Quantitative PCR technique for the identification of microrearrangements of the AZFc region

Purpose: The AZFc region spans about 3.5 Mb and contains many amplicons causing recombination events. Several papers have reported the occurrence of AZFc partial deletions resulting from non allelic homologous recombination (NAHR) (“gr-gr”, “b1-b3” or “b2-b3” deletions), particularly in infertile patients. DAZ genes are present in 4 copies and rearrangements involve a modification of the number of DAZ genes. Methods: In addition to STS plus/minus PCR, we developed a quantitative technique using real time PCR (Q-PCR) to determine the number of DAZ genes. Fourteen DNA controls were selected to validate the use of Q-PCR to detect AZFc microrearrangements, and sperm DNA samples from 30 fertile men were studied. Results: Rearrangements of 14 controls were well identified with Q-PCR, and 2 AZFc partial deletions were detected in fertile men (1 “gr-gr” and 1 “b2-b3”). Conclusion: Q-PCR represents a well-adapted method to detect microrearrangements of the Y-chromosome, complementary to STS analysis.     

A comparative study of Y chromosome microdeletions in infertile males from two Chinese populations

Purpose : To compare the prevalence and type of Y-microdeletions in Hong Kong and Shanghai men with severe male-factor infertility. Methods : Seven Y-linked sequence tagged site (STS) primers and seven gene-specific primers were screened in 293 infertile males (139 from Hong Kong and 154 from Shanghai) and 161 fertile men (61 from Hong Kong and 100 from Shanghai). Serum FSH, LH, and testosterone levels were also measured in these men. Results : The incidence of Yq microdeletions in nonobstructive azoospermic men from Hong Kong (8.5%) and Shanghai (6%) was similar. Yq microdeletions were observed in severe oligospermic patients (8.5%) from Hong Kong but not from Shanghai. Among the 9 Hong Kong men with Y-microdeletions, 8 had AZFc deletion and one had AZFb deletion. In contrast, 6 of 9 men from Shanghai with Y-microdeletions had AZFb deletion. The incidence of AZFb deletion among Y-microdeleted men was statistically different between the two populations. Two of the men with AZFb deletion also had AZFa and AZFc deletions. Conclusions : Regional variations in the type of Y-microdeletion existed between Hong Kong and Shanghai infertile males.     

Molecular analysis of the androgen receptor gene in Hong Kong Chinese infertile men

Purpose : To investigate the relationship between CAG repeat length in the androgen receptor gene and impaired spermatogenesis in Hong Kong Chinese population. Methods : The CAG repeat region was amplified by polymerase chain reaction (PCR) in 85 nonobstructive azoospermic or severe oligozoospermic men, and 45 fertile males. The number of CAG repeat was analyzed by DNA sequencing. Serum FSH, LH, and testosterone levels were also determined in these men. Results : Among nonobstructive azoospermic males, three men (5.7%) possessed short CAG repeats (<16), and three (5.7%) other men possessed long CAG repeats (>30). Short CAG repeats (<16) were also found in two severe oligozoospermic males (6.3%). The incidence of infertile men with short or long CAG repeats is significantly higher in the azoospermic group (p = 0.03) but not in the severe oligozoospermic group (p = 0.17) when compared with the fertile controls. Conclusion : Our data suggest an association between CAG repeat lengths and impaired spermatogenesis in azoospermic males in our population.     

High frequency of de novo DAZ microdeletion in sperm nuclei of subfertile men: possible involvement of genome instability in idiopathic male infertility

The occurrence and diagnosis of Y-chromosome microdeletions, specifically deletions of the DAZ (Deleted in Azoospermia) genes are an important issue in male infertility. Screening Y chromosome microdeletion is mainly done using polymerase chain reaction (PCR) on blood leukocytes. However, there is some evidence indicating that presence of DAZ in somatic cells might not be indicative of its presence in the germ cell lineage. Therefore, a total of 130 men with poor semen quality were examined for presence of DAZ microdeletion in their leukocytes. From these, sperm from 40 randomly selected men with no DAZ microdeletions in their leukocytes (n?=?10 oligozoospermia; n?=?10 asthenozoospermia; n?=?10 oligoasthenozoospermia; and n?=?10 near-azoospermia) were were compared to sperm from men of normal semen quality (n?=?10) using combined primed in situ labelling and fluorescent in situ hybridization (PRINS-FISH) technique as well as screening for sex chromosome aneuploidy. There was an increased frequency of DAZ microdeletion in blood samples from oligozoospermic (5%) (p?p?DAZ microdeletion was observed in the sperm of patients with no DAZ microdeletion in their leukocytes compared to control (p?DAZ microdeletion induction during spermatogenesis.     

