Genetics of human female infertility†

About 10% of women of reproductive age are unable to conceive or carry a pregnancy to term. Female factors alone account for at least 35% of all infertility cases and comprise a wide range of causes affecting ovarian development, maturation of oocytes, and fertilization competence, as well as the potential of a fertilized egg for preimplantation development, implantation, and fetal growth. Genetic abnormalities leading to infertility in females comprise large chromosome abnor - malities, submicroscopic chromosome deletion and duplications, and DNA sequence variations in the genes that control numerous biological processes implicated in oogenesis, maintenance of ovarian reserve, hormonal signaling, and anatomical and functional development of female reproductive organs. Despite the great number of genes implicated in reproductive physiology by the study of animal models, only a subset of these genes is associated with human infertility. In this review, we mainly focus on genetic alterations identified in humans and summarize recent knowledge on the molecular pathways of oocyte development and maturation, the crucial role of maternal-effect factors during embryogenesis, and genetic conditions associated with ovarian dysgenesis, primary ovarian insufficiency, early embryonic lethality, and infertility. Key words: female infertility, follicular development, genetics, oocyte development, preimplantation embryo, prema- ture ovarian failure, X chromosome. Overview Infertility is a disease of the reproductive system, encompasses a wide spectrum of conditions that affect the capacity of an individual to reproduce. A diagnosis of female infertility, defined as the inability to establish a clinical pregnancy after 12 months of regular unprotected sexual intercourse with a healthy partner, can also lead to anxiety and depression. Genetic, endocrine, physiological, anatomical, and immunological abnormalities of the reproductive system can affect a woman’s likelihood of becoming pregnant and delivering a living child. The high prevalence of reproductive disorders and infertility, af- fecting ∼10–15% of couples worldwide, is perhaps not surprising as successful reproduction requires the precise regulation of com- plex processes essential for development of functional gonads and other reproductive organs, sex determination, gametogenesis, neu- roendocrine competency, and ability to carry a pregnancy. Oogenesis is a process by which the mammalian egg becomes competent for fer- tilization and involves complex interaction between the oocyte and somatic cells that surround it, including the interplay of multiple transcriptional regulators. The disruption of these transcriptional C⃝ The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: [email protected] 549 550 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 Primordial germ cells Primordial follicle Primary oocyte (meiotic arrest) Primary follicle Secondary follicle Early antral follicle Preovulatory follicle Secondary oocyte Zygote Embryo Oogonia clusters Mitosis Cluster breakdown Meiosis I Ovarian determination Gonadotropin-dependent stimulation Gonadotropin-independent Fertilization Completion of meiosis Preimplantation development DSD, Gonadal dysgenesis, Streak gonads Depletion of ovarian reserve Embryonic arrest Reduced fetal viability NLRP7 KHDC3L Fetus PADI6 TLE6MEI1 TOP6BL REC114 Epigenetic modifications Cell cycle regulation Chromosome segregation Chromatin modification DNA repair Meiosis resumption Regulation of transcription Oocyte maturation Protein degradation Embryonic genome activation Implantation Placenta Maternal RNA degradation NLRP2 NLRP5 PATL2 TUBB8 WEE2 Response to hormonal stimulation Granulosa cell signalingZP1 ZP2 ZP3 MCM8 MCM9 XRCC4 ERCC6 MSH5 Follicular atresia NOBOX BMP15 GDF9 FIGLA LHX8 SOHLH1 SOHLH2 ESR1 ESR2 LHCGR DNA repair ANXA5 F2 F5 BUB1B HFM1 NUP107 PSMC3IP SGO2 STAG3 SYCE1 SYCP3 FMR1 EIF4ENIF1 RCBTB1 CPEB1 DIAPH2 SOX8 HAX1 MRPS22 POF1B POLR3H REC8 SMC1B SPIDR Chromatin remodeling Synthesis of cytoplasmic factors Zona pellucida formation P i di l Fetal ovarian development Pos t-pubertal folliculogenesis FOXL2 NR5A1 WNT4 LHX9 WT1 snoitidnoc cimoneg dna seneg detacilpmI Biological processes Birth X chromosome aneuploidy Female reproductive tract malformations EMX2 HNF1B HOXA13 LHX1 PAX2 PAX8 WNT4 WNT7A WNT9B Syndromic conditions AXL ATG7 ATG9A CCDC141 DUSP6 FEZF1 FGF17 FGF8 FGFR1 FLRT3 FSHB FSHR GNRH1 GNRHR HS6ST1 IL17RD KISS1 KISS1R LHB NSMF PROK2 PROKR2 SEMA3A SPRY4 TAC3 TACR3 WDR11 X chromosome structural alterations Autosomal structural alterations Observed phenotypes Menstrual irregularity Primary ovarian insufficiency Figure 1. Biological processes and genetic causes implicated in female infertility. Large X chromosome alterations