[Effect of complete Y chromosome AZFc microdeletion on embryo euploidy].

OBJECTIVE: Preimplantation genetic testing (PGT) was performed to analyze the embryo euploidy in patients with complete Y chromosome AZFc microdeletion. METHODS: The clinical data of complete AZFc microdeletion underwent PGT from January 2013 to December 2021 in Reproductive Medicine Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively analyzed. The patients with monogenic disease who underwent PGT during the same period were set as the control group. The basic characteristics, fertilization rate, Day 3 high quality embryo rate, blastocyst formation rate, embryo euploid rate, 45, X embryo ratio was compared between the two groups. RESULTS: A total of 220 patients were included, including 91 patients with complete AZFc microdeletion and 129 patients with monogenic disease. There was no significant difference in age between the two groups. In semen parameters, the sperm concentration, total sperm count and progressive motility in AZFc microdeletion group were lower than those in monogenic disease group, and the differences were statistically significant (P=0.001). The fertilization rate of AZFc microdeletion group was lower than that in monogenic disease group (P=0.012), and there was no significant difference in the number of MII oocytes, Day 3 high-quality embryo rate and blastocyst formation rate. A total of 933 blastocysts were successfully tested, including 496 blastocysts in AZFc deletion group and 437 blastocysts in monogenic disease group. The euploid, aneuploid and mosaic rates of the AZFc microdeletion group were 57.26%, 24.60% and 18.14%, respectively, while those of the monogenic disease group were 66.13%, 23.80% and 10.07%, with statistically significant differences between the two groups (P=0.001). Further analysis of the two groups of aneuploid embryos showed that aberrations occurred most commonly in chromosome16 (3.87%), X (3.46%), 13 (2.44%), 22 (2.24%) and 19 (2.03%) in AZFc microdeletion group, respectively, while the monogenic disease group was 22 (4.35%), 16 (2.97%), 7 (2.74%), 15(1.60%) and 2(1.60%), respectively. The proportion of sex chromosome abnormality in AZFc microdeletion group was higher than that in monogenic disease group (P=0.039), and there was no significant difference in the proportion of 45,X between the two groups. CONCLUSION: Compared with monogenic disease group, The embryo euploid rate in AZFc microdeletion patients decreased and the proportion of 45, X embryos did not increase significantly. It is recommended to select euploid female embryos by PGT, which not only avoids vertical transmission of AZFc microdeletion, but also reduces the risk of miscarriage due to aneuploid embryos.

[Outcomes of micro-TESE combined with ICSI for non-obstructive azoospermia].

OBJECTIVE: To investigate the clinicaleffects of micro-dissection of testicular sperm extraction (micro-TESE) and its combination with intracytoplasmic sperm injection (ICSI) in the treatment of non-obstructive azoospermia (NOA). METHODS: We retrospectively analyzed the clinical data on 130 NOA patients treated between December 2015 and December 2017, including36 cases of idiopathic NOA, 22 cases of idiopathic NOA with small testis, 18 cases of Klinefelter syndrome, 46 cases of surgically treated cryptorchidism and 8 cases of AZFc microdeletion.All the patients underwent micro-TESE and 29 of them with sperm received micro-TESE + ICSI. We observed the changes in the postoperative serum T level, analyzed the influences of different types of NOA and testicular pathology on the sperm retrieval rate (SRR) and the outcomes of ICSI. RESULTS: All the micro-TESE operations were successfully completed. The SRR was significantly higher in those surgically treated for cryptorchidism (60.9%) than in the other NOA groups (P<0.05), withstatistically significant differencesnot among the latter groups (P>0.05) but among differenttypes of testicular pathology (P<0.05), the highest in the hypospermatogenesis group (100%), very low in those with Sertoli-cell-only syndrome (11.8%), and the lowest in the cases of spermatogenesis arrest (0). The serum T level was remarkably decreased at 1 and 6 months after operation (P<0.01), but markedly higher at 6 than at 1 month (P<0.01). Of the 52 patients with sperm retrieved at micro-TESE, 29 of their spouses received ICSI, resulting in 17 pregnancies, 6 live births, and 6 spontaneous abortions. CONCLUSIONS: Micro-TESE is an effective method for the treatment of NOA, and its combination with ICSI can help the NOA patients obtain their genetic offspring.