Partial-AZFc deletions in Chilean men with primary spermatogenic impairment: gene dosage and Y-chromosome haplogroups

PurposeTo investigate the association of partial-AZFc deletions in Chilean men with primary spermatogenic failure and their testicular histopathological phenotypes, analyzing the contribution of DAZ dosage, CDY1 copies, and Y-chromosome haplogroups.Subjects and methodsWe studied 479 Chilean men: 334 infertile patients with histological examination (233 cases with spermatogenic defects and 101 normal spermatogenesis, obstructive controls, OC), and 145 normozoospermic controls (NC). AZFc subdeletions were detected by single-tagged sequences and single nucleotide variants analysis. DAZ-copy number was quantified by real-time qPCR. Y-chromosome haplogroups (Y-hg) were hierarchically genotyped through 16 biallelic-markers.ResultsThe prevalence of AZFc-partial deletions was increased in cases (6%) compared with NC (1.4%) (P = 0.035). There was no difference between 143 Sertoli-cell only syndrome, 35 maturation arrest, or 35 mix atrophy patients and controls. However, gr/gr deletions were more frequent in 16 subjects with hypospermatogenesis compared with NC (P = 0.003) and OC (P = 0.013). Y-hg R was the most prevalent (~ 50%), but decreased among gr/gr deletions (21%, P = 0.03). The prevalence of Y-hg M increased in cases versus controls, both in total and non-deleted men (3.9 and 3.7% versus 0.4%, P = 0.009 and P = 0.016, respectively). Among gr/gr deletions, Y-hg H increased compared with non-deleted men (14.3% versus 0.4%, P = 0.0047).ConclusionPartial-AZFc deletions in a Chilean admixed population are associated with secretory azo/oligozoospermia and might have a role in the development of hypospermatogenesis. Low represented haplogroups, Y-hg M and Y-hg H, show an association with the occurrence of spermatogenic failure and gr/gr deletions respectively; however, additional studies are required.Electronic supplementary materialThe online version of this article (10.1007/s10815-020-01957-6) contains supplementary material, which is available to authorized users.     

The association of trefoil factor 3 gene polymorphisms and haplotypes with unexplained female infertility: Molecular insights into TFF3 regulation in receptive phase endometrium

Abstract

This study examined the genetic variation within the gene trefoil factor 3 (TFF3) in relation to unexplained female infertility in a group of women where aberrant endometrial maturation was suspected. The study consisted of 113 women with a diagnosis of unexplained infertility and 289 healthy fertile volunteers. Five single nucleotide polymorphisms rs225439, rs533093, rs225361, rs11701143, and rs77436142 within TFF3 gene were analyzed using real-time PCR. The formed haplotype pattern within the TFF3 gene in relation to infertility was also assessed. TFF3 protein localization and expression in receptive stage endometrium at the time of implantation was measured in a subset of fertile (n = 7) and infertile (n = 12) women. Allele and genotype frequencies did not differ significantly between fertile and infertile women, nor did the formed haplotypes. TFF3 protein was expressed in all cell types in receptive stage endometria in fertile and infertile women. No significant association was observed between protein expression and analyzed genotypes. A significantly higher TFF3 expression in luminal epithelial cells was detected in women with unexplained infertility (p = 0.003).     

Useful marker for the estimation of a recombination pair in the partial azoospermia factor c (gr/gr) deletion using quantitative real-time polymerase chain reaction

