Introduction
Currently, selective high-quality embryo transfer is the most promising method available for attaining a consistently high pregnancy rate in assisted reproductive technology. A mature oocyte is the key to generating a normally fertilized, cleaved and high-quality embryo (Plachot and Mandelbaum, Reference Plachot and Mandelbaum1990; Moor et al., Reference Moor, Dai, Lee and Fulka1998). Once the luteinizing hormone (LH) level surges, oocytes resume the meiotic process of chromosomal reduction to a haploid after extruding the small first polar body (PB1), involving various morphological events such as spindle assembly, polarized differentiation of the oocyte cortex, and asymmetric division (Park et al., Reference Park, Su, Ariga, Law, Jin and Conti2004; Mehlmann, Reference Mehlmann2005; Coticchio et al., Reference Coticchio, Dal Canto, Mignini Renzini, Guglielmo, Brambillasca, Turchi, Novara and Fadini2015). Abnormalities in any of these events can cause primary female infertility due to the arrest of oocyte maturation, fertilization failure or early embryonic arrest (Matzuk and Lamb, Reference Matzuk and Lamb2008; Feng et al., Reference Feng, Sang, Kuang, Sun, Yan, Zhang, Shi, Tian, Luchniak, Fukuda, Li, Yu, Chen, Xu, Guo, Qu, Wang, Sun and Liu2016; Namgoong and Kim, Reference Namgoong and Kim2018).
Asymmetric meiotic division is carried out in conjunction with oocyte maturation to protect the precious ooplasm through the expulsion of a polar body of a minimal size (Sanders and Jones, Reference Sanders and Jones2018). Improper asymmetric division might result in the creation of a large polar body, making it difficult to determine whether these oocytes can be used for insemination or not (Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021). In the early years, it was believed that the presence of an enlarged PB1 was linked to lower rates of fertilization, poor quality of cleavage embryos, and lower blastocyst yield (Ebner et al., Reference Ebner, Yaman, Moser, Sommergruber, Feichtinger and Tews2000; Navarro et al., Reference Navarro, de Araújo, de Araújo, Rocha, dos Reis and Martins2009; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). It was even the consensus that insemination should not be performed on oocytes with an excessively large polar body (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). As a result, the application of this portion of large polar body oocytes has scarcely been reported to date. However, recently, several authors accepted that there was no correlation between PB1 morphology and pregnancy rate or implantation rate in fresh embryo transfer cycles (Halvaei et al., Reference Halvaei, Khalili, Soleimani and Razi2011; Zhou et al., Reference Zhou, Fu, Sha, Chu and Li2016). No obvious evidence was found on the significance of PB1 morphology in relation to oocyte aneuploidy (Verlinsky et al., Reference Verlinsky, Lerner, Illkevitch, Kuznetsov, Kuznetsov, Cieslak and Kuliev2003).
To date, we have not yet discovered any cases of live birth and successful perinatal development from oocytes with extraordinarily large polar bodies. Only a few sporadic cases represent the link between genetic variants and the large PB1 phenotype reported in previous studies (Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). However, for most clinical cases, large PB1 oocytes were just a small portion of those retrieved oocytes, and they were always not linked to genetic defects. The application of this part of the oocytes has scarcely been reported to date.
Here, we present a unique case of an extremely large polar body oocyte, which resulted in a successful full-term pregnancy and healthy childbirth. This may bring us a new understanding that will help us avoid wasting large PB1 oocytes by paying attention to their re-evaluation.
Materials and methods
This case report was taken from the Department of Reproductive Medicine and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (grant no. ZYYEC-ERK-2022–093). Additionally, the consent of the patient for this case report has been obtained.
A 29-year-old woman who had experienced primary infertility for more than 6 years came to our assisted reproduction department for fertility treatment. Despite having no exceptional family medical history, the woman was given the diagnosis of Hashimoto thyroiditis. Her body mass index was elevated (28.32 kg/m2). Her menarche was at the age of 14 and her menstrual cycle varied from 37 to 45 days. Her hysterosalpingography revealed adhesion of the fimbria end of the fallopian tube. Her antral follicle count was 15 and her baseline serum hormone levels were all within normal ranges as follows: serum follicle-stimulating hormone (FSH, 6.09 mIU/ml), LH (6.35 mIU/ml), estradiol (E2, 19.99 pg/ml), testosterone (T, 0.82 ng/ml), and anti-Müller hormone (AMH, 3.44 ng/ml). Her karyotype was 46, XX. Her husband’s sperm concentration was 43.5 × 106/ml, and the percentages of progressively motile sperm and normal sperm morphology were 15.5% and 2.5%, respectively. The husband had no history of smoking, potential toxic agents, radiation, or other environmental xenobiotics exposure. The peripheral blood sample karyotype analysis yielded normal results (46, XY).