[Differentiation of embryonic stem cells from the embryos of the couples with male asthenozoospermia and Robertsonian translocation into germ cells].

OBJECTIVE: To assess the risk of male infertility in the offspring conceived through assisted reproductive technology (ART) byin vitroinductionof the differentiation of embryonic stem cells (ESCs) derived from the embryos of the couples with male asthenozoospermia and Robertsonian translocation (RT) into germ cells. METHODS: We established a CCRM16ESC line with the karyotype of 46, XY, +14, rob(13; 14) (q10; q10) from the embryo donated by a patientwithasthenozoospermiaand RT and his wife by isolation of the inner cell mass of blastula, culturing, passaging, and amplification,followed by in vitro induction and differentiationof the ESCs into germ cells with ratinoic acid(RA) at 2 mol/L. Then, we analyzed the process of differentiation and the expressions of its related genes and compared them with those in the normal CCRM23ESCs. RESULTS: CCRM16 showed the typical characteristics of ESCs, expressing the pluripotency makers of NANOG, OCT4, TRA-1-181 and SSEA4, forming embryoid bodies, and differentiating into three germlayer tissues in vitro and in vivo. Intervention with 2 mol/LRAinduced direct differentiation of the ESCs into germ cells. The expressions of the primordial germ cell marker geneDAZLand the meiosis marker geneSCP3were markedly decreased in the CCRM16 as compared with those in the normal CCRM23 ESCs. CONCLUSIONS: The CCRM16ESC linewith the karyotype of46, XY, +14, rob(13; 14) (q10; q10) has thetypical characteristics of ESCs but an abnormal process of differentiation into germ cells in the early stage. In vitroinductionof the differentiation of ESCs into germ cells can be used for assessing the risk of male infertility in the offspring conceived through ART for asthenozoospermia patients.

Oocyte vitrification technology has made egg-sharing donation easier in China.

When infertile women undergoing IVF or intracytoplasmic sperm injection (ICSI) have more than 20 mature oocytes retrieved, at least 15 oocytes are inseminated by their husband's spermatozoa. The extra oocytes are cryopreserved by vitrification. If the patients became pregnant and have healthy live births, the patients are encouraged to donate their remaining cryopreserved oocytes. Forty-seven egg-sharing donors were recruited after having normal deliveries and they donated their remaining oocytes, totalling 395 cryopreserved oocytes, to 75 recipients. The survival rate of vitrified-warmed oocytes was 83.0%. Following insemination by ICSI, the fertilization and cleavage rates were 83.8% and 89.8%, respectively. Out of 75 recipients, 71 recipients completed the treatment cycles and 30 of them became pregnant with clinical pregnancy and implantation rates of 42.3% and 25.5%, respectively. The birthweight of the new-born infants (22 from singleton and two from one set of twins) were 3344.5 ± 669.1g and 2425.0 ± 742.5 g, respectively. No birth defects were observed for the live births. These results indicate that oocyte vitrification is an effective methodology for an egg-sharing donation programme, with acceptable pregnancy and implantation rates.

Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

BACKGROUND: Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified. METHODS: The expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot. RESULTS: Hsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9). CONCLUSIONS: Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.

[Correlation between serum progesterone level at the day with human chorionic gonadotrophin administration and the outcome of pregnancy in in-vitro fertilization].