Background and aims:  Azoospermia factor c (AZFc) microdeletions are associated with male infertility and are caused by intrachromosal recombination between homologous repetitive sequence segments. Partial AZFc deletion (gr/gr) has been reported in male factor infertility. In the present study, we established detecting the copy number using quantitative real-time polymerase chain reaction (qRT-PCR) with the genome DNA, and assessed the association of the recombination pair set of gr/gr deletion and deleted in azoospermia copies. Furthermore, we determined the clinical significance of differential recombination patterns of gr/gr deletion, and compared them with azoospermia and proven fertile volunteers, with both groups having gr/gr deleted Japanese subjects.
Materials and methods:  A total of 16 Japanese subjects with idiopathic azoospermia, and 13 proven fertile men with gr/gr deletion, were studied. qRT-PCR was used for the estimation of an identical site number.
Results:  The g1/g2 deletion was found in 69.2% (9/13) in proven fertile men and in 75% (12/16) of idiopathic infertile men. The gr/gr deletion could result in the recombination of g1/g2 segments. Furthermore, there was no difference in the position of deletion between azoospermic patients and controls ( P  = 0.59).
Conclusion:  There was no association between the loss of DAZ cluster and azoospermia in gr/gr deletion. This suggests that most of the partial deletions are neutral variants.     

Effects of life events on infertility diagnosis: comparison with presumably fertile men and women

Objective: To compare the occurrence and degree of stress attributed to life events during childhood/adolescence and adulthood between individuals diagnosed with infertility and presumably fertile individuals, and to examine the effect of life events occurrence and stress levels on an infertility diagnosis.

Background: Although stress has been explored as a consequence of the experience of infertility, its role as a predictor of this disease still lacks research, particularly regarding the use of adequate control groups composed of non-parents.

Methods: The final sample had 151 infertile subjects (74 males and 77 females) and 225 presumably fertile participants (95 males and 130 females), who completed a questionnaire indicating occurrence (y/n) and degree of stress of life events (1–5) during childhood/adolescence and adulthood.

Results: Significant differences regarding occurrence were found in seven stressful life events in men and in nine events in women, with infertile groups presenting higher occurrence than presumably fertile groups. Eleven stressful life events were rated differently by men and women regarding the degree of stress, with group significant differences observed in both directions. While most events were rated as more stressful by infertile men, infertile women reported less stress resulting from these events than presumably fertile women. After controlling for age, the degree of stress induced by life events in childhood/adolescence and adulthood were not significant predictors of infertility diagnosis, for both men and women.

Conclusion: The amount of stress associated with earlier or concurrent life events does not seem to be related with infertility. Further prospective research is needed to validate these findings.     

A new compound heterozygous mutation in the CYP17A1 gene in a female with 17α-hydroxylase/17,20-lyase deficiency

Abstract

Background: Congenital adrenal hyperplasia due to 17α-hydroxylase/17,20-lyase deficiency (OMIM #202110) is a rare autosomal recessive disorder, which is caused by mutations of the CYP17A1 gene located on chromosome 10q24.3. It has been reported that the type of mutation of the CYP17A1 gene was associated with the extent of 17α-hydroxylase/17,20-lyase deficiency, and the prevalence of common mutation was different among ethnic groups.

Case: A 21-year-old Korean female presented with primary amenorrhea and sexual infantilism, and intermittent hypokalemic episodes. Laboratory test was consistent with hypergonadotropic hypogonadism. The karyotype was 46,XX[20]. Genomic DNA was extracted from peripheral blood leukocytes. All the eight exons of the CYP17A1 gene including flanking regions of introns were amplified by PCR. The mutations of the CYP17A1 gene were detected by direct sequencing. A compound heterozygous mutation was identified; one allele had a missense mutation of c.1118A>T (p.His373Leu), which was reported previously and induced the complete loss of both 17α-hydroxylase/17,20-lyase activity. This mutation has been known to be one of the common mutation types in East Asia. The other allele had a novel 1-bp deletion c.1148delA causing frameshift, premature termination codon (p.Glu383fs) and induced truncated enzymes.

Conclusion: Our experience for stepwise clinical, laboratory and molecular approach would be helpful to diagnose these patients accurately and understand the genetic events in 17α-hydroxylase/17,20-lyase deficiency patients.     

