The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

Background: SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. Methods: We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). Results: SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. Conclusions: The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Identification of common genetic polymorphisms associated with down-regulated gonadotropin levels in an exome-wide association study.

OBJECTIVE: To investigate whether common genetic polymorphisms are associated with gonadotropin levels after down-regulation with daily gonadotropin-releasing hormone agonist and whether the polymorphisms of candidate variants influence the ovarian response to exogenous gonadotropins. DESIGN: Genetic association study. SETTING: University-affiliated in vitro fertilization center. PATIENTS: Subjects enrolled in an exploratory exome-wide association study (n = 862), a replication exome-wide association study (n = 86), and a classifier validation study (n = 148) were recruited from September 2016 to October 2018, September 2019 to September 2020, and January 2021 to December 2021, respectively. The included patients were aged ≤40 years and had a basal follicle-stimulating hormone (FSH) ≤12 IU/L. INTERVENTIONS: All participants received a luteal phase down-regulation long protocol. Genome DNA was extracted from the peripheral blood leukocytes. For the exploratory and replication cohorts, exome sequencing was conducted on a HiSeq 2500 sequencing platform. The multiplex polymerase chain reaction amplification technique and next-generation sequencing also were performed in the exploratory and replication cohorts. For the samples of the validation cohort, Sanger sequencing was performed. MAIN OUTCOME MEASURES: The primary endpoint was the gonadotropin levels after down-regulation, and the secondary endpoints were hormone levels and follicle diameters during stimulation, the total dose of FSH, duration of FSH stimulation, number of oocytes retrieved, and clinical pregnancy rate. RESULTS: In the exploratory cohort, we identified that FSHB rs6169 (P=2.71 × 10-24) and its single-nucleotide polymorphisms in high linkage disequilibrium were associated with the down-regulated FSH level. The same locus was confirmed in the replication cohort. Women carrying the C allele of FSHB rs6169 exhibited higher average estradiol level during stimulation (P=6.82 × 10-5), shorter duration of stimulation, and less amount of exogenous FSH (Pduration=0.0002; Pdose=0.0024). In the independent validation set, adding rs6169 genotypes into the prediction model for FSH level after down-regulation enhanced the area under the curve from 0.560 to 0.712 in a logistic regression model, and increased prediction accuracy by 41.05% when a support vector machine classifier was applied. CONCLUSION: The C allele of FSHB rs6169 is a susceptibility site for the relatively high level of FSH after down-regulation, which may be associated with increased ovarian FSH sensitivity.

Pregnancy outcomes in patients with polycystic ovary syndrome who conceived after single thawed blastocyst transfer: a propensity score-matched study.

BACKGROUND: It remains unclear whether polycystic ovary syndrome (PCOS) is an independent risk factor for pregnancy complications in women undergoing assisted reproductive technology (ART) treatment. For the integrative treatment of PCOS patients, it is still important to investigate the pregnancy outcomes of PCOS patients after adjusting for potential biases, such as body mass index, embryo quality and endometrial preparation method. METHODS: This retrospective cohort study ultimately included a total of 336 PCOS patients who conceived after single thawed blastocyst transfer in the PCOS group and 2,325 patients in the control group from January 2018 to December 2020. A propensity score matching (PSM) model was used, and 336 PCOS patients were matched with 336 patients in the control group. RESULTS: Before PSM, no differences in the miscarriage rate, pregnancy complication rate, preterm birth rate, or live birth rate were found between the PCOS group and the control group. After PSM, the late miscarriage rate of the PCOS group was significantly higher than that of the control group (3.3% vs. 0.6%, P = 0.040), although the early miscarriage rates were similar (14.0% vs. 13.7%). The rates of pregnancy complications, preterm birth and live birth in the PCOS group were comparable to those in the matched control group (P = 0.080, P = 0.105, P = 0.109, respectively). The neonatal weights of male infants and female infants were similar between the two groups (P = 0.219, P = 0.169). Subgroup analysis showed that PCOS patients with homeostasis model assessment of insulin resistance (HOMA-IR) levels ≥ 2.49 had a significantly increased risk of preterm birth compared with those with HOMA-IR levels < 1.26 and 1.26 ≤ HOMA-IR levels < 2.49 (26.0% vs. 6.0% vs. 9.8%, P = 0.005). PCOS patients with total testosterone levels ≥ 0.7 ng/ml had a higher early miscarriage rate but a lower late miscarriage rate than those with total testosterone levels < 0.7 ng/ml (29.4% vs. 12.3%, 0% vs. 3.6%, respectively, P = 0.032). CONCLUSIONS: PCOS is an independent risk factor for late miscarriage in patients conceived after a single thawed blastocyst transfer, even after adjusting for biases. Among PCOS patients, insulin resistance and hyperandrogenism are associated with a higher risk of preterm birth and early miscarriage, respectively.

