required to timely breakdown cyst and form follicles. Altogether, we propose the DNA damage response controls the number of oocytes present perinatally and is required to properly break down oocyte cysts and form follicles, highlighting the importance of the DNA damage response in setting the reserve of oocytes each female will use during their entire lifespan.