Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

ABSTRACT: Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1, the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure.

A DMD case caused by X chromosome rearrangement.

Duchenne/Becker muscular dystrophy (DMD/BMD) is one of the most common progressive muscular dystrophy diseases with X-linked recessive inheritance. It is mainly caused by the deletion, duplication and point mutation of DMD gene. In rare cases, it is also caused by the destruction of DMD gene by chromosomal structural rearrangement. Here, we report a case of Duchenne/Becker Muscular dystrophy (DMD/BMD) with typical symptoms but unknown genetic defects after MLPA and next generation sequencing tests in other hospitals. Interestingly, we find a pericentric inversion of X chromosome (Chr.X: g. [31939463-31939465del; 31939466-131765063 inv; 131765064-131765067del]) in this patient. We then use the karyotyping, FISH, long-read sequencing and Sanger sequencing technologies to characterize the chromosome rearrangement. We find that this chromosomal aberration disrupt both the DMD gene and the HS6ST2 gene. The patient present with typical DMD symptoms such as muscle weakness, but no obvious symptoms of Paganini-Miozzo syndrome. Our results suggest that the destruction of DMD gene by structural rearrangement is also one of the important causes of DMD. Therefore, we suggest to provide further genetic testing for those DMD patients with unknown genetic defects through routine genetic testing. Cost-effective karyotyping and FISH should be considered firstly to identify chromosome rearrangements. Long-read sequencing followed by Sanger sequencing could be useful to locate the precise breakpoints. The genetic diagnosis of this case made it possible for reproductive intervention in the patient's family.

One healthy live birth after preimplantation genetic testing of a cryptic balanced translocation (9;13) in a family with cerebral palsy and glaucoma: a case report.

BACKGROUND: Cryptic balanced translocations often evade detection by conventional cytogenetics. The preimplantation genetic testing (PGT) technique can be used to help carriers of balanced translocations give birth to healthy offspring; however, for carriers of cryptic balanced translocations, there is only one report about trying assisted reproduction using the PGT technique but with no pregnancy. CASE PRESENTATION: A couple had 3 births out of 4 pregnancies, and all died very young, with two of them having both cerebral palsy and glaucoma. The husband with oligoasthenospermia was found to be a cryptic balanced translocation carrier for t (9,13) (p24.3, q31.3) with G-banding, FISH (fluorescence in-situ hybridization), and MicroSeq techniques; live birth of a healthy baby girl was achieved with PGT/NGS (next-generation sequencing) for the couple. CONCLUSION: Here, we report for the first time a successful live birth of a healthy baby through the PGT technique for a family in which the husband is a carrier of the cryptic balanced translocation t (9,13) (p24.3, q31.3), presumably causative for cerebral palsy and glaucoma. Our study showed that the PGT/NGS technique can effectively help families with a cryptic balanced translocation have healthy offspring.

Whole-genome mate-pair sequencing of apparently balanced chromosome rearrangements reveals complex structural variations: two case studies.

BACKGROUND: Apparently balanced chromosome rearrangements (ABCRs) in non-affected individuals are well-known to possess high reproductive risks such as infertility, abnormal offspring, and pregnancy loss. However, caution should be exercised in genetic counseling and reproductive intervention because cryptic unbalanced defects and genome structural variations beyond the resolution of routine cytogenetics may not be detected. CASE PRESENTATION: Here, we studied two familial cases of ABCRs were recruited in this study. In family 1, the couple suffered two abortions pregnancies and underwent labor induction. Single nucleotide polymorphism (SNP) array analysis of the aborted sample from the second pregnancy revealed a 10.8 Mb heterozygous deletion at 10q26.13q26.3 and a 5.5 Mb duplication at 19q13.41-q13.43. The non-affected father was identified as a carrier of three-way complex chromosomal rearrangement [t (6;10;19)(p22;q26;q13)] by karyotyping. Whole-genome mate-pair sequencing revealed a cryptic breakpoint on the derivative chromosome 19 (der19), indicating that the karyotype was a more complex structural rearrangement comprising four breakpoints. Three genes, FAM24B, CACNG8, and KIAA0556, were disrupted without causing any abnormal phenotype in the carrier. In family 2, the couple suffered from a spontaneous miscarriage. This family had an affected child with multiple congenital deformities and an unbalanced karyotype, 46,XY,der (11) t (6;11)(q13;p11.2). The female partner was identified as a balanced translocation carrier with the karyotype 46,XX,t (6;11)(q13;p11.2) dn. Further SNP array and fluorescent in situ hybridization (FISH) indicated a cryptic insertion between chromosome 6 and chromosome 11. Finally, whole-genome mate-pair sequencing revealed an extremely complex genomic structural variation, including a cryptic deletion and 12 breakpoints on chromosome 11, and 1 breakpoint on chromosome 6 . CONCLUSIONS: Our study investigated two rare cases of ABCRs and demonstrated the efficacy of whole-genome mate-pair sequencing in analyzing the genome complex structural variation. In case of ABCRs detected by conventional cytogenetic techniques, whole genome sequencing (WGS) based approaches should be considered for accurate diagnosis, effective genetic counseling, and correct reproductive intervention to avoid recurrence risks.

Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells.

PURPOSE: This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes. METHODS: After sequential culture for 5-6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis. RESULTS: The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P < 0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes. CONCLUSIONS: Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.

Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.

The first patient with a pure 1p36 microtriplication associated with severe clinical phenotypes.

BACKGROUND: Copy Number Variants (CNVs) is a new molecular frontier in clinical genetics. CNVs in 1p36 are usually pathogenic and have attracted the attention of cytogeneticists worldwide. None of 1p36 triplication has been reported thus far. RESULTS: We present three patients with CNVs in 1p36. Among them one is the first 1p36 tetrasomy due to a pure microtriplication and the other two are 1p36 microdeletion. Traditional chromosome G-banding technique showed a normal karyotype. Single nucleotide polymorphism (SNP) microarray analysis combined with multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) were used to identify and confirm the chromosome microdeletion/microtriplication. The facial dysmorphisms of the patient with 1p36 tetrasomy differed from those two patients with 1p36 monosomy. The expression levels of B3GALT6, MIB2, PEX10 and PANK4 in the blood were determined, and differential expressions were observed between the patients and controls. CONCLUSIONS: Our study shows the first case of 1p36 tetrasomy due to a pure microtriplication in a patient with severe intellectual disability and seizures. The study provides a new resource for studying the mechanisms of microtriplication formation, and provides an evidence that overexpression of the specific genes might be related the specific phenotype of 1p36 microtriplication.

Karyotype determination and reproductive guidance for short stature women with a hidden Y chromosome fragment.

Two unrelated couples came to the Reproductive and Genetic Hospital of Citic-Xiangya to ask for reproductive guidance. One couple had an affected son and the other couple had secondary infertility. Conventional GTG banding showed that the women in both couples had a 46,X,add(X)(p22) karyotype. Further molecular cytogenetic studies showed that both women had a 46,X,der(X)t(X;Y)(p22;q11.2) karyotype and that the affected boy had inherited the derivative X chromosome, which resulted in an Xp contiguous gene syndrome. After an assessment of reproductive risk, the first couple conceived naturally and opted for prenatal diagnosis (PND) by amniocentesis. No abnormal karyotypes were found for the twin pregnancy and healthy twin girls were born after a full-term normal pregnancy. The second couple chose to undergo IVF with preimplantation genetic diagnosis (PGD). Two PGD cycles were performed by fluorescence in-situ hybridization. In the first PGD cycle, all three embryos had abnormal hybridization signals. In the second cycle, a male embryo with normal hybridization signals was transferred into the womb and a normal pregnancy was achieved. The results show the importance of detecting the derivative chromosome followed by PND or PGD if a woman carries an Xp;Yq translocation.

[A de novo partial 5p deletion and cryptic 18p duplication detected by SNP-Array in a boy featuring Cri du Chat syndrome].

OBJECTIVE: To determine the karyotype of a boy suspected to have Cri du Chat syndrome with severe clinical manifestations, and to assess the recurrence risk for his family. METHODS: High-resolution GTG banding was performed to analyze the patient and his parents. Fluorescence in situ hybridization (FISH) with Cri du Chat syndrome region probe as well as subregional probes mapped to 5pter, 5qter, 18pter, 18qter, and whole chromosome painting probe 18 was performed to analyze the patient and his parents. In addition, single nucleotide polymorphism-based arrays (SNP-Array) analysis with Affymetrix GeneChip Genome-wide Human SNP Nsp/Sty 6.0 were also performed to analyze the patient. RESULTS: Karyotype analysis indicated that the patient has carried a terminal deletion in 5p. FISH with Cri du Chat syndrome region probe confirmed that D5S23 and D5S721 loci are deleted. SNP-Array has detected a 15 Mb deletion at 5p and a 2 Mb duplication at 18p. FISH with 5p subtelomeric probes and 18p subtelomeric probe further confirmed that the derivative chromosome 5 has derived from a translocation between 5p and 18p, which has given rise to a 46,XY,der(5)t(5;18)(p15.1;p11.31)dn karyotype. CONCLUSION: A de novo 5p partial deletion in conjunction with a cryptic 18p duplication has been detected in a boy featuring Cri-du-Chat syndrome. His parents, both with negative findings, have a low recurrence risk. For its ability to detect chromosomal imbalance, SNP-Array has a great value for counseling of similar patients and assessment of recurrence risks.