Impact of metabolic disorders on endometrial receptivity in patients with polycystic ovary syndrome.

The present study investigated the expression of endometrial receptivity-related molecules in patients with polycystic ovary syndrome (PCOS) and different androgen status, insulin resistance (IR) levels, and body mass indexes (BMI) to identify the mechanism underlying their effects on pregnancy outcomes. The present study recruited 43 participants from November 2020 to January 2021, which were classified into five groups: i) Hyperandrogenemia (HA) combined with impaired glucose tolerance group (n=8); ii) HA combined with diabetes mellitus group (n=8); iii) HA combined with non-IR (NIR) group (n=10); iv) non-HA (NHA) androgen combined with IR group (n=8); and v) NHA combined with NIR group (n=9). In addition, according to their BMIs, patients were sub-grouped into lean/normal (n=27), overweight (n=8) or obese (n=8) groups. The mRNA expression levels of endometrial receptivity-related molecules were detected using reverse transcription-quantitative PCR. In addition, flow cytometry was used to determine the phenotype and percentage of uterine natural killer cells (uNK). According to the results, patients with PCOS and IR status, HA and obesity (BMI ≥24 kg/m2) demonstrated significantly decreased mRNA expression levels of adiponectin, adiponectin receptor (AdipoR)1, AdipoR2, adapter protein containing PH domain, PTB domain and leucine zipper motif 1, estrogen receptor (ER) α, ERß, progesterone receptor (PR), IL-15, integrin ß3 avß3, and insulin-like growth factor binding protein-1, but increased mRNA expression levels of IL-6 and IL-8 compared with NHA + NIR group or lean/normal group, respectively. In addition, obese patients with PCOS demonstrated increased mRNA expression levels of PR compared with overweight patients. This suggested that insulin resistant status, HA, and obesity could alter the endometrial receptivity of patients with PCOS, which may explain poorer embryo implantation and pregnancy outcomes in clinics.

Establishment of an evaluation model for human milk fat substitutes.

Fatty acid composition and distribution of human milk fat (HMF), from mothers over different lactating periods in Guangzhou, China, were analyzed. The universal characteristics were consistent with previously reported results although the fatty acid content was within a different range and dependent on the local population (low saturated fatty acid and high oleic acid for Guangdong mothers' milk fat). Based on the composition of the total and sn-2 fatty acids of mature milk fat, an efficient evaluation model was innovatively established by adopting the "deducting score" principle. The model showed good agreement between the scores and the degree of similarity by assessing 15 samples from different sources including four samples of HMF, eight samples of human milk fat substitutes (HMFSs) and infant formulas, and three samples of fats and oils. This study would allow for the devolvement of individual human milk fat substitutes with different and specific fatty acid compositions for local infants.

Establishment of human embryonic stem cell line from gamete donors.

BACKGROUND: Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. METHODS: Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed. RESULTS: Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types. CONCLUSIONS: HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

Cryopreservation of human embryonic stem cells by vitrification.

BACKGROUND: The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method. METHODS: Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified. RESULTS: Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers. CONCLUSIONS: Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.