Use of Failed-Fertilized Oocytes for Diagnostic Zona Binding Purposes After Sperm Binding Improvement with a Modified Medium

Purpose: Because the availability of prophase oocytes for zona binding testing is limited, we compared sperm binding to the zona of failed-fertilized intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) oocytes after incubation in a standard IVF medium and a specially composed binding improvement medium. Methods: Semen samples from nine patients and nine fertile donors were separated in parallel by the standard swim-up method in both media. Subsequently, hemizona assays were performed with prophase, failed-fertilized ICSI and IVF oocytes. Results: Sperm separation resulted in a significantly higher sperm count (P < 0.01) and progressive motility (P = 0.018) in binding improvement medium. Moreover, spermatozoa coincubated with hemizonae (prophase, failed-fertilized ICSI and IVF oocytes) in binding improvement medium bound significantly more to hemizonae than in the controls (P < 0.01). However, the hemizona index did not differ. Conclusions: Thus, the limited number of human zonae can be increased by the use of oocytes that failed to fertilize during ICSI or IVF. This will lead to a qualitative improvement of the diagnostic spectrum in male-factor infertility.     

High sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa in obstructive azoospermic men

Purpose: To evaluate the frequencies of sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa from obstructive azoospermic men and its impact on intracytoplasmic sperm injection (ICSI) outcomes. Methods: Epididymal spermatozoa retrieved from 24 obstructive azoospermic men and ejaculated spermatozoa from 24 fertile donors were analyzed using triple color fluorescence in situ hybridization (FISH) techniques, in order to investigate the rates of diploidy and aneuploidy for chromosomes 18, X and Y. Results: Epididymal spermatozoa from obstructive azoospermic men had total sex aneuploidy, disomy 18, and diploidy rates significantly higher than ejaculated spermatozoa from normozoospermic fertile controls (1.44% vs. 0.14%, 0.11% vs. 0.02%, and 0.18% vs. 0.02%, respectively; p < 0.005). There were no statistically significant differences in ICSI outcomes between the patients who had high and low epididymal sperm aneuploidy rate. Conclusions: Epididymal spermatozoa from obstructive azoospermic patients had an elevated sex chromosome aneuploidy and diploidy rate. The increased frequency of chromosomal abnormalities did not have a direct effect on the ICSI outcome.     

Relationship Between Standard Semen Parameters,Calcium, Cholesterol Contents,and Mitochondrial Activity in Ejaculated Spermatozoa From Fertile and Infertile Males

Purpose: To correlate levels of cholesterol (CH), calcium (Ca²⁺), and mitochondrial activity (MA) with the standard semen parameters and to compare them between fertile and infertile men.Methods: We studied 151 semen samples from infertile (n=60) or fertile (n=91) males. Basic sperm parameters were analyzed. Ca²⁺ and CH concentrations on seminal plasma were determined by enzymoimmunoanalysis. Intracellular Ca²⁺ and CH concentrations in the sperm plasma membrane and mitochondrial activity by fluorometry.Results: There was a significant positive correlation between sperm membrane CH and sperm morphology. Intracellular Ca²⁺ was lower in infertile patients compared to fertile. No differences were found regarding Ca²⁺ and CH concentrations in seminal plasma. MA is directly and strongly related with sperm motility.Conclusions: Intracellular concentrations of Ca²⁺ and the proportion of CH in the sperm membrane are two important markers of the sperm quality due to its direct relationship with sperm morphology and fertility potential.     

Leukemia Inhibitory Factor Expression in Different Endometrial Locations Between Fertile and Infertile Women Throughout Different Menstrual Phases

Purpose: The purpose was to demonstrate the leukemiainhibitory factor (LIF) expression in different endometriallocations between fertile and infertile women throughoutdifferent menstrual phases. The relationship betweenprogesterone level and LIF expression were evaluated. Methods: Endometrial biopsies were performed onidiopathic infertile and normal fertile women accepted theinfollicular, periovulatory, and luteal phases. The lutealprogesterone level was measured. Endometrial LIFimmunostaining of luminal epithelium, glandular epithelium, andstroma were detected. The relationship between luteal LIFexpression and progesterone level was evaluated. Results: Significant LIF expression was noted in theendometrium of fertile women rather than that of infertile women.The LIF expression was highest in the luminal epithelium,moderate in the glandular epithelium, and lowest in thestroma. The luminal and glandular epithelial staining werelowest in follicular phase, moderate in periovulatory phase,and strongest in luteal phase. The stromal LIF presentedwith a noncyclical manner. The LIF expression is not relatedwith the progesterone level. Conclusions: Endometrial LIF expression is related tohuman fertility. Endometrial LIF expression is dependenton cellular localizations and menstrual stages. Stronger LIFexpression presents in the endometrial epithelium duringluteal phase.     