are the frequent cause of ovarian dysgenesis. A less severe phenotype manifesting as primary or secondary amenorrhea as well as reduced fetal viability can be seen in patients with balanced rearran gements, submicroscopic alterations, and single gene defects. Reproductive potential in patients with genomic alterations and syndromic conditions great ly depends on diagnosis, genes affected, and concomitant manifestations. Causative genes are displayed beneath the key biological processes, however can be inv olved in multiple pathways, affecting several stages of oocyte development. regulators leads to ovarian dysgenesis or disorders of sex develop- ment. Newborn girls’ ovaries have, on average, 1–2 million of pri- mordial oocytes, and by the time of puberty, only about 400,000 remain. Individual oocytes are enveloped by somatic cells to form follicles and stay arrested at the diplotene stage of meiosis I (MI) until puberty. Completion of MI and ovulation are triggered by pi- tuitary gonadotropins (Figure 1). The initial endowment of follicles (ovarian reserve) determines a woman’s reproductive potential and, subsequently, her reproductive lifespan and age of menopause onset (between the ages of 40 and 58). Diminished ovarian reserve, either due to a low number of follicles at birth or puberty or to a rapid decline in the ovarian follicle pool after puberty, is associated with ir- regular menstruation, follicle exhaustion, and premature menopause (before age 40). Multiple genes have been implicated in the production and matu- ration of germ cells, defects in which can cause accelerated cell apop- tosis and follicle atresia, resulting in primary ovarian insufficiency (POI). Follicular atresia is one of the major mechanisms for eliminat- ing germ cells from the ovary. About 1–2% of women are affected by POI, a phenotypically and etiologically heterogeneous condition characterized by primary or secondary amenorrhea, infertility, de- creased estrogen production, elevated gonadotropins (follicle stim- ulating hormone and luteinizing hormone), and increased risk for osteoporosis and cardiovascular disease. POI is a term that is increas- ingly used and has been adopted to encompass diagnostically similar conditions, including hypergonadotropic hypogonadism, premature ovarian failure, and ovarian dysgenesis. POI is etiologically due to intrinsic problems within the ovary itself, which may include iatro- genic causes, such as surgery or radiation, or disruption of genes that primarily affect ovarian function. In a more severe presentation, POI presents with streak ovaries due to the depletion of germ cells and surrounding somatic, granulosa cells. Other causes of POI include abnormal chromosome segregation, deficiency in DNA repair, in- sufficient hormonal synthesis or signaling, defects in pathways that affect folliculogenesis, oocyte maturation and/or ovulation. Functional disruption at the level of the hypothalamus or pi- tuitary glands leads to hypogonadotropic hypogonadism, small ovaries, amenorrhea, and infertility. Infertility can also result from fertilization failure, either due to oocyte maturation arrest and the inability to produce a haploid zygote or to a defective zona pellucida. Embryo development is initially regulated by maternal transcripts that are synthetized and stored in the cytoplasm during oocyte mat- uration. The euploid maternal genome and the cytoplasmic com- ponents are essential for normal embryonic development. Other pathologies, such as disruption of reproductive tract development, endometriosis, uterine fibroids, polycystic ovary syndrome, or au- toimmune factors, may have a negative impact on implantation and pregnancy, leading to infertility or recurrent pregnancy loss. Numerous genetic studies in animal models have identified thou- sands of genes that are essential for mammalian reproduction. Only a small proportion (less than 200) of these genes shows strong as- sociation with human infertility. Recent applications of whole ex- ome and genome sequencing to families with infertility, have led to unbiased approaches and discovered new genes and novel variants involved in human infertility. These family-based approaches and re- cent genome-wide association studies have unearthed a small number of human infertility genes, and more discoveries are expected in the near future. Genetics female infertility, 2019, Vol. 101, No. 3 551 X Chromosome and female infertility In the normal ovary, the primordial germ cells carry two X chro- mosomes, one of which is initially inactivated similarly to any other somatic cell. Importantly, the second X chromosome is reactivated prior to meiosis as the presence of two transcriptionally active X chromosomes is essential for oogenesis [ 1, 2]. In females, both X chromosomes must be active during