[Identification of a cryptic 1p36.3 microdeletion in a patient with Prader-Willi-like syndrome features].

OBJECTIVE: To determine the karyotype of a patient with Prader-Willi-like syndrome features. METHODS: Chromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region. RESULTS: No abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient. CONCLUSION: Molecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.

[Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR].

OBJECTIVE: To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD). METHODS: Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed. RESULTS: The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods. CONCLUSION: PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.

Mutation Analysis and Prenatal Exclusion of Fibrodysplasia Ossificans Progressiva in a Chinese Fetus.

Aims: Fibrodysplasia ossificans progressiva (FOP) is a rare and severely disabling autosomal dominant disorder characterized by congenital malformations of the great toes and progressive postnatal heterotopic ossification. A point mutation in the activin receptor IA (ACVR1) gene is the cause of FOP. Most of the reported cases of FOP are sporadic and caused by de novo mutations; however, some rare cases can also result from parental germline mosaicism associated with a greater risk of recurrence in successive pregnancies. Therefore, once the pathogenic mutation has been identified in the proband, it is relative cheaper and important to perform prenatal diagnostic tests to exclude the recurrence risk of FOP in subsequent pregnancies. In this study, we first investigated the mutation in the ACVR1 gene in a Chinese FOP patient and then performed prenatal tests to exclude the risk of recurrence in the patient's unborn sibling. Methods: A couple visited our clinic with their 4-year-old son, who was clinically diagnosed with FOP, for genetic counseling. Genetic testing was performed by amplifying all the nine exons of the ACVR1 gene using the conventional polymerase chain reaction. Further, DNA sequencing was used to determine the mutation based on the results of a mutation screening using denaturing high-performance liquid chromatography. Subsequently, a prenatal test was performed using the same technique as that used for the proband. Results: A recurrent single nucleotide mutation c.617 G>A (R206H) of the ACVR1 gene was identified in the patient; however, both the parents had a normal ACVR1 gene. Prenatal tests showed that the fetus did not carry the pathogenic mutation. Conclusion: The results confirmed that a recurrent single nucleotide mutation c.617 G>A (R206H) was the genetic cause of FOP and explored the utility of prenatal testing in excluding the risk of recurrence in the successive pregnancy.

Crypt Y chromosome fragment resulting from an X;Y translocation in a patient with premature ovarian failure.

OBJECTIVE: To identify a cryptic Y chromosome fragment that resulted from a X;Y translocation in a patient with premature ovarian failure (POF) and analyze the karyotype-phenotype correlation. DESIGN: Case report. SETTING: A university-based reproductive medicine center. PATIENT(S): A 33-year-old woman with POF. INTERVENTION(S): Karyotyping analysis, comparative genomic hybridization, fluorescence in situ hybridization, and polymerase chain reaction (PCR) analysis for the patient. MAIN OUTCOME MEASURE(S): Karyotype determination of the patient. RESULT(S): The patient was suspected to carry an abnormal X chromosome by traditional cytogenetic analysis. A Y chromosome hybridization signal was found in the patient's genome by comparative genomic hybridization analysis. The fluorescence in situ hybridization result showed that the Y chromosome material resulted from a translocation between Xq and Yq. Using the specific sequence-tagged sites, the breakpoints on the X and Y chromosomes were located at Xq26.3 and Yq11.223, respectively. Combined with chromosome G banding and C banding, the karyotype of the patient was determined as 46,X,der(X)t(X;Y) (q26.3;q11.223). CONCLUSION(S): The advanced molecular cytogenetic techniques are helpful to detect cryptic chromosome aberrancies in patients with POF. This rare case supports that Xq26-q28 is the critical region of POF, and is helpful to analyze the risk of gonadoblastoma in patients with POF with Y chromosomal material.

Tumor progression of culture-adapted human embryonic stem cells during long-term culture.

