• Clinical and Experimental Reproductive Medicine
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• 제46권1호
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• pp.36-40
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• 2019

A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients' semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.

• Clinical and Experimental Reproductive Medicine
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• 제40권1호
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• pp.7-11
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• 2013

Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

• Journal of Yeungnam Medical Science
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• 제38권1호
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• pp.53-59
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• 2021

Background: There are no guidelines for the optimal incubation time or temperature to improve pregnancy outcomes in testicular sperm extraction-intracytoplasmic sperm injection (TESE-ICSI) cycles. We aimed to evaluate whether a 24-hour in vitro culture of testicular spermatozoa affects pregnancy outcomes in TESE-ICSI cycles. Methods: This was a retrospective study of 83 TESE-ICSI cycles using testicular spermatozoa in 46 couples with male partners suffering from nonobstructive or obstructive azoospermia. Sperm retrieval was performed either on the oocyte retrieval (OR) day (65 cycles in 33 couples; group A) or on the day before OR (18 cycles in 13 couples; group B) followed by in vitro culture for 24 hours. The clinical characteristics and pregnancy outcomes, including the number of retrieved oocytes, fertilization rates, embryo transfer rates, implantation and clinical pregnancy rates, were compared between the two groups. Results: There were no differences in terms of clinical characteristics except for the levels of luteinizing hormone (LH) in males. Group B had higher LH levels than group A (4.56±1.24 IU/L vs. 3.67±1.07 IU/L, p= 0.017). Group B showed higher fertilization rate (72.4%±32.1% vs. 59.2%±21.7%, p=0.045), implantation rate (35.0%±34.1% vs. 14.0%±21.5%, p=0.010), pregnancy rate per cycle (80% vs. 39%, p=0.033), and clinical pregnancy rate per cycle (80% vs. 37.5%, p=0.024) than those of group A. Conclusion: Testicular sperm retrieval performed on the day before OR followed by in vitro culture can potentially improve pregnancy outcomes.

• 한국수정란이식학회지
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• 제20권3호
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• pp.201-206
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• 2005

본 연구는 ICSI후 돼지 난자의 활성화 처리와 ICSI전 주입정자의 수정 능력 획득 유기효과를 구명하기 위하여 실시하였다. ICSI후 ethanol, cycloheximide 그리고 ethanol과 cycloheximide를 병용처리 시 난할율과 배반포배 발달율이 대조구와 처리 구간에 유의적인 차이가 없었다(p<0.05). 그러나 ICSI전 caffeine과 Ca-ionpphre로 주입정자의 수정능력 획득 유기 처리 시 난할율과 배반포배발달율 모두 처리구에서 유의적으로 놓았다(p<0.05). 한편 ICSI후 난자활성화처리 시 전핵형성율과 총 난자 활성화율은 대조구와 처리구간에 유의차가 없었지만(p<0.05) 정자의 수정능력 획득 유기 처리 시 전핵형성율과 총 난자 활성화율은 처리구에서 유의적으로 높게 나타났다(p<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제50권3호
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• pp.185-191
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• 2023

Objective: Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. Methods: In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. Results: DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. Conclusion: The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.95-99
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• 1997

Irreparable obstructive azoospermic patients can be treated successfully with microsurgical epididymal sperm aspiration(MESA) or testicular sperm extraction (TESE) by intracytoplasmic sperm injection(ICSI). Obstructive azoospermic patients generally have normal spermatogenesis. The aim of this study was to see if any spermatozoa could be retrieved from non-obstructive azoospermia and to assess the efficacy of ICSI with TESE in germinal failure. 42 non-obstructive azoospermic patients revealed no spermatozoa at all in their ejaculates, even after centrifuge. The histology of 42 patients revealed 15 Sertoli cell only Syndrome, 4 maturation arrest and 23 severe hypospermatogenesis. All patients underwent extensive multiple testicular biopsy for sperm retrieval. These patients were scheduled for ICSI using testicular spermatozoa. In 25 out of 42 non-obstructive azoospermic patients, spermatozoa were recovered from multiple testicular biopsy specimen and 11 ongoing pregnancies were achieved. There are usually some tiny foci of spermatogenesis which allow TESE with ICSI in non-obstructive azoospermia. Also these patients may have sufficient sperm in the testes for ICSI, despite extremely high FSH level and small testes.

• Clinical and Experimental Reproductive Medicine
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• 제43권4호
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• pp.221-227
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• 2016

Objective: The aim of this study was to evaluate the influence of maternal age on fertilization, embryo quality, and clinical pregnancy in patients undergoing intracytoplasmic sperm injection (ICSI) using testicular sperm from partners with azoospermia. Methods: A total of 416 ICSI cycles using testicular spermatozoa from partners with obstructive azoospermia (OA, n = 301) and non-obstructive azoospermia (NOA, n = 115) were analyzed. Female patients were divided into the following age groups: 27 to 31 years, 32 to 36 years, and 37 to 41 years. The rates of fertilization, high-quality embryos, clinical pregnancy, and delivery were compared across maternal age groups between the OA and NOA groups. Results: The rates of fertilization and high-quality embryos were not significantly different among the maternal age groups. Similarly, the clinical pregnancy and delivery rates were not significantly different. The fertilization rate was significantly higher in the OA group than in the NOA group (p< 0.05). Age-group analysis revealed that the fertilization and high-quality embryo rates were significantly different between the OA and NOA groups in patients aged 27 to 31 years old, but not for the other age groups. Although the clinical pregnancy and delivery rates differed between the OA and NOA groups across all age groups, significant differences were not observed. Conclusion: In couples using testicular sperm from male partners with azoospermia, pregnancy and delivery outcomes were not affected by maternal age. However, women older than 37 years using testicular sperm from partners with azoospermia should be advised of the increased incidence of pregnancy failure.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.385-391
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• 1997