meiosis to pair efficiently like autosome homologues [ 3]. Moreover, ∼20% of X-linked genes es- cape inactivation and continue to be expressed from the inactive X chromosome, maintaining the dosage of female-specific transcripts in surrounding somatic cells [ 1–3]. In humans, chromosomal abnormalities such as monosomy X (Turner syndrome), cytogenetically visible deletions and duplica- tions, and balanced and unbalanced X-autosome rearrangements are associated with an accelerated loss of primordial oocytes during female fetal development, resulting in streak gonads at birth [ 4, 5]. They account for close to 10% of cases of POI [ 6]. The mechanism of such massive germ cell loss is not well understood; however, some studies suggest the presence of a surveillance mechanism that may detect and eliminate cells with unpaired or unsynapsed chromatin, such as cells with monosomy X [ 7, 8]. It is also possible that accelerated germ cell depletion is caused by inadequate signaling from surrounding somatic granulosa cells that also carry a single X chromosome [ 9]. Failure in either dosage of X-linked genes or the X chromosome structural integrity can lead to POI [ 10]. Despite great importance of X chromosome in ovarian biology, only few X-linked genes have been implicated in ovarian function. BMP15, FMR1,a n d PGRMC1 genes are located on the human X chromosome and have been linked with ovarian development and POI. Structural X chromosome abnormalities, rather than gene-specific abnormalities, may be the major reason for germ cell loss. Beside POI, X chromosome abnormalities such as deletions, duplications, inversions, complex rearrangements, X-autosome translocations, and single-gene sequence variants, carried by at least 5–6% of women, can lead to recurrent fetal losses [ 11, 12]. In females, X chromosome abnormalities may result in early embry- onic or fetal lethality, particularly of male conceptuses. To date, ∼30% of X-linked genes are associated with a specific phenotype in humans, while pathogenic variants in a significant number of X- linked genes, such as BCOR, EBP, FLNA, HCCS, IKBKG, MECP2, OFD1, OTC, and REP1, are presumed to be male-lethal [ 12–14]. In addition, germline mosaicism is another explanation of infertility and recurrent miscarriages of affected fetuses with X-linked or au- tosomal dominant lethal alterations. Maternal germline mosaicism for pathogenic variants has been reported for male-lethal genes, in- cluding IKBKG [15], FLNA [16], and MECP2 [17], and is likely an under-recognized condition. Female fetuses can also be affected by male-lethal X-linked disorders in cases of skewed X chromosome inactivation. Genetic causes of gonadal dysgenesis due to defects in sex determination Sexual development includes two processes: “sex determination,” in which the undifferentiated gonad develops into a testis or ovary based on XY or XX chromosomal complement; and “sex differentiation,” which leads to sex-specific hormone production governing anatomical and psychological differences between males and females. XY gonadal dysgenesis Phenotypic females with defects in sex determination, also known as Swyer syndrome, commonly present as XY individuals with female- appearing external genitalia and M ¨ullerian structures (uterus and fal- lopian tubes), but dysgenic and non-functional gonads, lack of spon- taneous pubertal development, absent breast development, primary amenorrhea, and infertility [ 18–20]. Approximately 10–15% of in- dividuals with XY gonadal dysgenesis have deletions or pathogenic sequence variants affecting the SRY gene on the Y chromosome. Other causes comprise Y chromosome structural alterations, mo- saicism for a cell line with monosomy X, heterozygous deletions, and sequence variants in the NR5A1 gene, duplications in the Xp21 re- gion involving NR0B1, WNT4 gene duplications, and deletions and loss-of-function pathogenic variants in the AR, MAP3K1, GATA4, DMRT1, DMRT2, ZNRF3, and DHH genes [21–27]. Disorders of sexual development are also often seen as a manifestation of other syndromic conditions. XX gonadal dysgenesis While many genes have been identified as key regulators of the testis developmental pathways, less is known about the mechanisms trig- gering ovarian development. Most of the current knowledge of key regulators involves genes predominantly expressed in the somatic compartment of the gonad. In XX individuals, the absence of SRY is not a default mechanism for ovarian differentiation and requires the activation of genes associated with female pathway development and suppression of male development factors [28–30]. Our knowledge of the genetic regulators of ovarian developments is based on studying animal models, as well as human families afflicted with ovarian dys- genesis. Animal, mainly mouse studies, imply