Prenatal diagnosis of a distal 3p deletion associated with fetoplacental chromosomal discrepancy and confined placental mosaicism detected by array comparative genomic hybridization

ObjectiveThis study is aimed at prenatal diagnosis of a distal 3p deletion associated with fetoplacental chromosomal discrepancy and confined placental mosaicism, and providing evidence for the limitation of array comparative genomic hybridization (aCGH) on placental tissues for molecular cytogenetic characterization of prenatally detected aneuploidy.Case ReportA 30-year-old woman underwent amniocentesis at 18 weeks of gestation because of maternal anxiety. Results of amniocentesis revealed a distal deletion of chromosome 3p. A malformed female fetus was delivered at 20 weeks of gestation with brachycephaly and facial dysmorphisms, and a cytogenetic analysis of the cord blood revealed a karyotype of 46,XX,del(3)(p26.1),inv(9)(p12q13). A whole-genome aCGH on uncultured cord blood and placental tissue was performed. The aCGH on cord blood revealed a 7.4-Mb deletion at 3p26.3-p26.1. However, the aCGH on placental tissue revealed a 32.42-Mb gene dosage increase at 3p26.1-p22.1 and a 26.28-Mb gene dosage increase at 1p36.33-p36.11 in addition to a 7.4-Mb deletion at 3p26.3-p26.1, indicating confined placental mosaicism for partial trisomy 3p (3p26.1→p22.1) and mosaicism for partial trisomy 1p (1p36.33→p36.11). The 7.4-Mb deleted region of 3p26.3-p26.1 contained the following genes: CHL1, CNTN4, CRBN, LRRN1, and ITPR1.ConclusionFetal tissue and amniocytes offer more reliable resources for aCGH characterization of prenatally detected aneuploidy compared with placental tissues. A molecular cytogenetic evaluation of prenatally detected aneuploidy using placental tissue should raise concerns of confined placental mosaicism and fetoplacental chromosomal discrepancy.     

Quality of life and sexual functioning of Polish infertile couples

Objectives To evaluate the influence of infertility on the quality of life (QoL) and sexual functioning of infertile couples.

Methods The research group consisted of 206 infertile couples and the control group of 190 fertile couples. A specific questionnaire was used as a research tool. It gathered information about socio-demographic features and infertility status, and included validated scales: Short Form-36 Health Survey, Female Sexual Function Index and International Index of Erectile Function.

Results The QoL parameters in all categories were generally lower for infertile women than for those of the control group. Clinical sexual dysfunctions were not significantly more common among infertile than fertile women (17.5% versus 12.1%, p = 0.13). Clinically relevant erectile dysfunctions were diagnosed in 23.9% of infertile men and in 13.7% of the controls. Male infertility had the most significant negative effect on men's sexual functioning.

Conclusions The risk groups for decreased QoL are infertile women and older subjects with lower education and occupationally inactive. Clinically relevant sexual disorders in the infertile population most frequently affect older men, with a lower educational level and with previously diagnosed male infertility.     

A new approach for screening for Y microdeletions: capillary electrophoresis combined with fluorescent multiplex PCR

Purpose : To apply capillary electrophoresis for rapid screening for Y microdeletions. Methods : A set of 25 specific sequence tagged sites that cover the azoospermia factor a, b, and c regions of the Y chromosome was amplified in 5 fluorescent multiplex sets each including 5 primer pairs. One of the primers of each pair was labeled with a fluorescent tag attached to the 5-end. After PCR amplification, analysis of the obtained PCR products was performed using capillary electrophoresis (ABI Prism 3100 Genetic Analyzer). The method was employed to determine Y microdeletions in azoospermic (n = 49) and severe oligozoospermic (n = 149) men. Results : The number of PCR cycle (from 45 to 30) and the amount of DNA template (20-fold) used in fluorescent multiplex PCR were reduced because of the high sensitivity of capillary electrophoresis. Approximately 1000 multiplex PCR sets from 198 patients were analyzed simultaneously within 50 h. Y microdeletions were found in 3 out of the 198 azoospermic or severe oligozoospermic men. Conclusions : Application of capillary electrophoresis for detection of PCR products provides a semiautomated, high throughput method for rapid screening for microdeletions on the Y chromosome.     