The first cycle was attempted in May 2020. On the first day of menstruation, the woman was downregulated with a 3.75 mg intramuscular injection of gonadotropin-releasing hormone (GnRH) agonist (Diphereline, Ipsen, France), and then stimulated for 10 days with recombinant FSH (Gonal F, Serono, Switzerland) and recombinant human LH (Luveris, Merck, USA). Here, 2400 IU of gonadotropins were administered overall. Her terminal E2 was 1153.55 pg/ml, P was 0.48 ng/ml, and endometrial thickness was 11 mm on the day 10,000 IU human chorionic gonadotropin (hCG; Novarel) was administered to trigger the final oocyte maturation. Oocytes were retrieved from the patient while being guided by transvaginal ultrasound.
The semen sample was prepared using the gradient method with a centrifuge for 10 min at 300 g. After sperm extraction, his sperm sample had a concentration of 7.0 × 106/ml, and the percentage of progressively motile sperm was 98.0%. Semen parameters were measured according to the protocol recommended by the WHO manual (Cardona Maya, Reference Cardona Maya2010). Due to previous semen parameters in the husband’s history and semen quality on the day of oocyte collection, it was recommended to perform short-term insemination with mild or moderate male infertility. A final concentration of 10.0 × 104 motile sperm/ml was used for insemination. After 4 h of sperm and oocyte coincubation, the granulosa cells around the oocytes were removed and assessed for the presence of the second polar body to facilitate signs of fertilization. If the second polar body was not detected or was present in fewer than 30% of all oocytes, r-ICSI was performed for those oocytes without a second polar body after 6 h of coincubation. The prognostic value of the first polar body morphology was assessed according to Ebner’s grading standard (Ebner et al., Reference Ebner, Yaman, Moser, Sommergruber, Feichtinger and Tews2000); the blastocyst was classified according to Gardner’s method (Gardner and Schoolcraft, Reference Gardner and Schoolcraft1999). Photographs were taken daily at the time of the IVF procedure, using imaging software on a Nikon inverted microscope.
Results
Six oocytes were ultimately retrieved from the woman. Three metaphase II oocytes (MII) were examined following the short-term insemination, although only one of them presumably had the second polar body extruded while the other two did not. r-ICSI was performed for those oocytes without a second polar body. In addition, two immature oocytes (germinal vesicle stage and metaphase I stage) and one oocyte with a remarkably large PB1 were also obtained from the woman. The size of the abnormal PB1 was almost 80 μM long and 40 μM wide, and was broken into three spherical parts (Figure 1), which belonged to grade 4. Due to unexpected fertilization signs (cytoplasmic movement was not observed), and no sperm was seen on the zona pellucida surface, the large PB1 oocyte was also performed with r-ICSI. On the following day, only the oocytes that had received r-ICSI treatment were observed to have two pronuclei (Figure 1; Figure S1). Finally, only the large PB1 oocyte was able to form into an available blastocyst on day 5 (classified 3BB), whereas the other two zygotes were both arrested on day 3. The cycle was cancelled due to evidence suggesting that large polar bodies might suffer chromosome segregation errors. So, with the patient’s permission, the blastocyst was frozen.
Figure 1. Embryo development derived from an oocyte with an extremely large polar body.
(A) Stripped oocyte with an extremely large polar body before rescue intracytoplasmic sperm injection (r-ICSI). (B) Day 1 after r-ICSI. (C) Day 2 after r-ICSI. (D) Day 3 after r-ICSI. (E) Day 5 embryo before freezing. (F) Day 5 frozen–thawed embryo.
After fully informing the patient of the pregnancy risks, the couple insisted on the transplantation of the blastocyst derived from the large PB1 oocyte for reason of economic difficulties. Then the woman underwent a subsequent FET, using a hormonal replacement treatment cycle for follicular preparation of the uterine lining.