numerous biological processes that are essential for ovarian determination, differentia- tion, and maintenance. Ovarian development is orchestrated by sev- eral transcription factors, including SOHLH1, SOHLH2, NOBOX, LHX8, FIGLA, GATA4, WNT4, WT1, LHX9, FOXL2, and NR5A1 [31–37], and comprises mitotic proliferation of XX primordial germ cells, their progression into meiosis, and the formation of primordial follicles (Figure 1). Mouse studies show that Sohlh1 and Sohlh2 af- fect differentiation of both male and female gametes, while Nobox, Lhx8,a n d Figla are gamete-specific genes known to regulate oogen- esis, without affecting male germ cell differentiation. In humans, the gonadal development in females and males is identical within first four weeks after conception. In fact, orthologues of above mouse genes are present in the human genome. Heterozygous and bial- lelic pathogenic variants in SOHLH1, SOHLH2, NOBOX, FIGLA, WNT4, WT1, FOXL2 ,a n d NR5A1 genes were found in women with a wide spectrum of reproductive abnormalities, including iso- lated as well as syndromic conditions associated with complete go- nadal dysgenesis and less severe forms of POI [ 37–40]. Phenotypic expression and severity of impaired gonadogenesis and oogenesis may depend on the type of alteration (loss of function vs. gain of function) and the effects on specific protein domains. Defects in early oogenesis and chromosome segregation as cause of infertility Once primordial germ cells cease proliferation, they become oogo- nia, and enter meiosis circa 12 weeks of human gestation. During the initial stages of meiosis, germ cells undergo a set of highly or- ganized processes that include homologous chromosome pairing, 552 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 alignment, synapsis, and crossover [ 41–43]. The accurate segrega- tion of chromosomes involves multiple processes to establish a phys- ical connection of the homologous chromosomes via centromere interaction and recombination, to ensure correct positioning (chro- mosome alignment) at the spindle equator at metaphase I and to control a proper meiotic chromosome segregation [ 44, 45]. During MI prophase, homologous chromosomes pair and undergo recom- bination, that consists of programmed DNA double-strand breaks (DSB) governed by SPO11 protein, a crossover event resulting in an exchange of large chromosomal segments, and a repair of DNA breaks. In humans, a total number of DSB repair events outnumber the count of crossover events by more than 10-fold. Cell cycle check- point responds to incorrectly repaired/unrepaired DNA breaks and to a non-disjunction of chromosomes by inducing cell cycle arrest and activating apoptotic programs. Programmed cell death is a naturally occurring mechanism to eliminate abnormal, damaged or excess cells at every stage of oogen- esis. The fetal ovary contains ∼7 million germ cells, only ∼400 000 (5%) of which will remain at puberty, and ∼400 oocytes (0.005% from the original germ cell pool) will ovulate during a female re- productive lifespan [ 46–48]. Accelerated apoptosis and disruption in signaling can lead to depletion of the primordial follicle pool, incomplete development of follicles, or failure of normal sexual dif- ferentiation, resulting in dysgenic or streak gonads. Premature loss of germ cells is a common cause of human gonadal dysgenesis, primary amenorrhea, and infertility in human females with defects in genes implicated in germ cell mitosis and meiosis (Table 1) and genes important in chromosome segregation, such as BUB1B, STAG3, SYCE1, SYCP3, HFM1, PSMC3IP, and SGO2, as well as genes involved in DNA damage repair, such as MCM8, MCM9, XRCC4, ERCC6, and BRCA2 [49–60]. There is compelling evidence that infertile women have a higher incidence of aneuploid oocytes [ 61, 62]. Rare deleterious alterations in genes required for chromosome synapsis and segregation during meiosis, such as SYCP3 [49], STAG3 [50], HFM1 [51], SYCE1 [52, 53], NUP107 [54, 55], PSMC3IP [56, 57], SGO2 [58], REC8 [59], SMC1B [59], and BUB1B [60], are the cause of meiotic disturbances, aneuploidy, streak gonads, primary ovarian insufficiency, embryonic arrest, recurrent miscarriage, risk of affected offspring with chromo- somal abnormalities, and infertility (Table 1). Meiosis-specific co- hesin complexes consist of two structural proteins (SMC1 α/SMC1β and SMC3), encoded by SMC1A and SMC1B, respectively; an α- kleisin protein (RAD21, RAD21L, or REC8); and a stromal anti- gen protein (STAG1, STAG2, or STAG3) [ 63]. Pathogenic variants in REC8, SMC1B ,a n d STAG3 are implicated in human infertility [50, 58]. Defects in assembly of the meiotic cohesion and/or synap- tonemal complex lead to multi-chromosome aneuploidy during the second meiotic division, resulting in follicle atresia. Such genetic