Human embryonic stem cells (hESCs) during long-term culture acquire chromosomal changes similar to those occurring in tumorigenesis. This was raised concerns about the progression from hESCs to malignant cells. This study aimed to investigate the changes in chromosomes, cell phenotype, and genes in culture-adapted hESCs to ascertain whether tumorigenic transformation occurred. By cytogenetic analysis we found progressive karyotypic changes from simple to complex in chHES-3, one of the hESC lines established in our laboratory, during a long-term suboptimal culture. We further compared chHES-3 cells at different karyotypic stages in cell surface markers, in vivo differentiation, cell cycle, apoptosis, and gene expression profiles. We found that the karyotypically aberrant chHES-3 had higher S-phase fraction in cell cycle distributions and antiapoptosis ability. In vivo differentiation of karyotypically normal chHES-3 resulted in relatively mature teratoma, whereas karyotypically aberrant chHES-3 formed immature teratoma (grade III), in which more primary neural epithelium was revealed by pathological analysis. The microarray analysis and real-time PCR results showed that some oncogenes were upregulated in karyotypically aberrant chHES-3 cells, whereas the genes related to differentiation were downregulated, and that Wnt signal pathway was activated. In conclusion, chHES-3 cells underwent deregulation of self-renewal and dysfunction of related genes in long-term culture adaptation, leading to malignant transformation.

[Delineating a supernumerary marker chromosome by combining several cytogenetic and molecular cytogenetic techniques].

OBJECTIVE: To characterize a supernumerary marker chromosome (SMC) by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH) and traditional cytogenetic techniques, and to explore the clinical application of these techniques in delineating de novo marker chromosomes. METHODS: A mental retardation patient received chromosome test by ordinary G banding. CGH and FISH techniques were used to analyze the origin of the de novo SMC, and N banding technique and C banding techniques were used to analyze the SMC structure. The phenotypic effects of the SMC were analyzed after the karyotype was determined. RESULTS: By G banding technique, the patient was showed to have a mosaic karyotype with SMC: mos.47, XX, +mar [31]/48, XX, +2mar[29]. CGH analysis showed a gain of 15q11 --> q14, and the result was confirmed by FISH with chromosome 15 painting probe. The further FISH analysis showed the SMC had two signals with UBE3A probe for detecting Prader-willi syndrome/Angelman syndrome (PWS/AS). N banding and C banding analysis showed the SMC had a double satellite and double centromere, respectively. Combined with the above results, the karyotype of the patient was: mos.47, XX, +der (15) (pter --> q14::q14 --> pter) [31]/48, XX, +2der (15) (pter --> q14::q14 --> pter) [29]. ish der(15)(WCP15+, UBE3A++, PML-). CONCLUSION: CGH is a valuable method to detect imbalanced chromosomal rearrangement. Combined with FISH and the traditional cytogenetic technique, it provides a valuable technique platform for characterizing the structure of the de novo SMC, and a basis for exploring the relation between karyotype and phenotype, prognosis and recurrent risk.

[Identification and characterization of marker chromosome in Turner syndrome].

OBJECTIVE: To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome, and study the phenotypic effects resulting from the abnormal karyotype. METHODS: Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome, and 6 marker chromosomes were ring chromosomes. Their karyotypes were showed as mos. 45, X/46, X, + mar or mos. 45, X/46, X, + r. Fluorescence in situ hybridization (FISH) technique with X/Y centromere probes was performed to determine the origin of the marker chromosome. Reverse chromosome painting technique was used to identify the breakpoints of two largest markers. Phenotype effects with different chromosome breakpoints were compared. RESULTS: All the 11 marker chromosomes were ring X chromosomes. The breakpoints of the r(X) were involved in Xp22, Xq22, Xq24 and Xq26, etc. CONCLUSIONS: The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X. Each r(X) in our patients was mosaic, indicating it was originated from mitosis error during early embryo development. To analyze the origin of the marker chromosome and the breakpoint of r(X) will provide guidance for the therapy and prognosis of the Turner syndrome patient.

[Advance in mutation analysis of the candidate genes in premature ovarian failure].

Premature ovarian failure (POF) is a complicated and heterogeneous disease. In majority of cases the underlying cause is not identified. Among the known causes, genetic aberration plays very important role. POF not only causes infertility, also adds the risk of osteoporosis and coronary heart disease because of the low level estrogen. The major therapy measures in present include hormone replacement therapy and infertility treatment with donated oocytes, but the effect is not ideal. To identify the genes of POF is the basis for treating and preventing this disease. A large number of candidate genes of POF are found on X chromosome and autosomes. The present paper reviewed the advance in mutation analysis on the candidate genes of POF, which is aimed to provide a basis to explore its molecular mechanism.