Pregnancy was first detected according to serum β-hCG level (1019 IU/l) 12 days following transfer, and clinical pregnancy was confirmed at 5 weeks (33 days) by transvaginal ultrasonography. The woman was closely monitored during her antenatal period, and this went well without any complications. A live delivery by caesarean section at 37 weeks and 5 days of gestation was performed, resulting in a live, healthy, male baby. The newborn’s weight at delivery was 3100 g, and his length was 48 cm. The outcome of the perinatal period of the pregnant woman was uneventful. Moreover, a 1-year follow-up survey revealed that the child’s behaviour and mental health were both normal. The couple refused to perform examinations on chromosome karyotype and related gene mutation detection on their baby.
Discussion
It is not uncommon that large PB1 morphology may reflect poor embryo quality, which may be valuable for embryo selection (Ebner et al., Reference Ebner, Yaman, Moser, Sommergruber, Feichtinger and Tews2000; Navarro et al., Reference Navarro, de Araújo, de Araújo, Rocha, dos Reis and Martins2009; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021), but nevertheless it is worth reporting this rare case of a healthy childbirth after r-ICSI from such a remarkably large PB1 oocyte. To our knowledge, there are no reports of live births from large PB1 oocytes that shared an unusual or interesting antenatal period or perinatal outcomes.
After the short-term IVF insemination in our case, total failure of fertilization was found for a variety of reasons. First of all, the husband’s semen parameter was mild to moderate, with a sperm concentration of 43.5 × 106/ml(≥15 × 106/ml), progressive motility of 15.5% (≥32%), and morphologically normal forms of 2.5% (≥4%).
Secondly, oocyte abnormality (such as aberrant nuclear maturation and ageing of oocytes) can occasionally cause spindle destruction, chromosomal segregation, disruptions in sperm–oocyte interaction, and ultimately reduced fertilization (Zhang et al., Reference Zhang, Wang, Quan, Huang, Tong, Ma, Guo, Wei, Ouyang, Hou, Xing and Sun2011, Reference Zhang, Liu, Ji, Sha, Zhang and Fan2015; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). In our case, the proportion of immature oocytes, total failure of fertilization, the occurrence of large PB1 oocytes and the outcomes of earlier embryonic arrest (three-cell stage) of mature oocytes may manifest their abnormalities of nuclear maturation and synchronization with cytoplasmic development. Our case’s results are in line with earlier research that oocytes with large PB1s had reduced rates of fertilization and poorer quality embryos on day 2 (Ebner et al., Reference Ebner, Yaman, Moser, Sommergruber, Feichtinger and Tews2000; Navarro et al., Reference Navarro, de Araújo, de Araújo, Rocha, dos Reis and Martins2009). Numerous other investigations have suggested that the fragmentation and aberrant size of an oocyte’s PB1 may be related to the asynchronous maturation of the nucleus and cytoplasm (Eichenlaub-Ritter et al., Reference Eichenlaub-Ritter, Schmiady, Kentenich and Soewarto1995). Poor nuclear maturation can lead to failure of the PB1 discharge, and improper asymmetric division can cause the formation of a large polar body (Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021; Zou et al., Reference Zou, Shan, Wang, Pan, Pan, Xu, Ju and Sun2021). The production of an abnormally large PB1 may conceal significant developmental perturbations and may have contributed to a trend of declining fertility and embryo quality, according to experiments carried out in animal models (Verlhac et al., Reference Verlhac, Lefebvre, Guillaud, Rassinier and Maro2000).
We are aware that ovarian stimulation is unquestionably fundamental in the treatment of infertility, yet when controlled superovulation occurs, the oocytes induced by hCG can result in various degrees of nuclear maturation and produce different grades of first polar bodies (Gitlin et al., Reference Gitlin, Gibbons and Gosden2003). Patients with low ovarian response might have a high proportion of overmature oocytes, which would also affect embryonic quality and development (Morita and Tilly, Reference Morita and Tilly1999).