Study of Microdeletions in the Y Chromosome of Infertile Men with Idiopathic Oligo- or Azoospermia

Purpose: To determine the relationships between idiopathic oligo- or azoospermia and microdeletions of the Y chromosome. Methods: Eighteen Y-linked sequence-tagged sites (STSs) in AZF (Azoospermia Factor) region were screened by means of multiplex PCR (Polymerase Chain Reaction) in 50 idiopathic infertile men, including 16 patients with azoospermia, 13 severe oligospermia, and 21 oligospermia. Results: Microdeletions in the genomic DNA were observed in 8 of 50 cases, 3 with azoospermia, 1 severe oligospermia, and 4 oligospermia. Total deletion rate was 16.0% (8/50). The deletion regions were concentrated on AZFd and AZFc. Conclusions: Microdeletions of the Y chromosome are an important cause for idiopathic oligo- or azoospermia. Multiplex PCR is a useful technique for detecting the microdeletions. To avoid transmission to their offspring, patients with idiopathic oligo- or azoospermia should be screened for microdeletions of the Y chromosome before ICSI treatment for infertility.     

Y chromosome structural variation in infertile men detected by targeted next-generation sequencing

Purpose

To provide a validated method to identify copy number variation (CNV) in regions of the Y chromosome of infertile men by next-generation sequencing (NGS).

Methods

Semen analysis was used to determine the quality of semen and diagnose infertility. Deletion of the azoospermia factor (AZF) region in the Y chromosome was detected by a routine sequence-tagged-site PCR (STS-PCR) method. We then used the NGS method to detect CNV in the AZF region, including deletions and duplications.

Results

A total of 326 samples from male infertility patients, family members, and sperm donors were studied between January 2011 and May 2017. AZF microdeletions were detected in 120 patients by STS-PCR, and these results were consistent with the results from NGS. In addition, of the 160 patients and male family members who had no microdeletions detected by STS-PCR, 51 cases were found to exhibit Y chromosome structural variations by the NGS method (31.88%, 51/160). No microdeletions were found in 46 donors by STS-PCR, but the NGS method revealed 11 of these donors (23.91%, 11/46) carried structural variations, which were mainly in the AZFc region, including partial deletions and duplications.

Conclusion

The established NGS method can replace the conventional STS-PCR method to detect Y chromosome microdeletions. The NGS method can detect CNV, such as partial deletion or duplication, and provide details of the abnormal range and size of variations.

     

DYZ1 copy number variation, Y chromosome polymorphism and early recurrent spontaneous abortion/early embryo growth arrest

Objectives

To find the association between recurrent spontaneous abortion (RSA)/early embryo growth arrest and Y chromosome polymorphism.

Study design

Peripheral blood samples of the male patients of big Y chromosome, small Y chromosome and other male patients whose partners suffered from unexplained RSA/early embryo growth arrest were collected. PCR and real-time fluorescent quantitative PCR were used to test the deletion and the copy number variation of DYZ1 region in Y chromosome of the patients. A total of 79 big Y chromosome patients (48 of whose partners suffered from RSA or early embryo growth arrest), 7 small Y chromosome patients, 106 other male patients whose partners had suffered from unexplained RSA or early embryo growth arrest, and 100 normal male controls were enrolled.

Results

There was no fraction deletion of DYZ1 detected both in big Y patients and in normal men. Of RSA patients, 1 case showed deletion of 266 bp from the gene locus 25–290 bp, and 2 cases showed deletion of 773 bp from 1347 to 2119 bp. Of only 7 small Y chromosome patients, 2 cases showed deletion of 266 bp from 25 to 290 bp, and 4 cases showed deletion of 773 bp from 1347 to 2119 bp and 275 bp from 3128 to 3420 bp.The mean of DYZ1 copies was 3900 in normal control men; the mean in big Y patients was 5571, in RSA patients was 2655, and in small Y patients was 1059. All of the others were significantly different (P < 0.01) compared with normal control men, which meant that DYZ1 copy number in normal control men was less than that of big Y chromosome patients, and was more than that of unexplained early RSA patients and small Y patients.

Conclusions

The integrity and copy number variation of DYZ1 are closely related to the Y chromosome length under microscope. The cause of RSA/early embryo growth arrest in some couples may be the increase (big Y patients) or decrease of DYZ1 copy number in the husbands’ Y chromosome.