OBJECTIVE: To investigate the relationship between serum progesterone level at the day with human chorionic gonadotrophin (hCG) administration and pregnant outcome from in in-vitro fertilization-embryo transfer (IVF-ET). METHODS: From Mar. 2002 to Apr. 2007, 786 cycles with serum progesterone measurement on the day of hCG administration for final oocyte maturation in IVF were analyzed retrospectively in Reproductive Medicine Center in First Affiliated Hospital of Nanjing Medical University. All stimulations were down-regulated with gronadotrophin release hormone agonist (GnRH-a) in both long protocols and short protocols before gonadotrophin stimulation. When the thresholds of serum progesterone were set at 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 nmol/L, respectively. If the level of progesterone was less than the thresholds, those patients were in lower progesterone group, on the contrary, more than the threshold value, those patients were in higher progesterone group. The laboratory results and the clinical outcomes between all patients at lower and higher progesterone group at different thresholds value were analyzed. RESULTS: The rate of normal fertilization, quality embryos, successful implantation, chemical pregnancy, clinical pregnancy and live birth did not exhibit remarkable difference between patients with higher and lower serum progesterone level at multiple thresholds on the day of hCG administration in the 786 cycles (P > 0.05). However, when the thresholds of serum progesterone were at 8.5 and 9.0 nmol/L, early abortion rates of 27.3% (3/11) and 3/7 in higher progesterone group were significantly higher than 8.8% (26/297) and 8.6% (26/301) in lower progesterone group (P < 0.05). And the total abortion rates of 3/7 in higher progesterone group were significantly higher than 11.0% (34/301) in lower progesterone group when the thresholds of serum progesterone were 9.0 nmol/L (P < 0.05). CONCLUSIONS: This study did not prove the correlationship between progesterone level at the day with hCG administration and the probability of clinical pregnancy or live birth. However, early abortion rates or the total abortion rates were associated with higher progesterone level when the thresholds of serum progesterone were at 8.5 nmol/L or 9.0 nmol/L.

Peroxiredoxin 4, a new oxidative stress marker in follicular fluid, may predict in vitro fertilization and embryo transfer outcomes.

BACKGROUND: Peroxiredoxin 4 (Prdx4), a member of the Prdx family, can catalyze the reduction of reactive oxygen species. This study aims to explore whether Prdx4 can serve as an effective marker in follicular fluid (FF) for predicting in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle outcomes. METHODS: In this prospective study, all participants were recruited from the center of clinical reproductive medicine from 2017 September to 2018 December. Women with tubal or male factor infertility undergoing their first IVF/ICSI cycle were recruited (n=138). FF samples from each patient were collected on the day of oocyte retrieval. Prdx4 concentrations were measured, and the correlation between Prdx4 levels and IVF outcomes was analyzed. RESULTS: The results showed that pregnant women had higher levels of Prdx4 than nonpregnant women. Prdx4 was positively correlated with the oocyte fertilization rate (r =0.334; P=0.011) and good quality embryo rate (r =0.326; P=0.013). Furthermore, we found that the clinical pregnancy rate was positively correlated with Prdx4 levels in a concentration-dependent manner in the Prdx4 quartiles (<13.38, 13.83-16.93, 16.93-22.93, >22.93 ng/mL). The fertilization rates, clinical pregnancy rates and live pregnancy rates were all significantly higher in the highest Prdx4 quartile group than in the lowest quartile. Moreover, the results indicated that Prdx4 had an area under the receiver operating characteristic curve (AUC) of 0.754, corresponding to an optimal cutoff point of 22.30 ng/mL. CONCLUSIONS: Our results provide evidence that higher expression of antioxidants, such as Prdx4, in the FF of IVF patients tends to indicate a higher likelihood of pregnancy through an oocyte quality mechanism.

[Retrospective analysis of the clinical outcomes of repeated IVF/ICSI cycles].