Recently, several studies have discovered novel hereditary causes of female infertility in humans that involve aberrant oocyte morphology and the production of large first polar bodies. Most of them identified TUBB8 variants as major genetic determinants of human oocyte maturation arrest (Feng et al., Reference Feng, Sang, Kuang, Sun, Yan, Zhang, Shi, Tian, Luchniak, Fukuda, Li, Yu, Chen, Xu, Guo, Qu, Wang, Sun and Liu2016; Chen et al., Reference Chen, Li, Li, Yan, Mao, Xu, Mu, Li, Jin, He, Kuang, Sang and Wang2017; Cao et al., Reference Cao, Guo, Xu, Lin, Deng, Cheng, Zhao, Jiang, Gao, Huang and Xu2021b; Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). One of the highly preserved tubulin isotypes, TUBB8, was presumably dominant during the construction of the meiotic spindle in human oocytes. A multiplicity of phenotypes with large polar bodies in the oocytes could result from various TUBB8 mutations in independent patients (Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). In addition, the decrease in cytoplasmic actin filaments by depleting leucine-rich-repeat kinase 2 (LRRK2) also causes the failure of spindle migration and induces large polar bodies in mouse oocytes (Pan et al., Reference Pan, Liu, Ju, Wang and Sun2022). Moreover, Mos, a specific upstream regulator of mitogen-activated protein kinase (MAPK), plays an extremely important role in meiotic maturation progression, and its mutation impairs the symmetrical cleavage and produces an abnormally large polar body (Choi et al., Reference Choi, Fukasawa, Zhou, Tessarollo, Borror, Resau and Vande Woude1996; Zhang et al., Reference Zhang, Zheng, Ren, Jin, Hu, Liu, Fan, Gong, Lu, Lin, Zhang and Tong2022). Furthermore, other maternal effect genes, including PATL2 (Cao et al., Reference Cao, Zhao, Wang, Cai, Xia, Zhang, Han, Xu, Zhang, Ling, Ma and Huo2021a), Centriolin (Sun et al., Reference Sun, Wang, Kwon, Yuan, Lee, Cui and Kim2017), RAB14 (Zou et al., Reference Zou, Shan, Wang, Pan, Pan, Xu, Ju and Sun2021), and GM130 (Zhang et al., Reference Zhang, Wang, Quan, Huang, Tong, Ma, Guo, Wei, Ouyang, Hou, Xing and Sun2011) have been linked to a similar oocyte phenotype of a large polar body. Nevertheless, there are still many underlying genetic factors behind this phenotype that remain largely unknown.
Oocyte aneuploidy has been linked to early non-equilibrium segregation, sister chromatid equilibrium, and homologous chromosomal non-segregation, according to various reports (Bennabi et al., Reference Bennabi, Terret and Verlhac2016; Sun et al., Reference Sun, Wang, Kwon, Yuan, Lee, Cui and Kim2017). Once the formation of spindles is damaged and the dispersion of sister chromatids is exchanged, aneuploidy anomalies might occur, resulting in chromosome loss, which is related to maturation of the oocyte and oocyte ageing (Wang et al., Reference Wang, Schatten and Sun2011). According to some earlier research, the oocytes of enlarged polar bodies had a higher incidence of aneuploidy than those of complete, round and smooth polar bodies, which was consistent with a lower fertilization rate (Liu et al., Reference Liu, Xi, Zhu, Yang, Jin, Wang, Zhang, Zhou, Zhang, Peng, Luo, Li and Zhang2021; Zheng et al., Reference Zheng, Hu, Zhang, Xu, Gao, Gong, Lu and Lin2021). However, other authors have disputed these studies. In particular, Verlinsky et al. (Reference Verlinsky, Lerner, Illkevitch, Kuznetsov, Kuznetsov, Cieslak and Kuliev2003) did not discover a correlation between PB1 morphology and chromosomal aneuploidy.
Without a doubt, this is an unusual and interesting case that may bring us new understanding and prompt us to reconsider the significance of large PB1 oocytes. This finding might be beneficial to patients who do not have an available embryo for transfer and prevent the waste of embryos. As this study only reports a case of live birth derived from a remarkably large PB1 oocyte, the contributory genetic factors for large PB1s largely remain unknown. It is unfortunate that preimplantation genetic testing techniques are not carried out in our department. Therefore, relevant gene sequencing, variant screening, and validation should be carried out in our future study.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0967199424000054
Financial support
This work was supported by the Medical Scientific Research Foundation of Guangdong Province, China (grant number A2023401).
Competing interests
The authors declare that they have no conflict of interest.
Ethics approval
All procedures followed were in accordance with the ethical guidelines of the Helsinki Declaration and were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (Grant no.ZYYEC-ERK-2022–093).
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.
Consent for publication
Written informed consent for publication was obtained from all participants.