The purpose of this study was to examine the effects of anti-sperm antibody (ASA) on the fertilization processes using conventional IVF and ICSI procedure in human and hamster oocytes. In human IVF, we have observed restricted fertilization with sperm testing positive for ASA. ($23{\sim}90%$ IgA, 60-97 % IgG). However, if ICSI was perform in the next IVF cycle with the same patients, we could successfully fertilize the oocytes (37%; p<0.001), thus achieving pregnancy and delivery. When the sperm were cocultured in medium containing ASA, there were binding of ASA to sperm surface. In addition, the mean rate of the acrosomal reaction in an in vitro acrosome reaction test was lower for Ab-bound sperm (43.5%) than for Ab-free sperm group (51.3%, p<0.05). We used human sperm and hamster oocytes to confirm the negative effects of the ASA on fertilization. The sperm and/or oocytes have been expose to medium containing ASA before IVF and ICSI. In this experiment, the ASA was bound to the oocyte and sperm surface. The following results were obtain by using various combinations of ASA free or ASA bound sperm with ASA free or ASA bound oocytes for IVF. When ASA free sperm were inseminate with ASA free and ASA bound hamster oocytes, the fertilization rates are 89.6% and 74.3% respectively. However, when ASA bound human sperm were use the results were 62.5% and 55.6% respectively. These shows the fertilization rate was significantly decreased in both ASA bound and ASA free oocytes when using ASA bound sperm. No difference found when ASA are present on the oocyte surface. When the hamster oocytes was treated by ICSI with ASA free or ASA bound human spermatozoa, no significant difference was found. These results showed that ICSI is the most promising method for couples who fertilization was not possible by conventional IVF because of ASA.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.301-309
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• 1997

본 연구에서는 체외수정, 난자내 정자 직접주입, 난자내 정자 두부 미두 주입 후의 핵과 미세소관의 변화를 관찰하였다. 핵과 미세소관의 움직임은 형광염색을 실시한 후 공초점주사현미경을 이용하여 관찰하였다. 체외수정에서 관찰된 바와 동일하게 정자를 난자에 직접주입 한 직후 정자 중편부에서 성상체가 형성되었고, 이 성상체에 의해 자성 웅성 전핵이 융합되는 것으로 관찰되었다. 그러나 난자내 정자를 직접주입하였을 경우 웅성전핵으로 발달하는 비율이 낮았다. 이는 주입된 정자가 원형질막과 perinuclear theca에 싸인 체 난자내로 들어가 난자내의 sperm nucleus decondensing factor와 정자 핵과의 반응이 억제되기 때문으로 생각된다. 정자 두부 만을 주입하였을 경우 성상체가 형성되지 않았지만 자성 웅성 전핵 사이 또는 그 주위에서 두터운 미세소관층이 관찰이 되었다. 따라서 소에 있어서는 정자의 중편부에 위치하여 microtubule organizing center (MTOC)의 역할을 하는 중심립 또는 중심체 없이도 모계에서 유래된 미세소관이 형성되어 이것이 전핵의 융합과 세포분열에 관여하는 것으로 생각된다. 정자의 미부 만을 주입하였을 경우 성상체가 형성이 되지 않았으며, 자성핵 사이에 형성된 미세소관과 떨어져서 관찰되었다. 따라서 주입된 정자의 꼬리는 미세소관형성과 관련이 없는 것으로 생각된다. 이러한 결과는 소에 있어서, 수정 시 정자로부터 유래되는 중심립 또는 중심체가 없이도 미세소관을 형성하여 미세소관에 의해 이후의 배발달이 정상적으로 일어남을 보여주고 있다.

• 대한의생명과학회지
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• 제10권1호
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• pp.31-34
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• 2004

White blood cells (WBCs) are present in most human ejaculates, but abnormally high concentration of seminal leukocytes may reflect an underlying pathological condition. The World Health Organization (WHO) has defined leukocytospermia as status of more than $10^6$ WBC/mL of semen. The purpose of this study was firstly, to compare the outcomes between conventional IVF and ICSI in leukocytospermia, and secondly, to investigate whether ICSI may be an alternation treatment for patients with leukocytospermia. Total 346 cycles of conventional IVF and ICSI candidates underwent IVF cycles at Eulji Hospital Infertility Clinic. Semen Parameters including concentration, motility, morphology of spermatozoa and concentration of leukocytes were assessed from the raw ejaculates. There was no difference in sperm concentration, motility and morphology. The rates of fertilization and good embryo development from ICSI were significantly higher than those from conventional IVF in leukocytospermia (60.4% ＆ 32.5%, respectively for ICSI group and 44.4% ＆ 28.5%, respectively for IVF group, P<0.00l). The pregnancy rate after ICSI was also higher than that from conventional IVF (34.0% vs 29.1 %, P<0.05). These results indicate that the presence of seminal leukocyes (> 1$\times10^6$ WBC/mL of semen) is adversely related with fertilization, embryo development and pregnancy rate. Therfore the measurement of seminal leukocytes in routine semen analysis appears to be of prognostic value with regard to male fertilizing potential. In conclusion, it is suggested that ICSI is an alternative choice of treatment for patients with leukocytospermia.