OBJECTIVE: To study whether the rates of pregnancy and implantation decline in repeated IVF/ICSI cycles and whether the decline is associated with the availability of embryo cryopreservation. METHODS: We retrospectively analyzed the pregnancy and implantation rates of 1,033 IVF/ICSI cycles accomplished in our center to determine the association of the clinical outcomes with the availability of embryo cryopreservation. RESULTS: The rates of pregnancy and implantation declined slightly in the cycles with embryo cryopreservation (43% vs 43%, P > 0.05; 29% vs 24% , P > 0.05), but significantly in the repeated IVF/ICSI cycles without embryo cryopreservation (43% vs 35%, 43% vs 22% , P < 0.05; 29% vs 23% , 29% vs 16% , P < 0.05). CONCLUSION: The rates of pregnancy and implantation remain similar in the following cycles for those with embryo cryopreservation in the first IVF/ICSI cycles, but decline significantly in the repeated IVF/ICSI cycles for those without.

[Polypronuclear zygotes and clinical pregnancy after IVF].

OBJECTIVE: To study the relationship between the percentage of polypronuclear zygotes and clinical pregnancy following IVF. METHODS: We collected the data of 954 IVF cycles, and according the percentage of polypronuclear zygotes in the IVF cycles, allocated them to Groups A (without polypronuclear zygotes) , B (with < 30% polypronuclear zygotes) and C (with > or = 30% polypronuclear zygotes). Then we analyzed the relationship between the percentage of polypronuclear zygotes and the rate of clinical pregnancy. RESULTS: Compared with Group A, Group C showed a significantly lower rate of clinical pregnancy (43.2% vs 28. 1%, P < 0.05), while Group B exhibited a markedly higher rate (43.2% vs 52.36%, P < 0.05) and obviously decreased polypronuclear zygote formation with the increase of age (35.6% vs 24.1%, P < 0.05). CONCLUSION: The percentage of polypronuclear zygotes in IVF cycles may serve as a prognostic indicator of the clinical outcome.

[Clinical effects of oocyte cryopreservation in assisted reproduction technology].

OBJECTIVE: To investigate the clinical effects of oocyte cryopreservation in assisted reproduction technology (ART). METHODS: 258 patients undergoing retrieval of more than 20 oocytes during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) were divided into 2 groups:Group A, undergoing surplus oocytes cryopreservation (84 cycles) and Group B undergoing embryo cryopreservation (174 cycles) according to the patients' choices. Fertilization rate and clinical pregnancy rate of fresh embryo transfer cycle were compared between these two groups. Twenty-three infertile couples' frozen oocytes were thawed for further ART treatment. Among them, fifteen couples received embryo transfer using their own frozen-thawed oocytes, and other four couples used donated frozen-thawed oocytes. The survival rate, fertilization rate, cleavage rate, implantation rate, and clinical pregnancy rate of these 19 cycles were compared to the outcome of 56 frozen-thawed embryo transfer cycles. RESULTS: The fertilization rate of Group A who underwent IVF was 65.9%, not significantly different from that of Group B who received IVF (66.9%, P > 0.05), and the fertilization rate of Group A who underwent ICSI was 71.6%, not significantly different from that of Group B who received ICSI (64.1%, P > 0.05). The clinical pregnancy rate (per embryo transfer cycle) of Group A who received IVF was 52.9%, not significantly different from that of Group B who received IVF (42.3%, P > 0.05), and the clinical pregnancy rate (per embryo transfer cycle) of Group A who received ICSI was 35.5%, not significantly different from that of Group B who received ICSI (34.4%, P > 0.05). The clinical pregnancy rate of frozen-thawed oocyte group (per embryo transfer cycle) was 47.4% (9/19). Four couples used donated frozen-thawed oocytes, two of them got clinical pregnancy and one of them had term delivery. CONCLUSION: For women who undergo retrieval of more than 20 oocytes in IVF/ICSI, the clinical outcome of fresh embryo transfer cycle, such as fertilization rate and clinical pregnancy rate, are not influenced by oocyte cryopreservation and embryo cryopreservation. There is no significant difference in the clinical pregnancy rate (per embryo transfer cycle) between frozen-thawed oocyte group and frozen-thawed embryo group. Compared with embryo cryopreservation, oocyte cryopreservation has obvious advantages in fertility preservation and oocyte donation.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa.

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

[Intracytoplasmic sperm injection can improve the fertilization limitation following in vitro fertilization during previous cycles].

OBJECTIVE: To evaluate whether or not intracytoplasmic sperm injection (ICSI) can improve previous fertilization limitation on conventional in vitro fertilization (IVF). METHODS: One hundred and thirty-six completed cycles in 113 patients with ICSI treatment were grouped. Group 1 was 106 cycles perform ICSI because of male factor, and group 2 was other 30 cycles with the history of fertilization failure and fertilization rate < 20% on conventional IVF, also assembling the cycles in group 2 according to the fertilization rate. RESULTS: There was no significant difference between two groups in the rates of normal fertilization(70.49% vs 72.02%), good quality embryos (38.28% vs 38.81%), and clinical pregnancy (40.57% vs 40.00%) (P > 0.05). The fertilization rate of a majority of cycles (70.00%, 21/30) in group 2 was higher than 50%, and the mean of fertilization rates was 79.79%. CONCLUSION: ICSI can improve the fertilization limitation following IVF during previous cycles, and the fertilization rates was similar to those treated ICSI because of male factor.

Downregulated expression of peroxiredoxin 4 in granulosa cells from polycystic ovary syndrome.

Peroxiredoxin 4 (PRDX4), a member of Peroxiredoxin (PRDX) family, is a typical 2-Cys PRDX. PRDX4 monitors the oxidative burden within cellular compartment and reduces hydrogen peroxide and alkyl hydroperoxide related to oxidative stress and apoptosis. Antioxidant, like PRDX4, may promote follicle development and participate in the pathophysiology of PCOS. In our previous study, we found that PRDX4 was expressed in mice oocyte cumulus oophorus complex, and that PRDX4 could be associated with follicle development. In this study, we explored the expression of PRDX4 in human follicles and possible role of PRDX4 in PCOS pathophysiology. Our data showed that PRDX4 was mainly expressed in granulosa cells in human ovaries. When compared to control group, both PRDX4 mRNA level and protein level decreased in PCOS group. The lowered levels of PRDX4 may relate to oxidative stress in the pathophysiologic progress of PCOS. Furthermore, expression of PRDX4 in the granulosa cells of in vivo or in vitro matured follicles was higher than that in immatured follicles, which suggested that PRDX4 may have a close relationship with follicular development. Altogether, our findings may provide new clues of the pathophysiologic mechanism of PCOS and potential therapeutic strategy using antioxidant, like PRDX4.

Delivery after transfer of frozen-thawed embryos from in vitro-matured oocytes in a woman at risk for ovarian hyperstimulation syndrome.

The present report describes the birth of a healthy infant after cryopreservation of embryos produced from in vitro-matured oocytes retrieved from a woman at risk of developing ovarian hyperstimulation syndrome (OHSS) during conventional in vitro fertilization (IVF) cycles. A conventional long protocol including gonadotropin-releasing hormone agonist (GnRHa) and gonadotropins induced a risk of OHSS. Oocyte retrieval was performed on day 11 of the cycle, and 27 immature oocytes were obtained. Following incubation for 24 h in maturation medium, 74.1% (20/27) of the oocytes were at the metaphase II stage. Fourteen oocytes (14/20, 70.0%) were fertilized after intracytoplasmic sperm injection (ICSI) with her husband's spermatozoa and cultured for 3 days. On day 4 following oocyte retrieval, three embryos at the 8-16 cell stage were transferred into the woman's uterus, and five spare embryos were frozen. Since the fresh embryo transfer failed to result in pregnancy, three post-thaw embryos were transferred into the woman three months later. Transfer of the frozen embryos resulted in pregnancy with delivery of a healthy infant girl.

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa.

PURPOSE: In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems. METHODS: Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA. RESULTS: Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients. CONCLUSIONS: This result demonstrates that the successful pregnancies and live births can be established from embryos produced from in vitro matured oocytes that fertilized with testicular sperm.