de- fects have been observed in patients with idiopathic infertility and recurrent miscarriages. Female meiosis is highly prone to chromosome segregation er- rors, particularly in older women, resulting in a high proportion of aneuploid zygotes and at least 10% of aneuploid human fetuses [64–66]. The gradual loss of cohesins or cohesin-related proteins such as SGO2, has been implicated in meiotic non-disjunction and age-related aneuploidy and infertility [67]. The BUB1B gene encodes a mitotic checkpoint serine/threonine kinase B, a protein that plays an important role in regulation of the spindle-assembly checkpoint. Heterozygous and biallelic alterations in BUB1B are rare findings among fetuses and liveborn individuals affected by mosaic varie- gated aneuploidy [ 68, 69]. Similarly, chromosome mis-segregations are observed in patients with homozygous pathogenic variants in the CEP57 gene [ 70, 71]. Impaired spindle checkpoint function re- sults in aneuploidies involving multiple different chromosomes and is likely to cause oocyte maturation arrest, early embryonic lethality, or miscarriage [ 60, 72]. The presumed inheritance of these genes is listed in Tables 1 and 2. DNA repair genes and their role in POI and human female infertility Many studies implicate DNA repair genes in follicle maturation and quality, reproductive aging, and subsequent age of menopause on- set [ 73–84]. Beside their role in meiosis, many DNA damage re- sponse genes also play essential roles in cell cycle control during mi- tosis. Diversity of DNA repair mechanisms, differential expression in various tissues, existence of the alternative tissue-specific DNA repair pathways, incomplete penetrance, environmental exposures, and other risk factors are the explanations for phenotypic variabil- ity in patients with defects in DNA repair genes. Thus, affected individuals may present with a spectrum phenotype manifesting as a syndromic condition or isolated tissue-specific phenotypes. Bial- lelic defects in DNA repair genes are the cause of syndromic condi- tions such as Fanconi anemia ( FANCA, FANCB, FANCC, BRCA2, FANCD2, FANCE, FANCF, XRCC9, FANCI, BRIP1, PHF9, PALB2, RAD51C, SLX4, ERCC4, RAD51, BRCA1, UBE2T, XRCC2, MAD2L2, RFWD3 ), Werner syndrome ( RECQL2), War- saw breakage syndrome ( DDX11), LIG4 syndrome ( LIG4), Bloom syndrome ( BLM), Nijmegen breakage syndrome ( NBN), ataxia- telangiectasia (ATM), trichothiodystrophy 1 (ERCC2, ERCC3), and xeroderma pigmentosum ( XPA, XPC, ERCC2, ERCC3, ERCC4 ). These syndromes are rare genetic conditions characterized by suscep- tibility to chromosome breakage and genome instability, triggering permanent cell cycle arrest, and apoptosis of the affected cells. As a result, patients with pathogenic variants present with growth retar- dation, skin lesions, bone marrow and immune deficiencies, neurode- velopmental problems, endocrine and early gonadal dysfunction, and predisposition to cancers. There are also a number of patients with biallelic variants in DNA repair genes such as MCM8, MCM9, XRCC4 ,a n d MSH5 who present with a non-syndromic primary ovarian insufficiency [78–83]. Homozygous pathogenic variants in the MCM8 or MCM9 genes were discovered in women with hypergonadotropic primary amenorrhea, hypothyroidism, growth retardation, and absent or very small ovaries. None of the affected women had cancer at the time of investigation; however, excessive chromosomal breakage was observed in their somatic cells [ 78, 79]. A single report indicated an affected family with MCM9 pathogenic variants in association with primary hypergonadotropic hypogonadism with early-onset colorec- tal cancer [84]. The heterozygous mutation carriers appeared healthy and fertile, although they also had an increased number of chromo- some breakages in comparison to wildtype MCM8 and MCM9 indi- viduals. Interestingly, population-based genome-wide studies found the strongest association with a non-synonymous single nucleotide polymorphism (rs16991615) in coding exon 9 of the MCM8 gene and the age of menopause in Caucasian, European, African Ameri- can [ 85], and Hispanic women. These results indicate that MCM8, and probably other DNA repair genes, regulate the reproductive span from gonadal formation to menopause, and that heterozygous variants may confer risk loci for primary ovarian insufficiency, di- minished ovarian reserves, and infertility [ 85]. Variants in MCM8 Genetics female infertility, 2019, Vol. 101, No. 3 553 Ta b l e 1 .Genes implicated in non-syndromic POI, gonadal dysgenesis, and female infertility. Gene Phenotype MIM/[ref] Phenotype, reproductive outcomes Genetic defects Mode of inheritance ANXA5 #614391 Susceptibility to recurrent pregnancy loss, RPRGL3 Sequence variants in the promoter AD ATG7, ATG9A [199] Primary ovarian insufficiency Sequence variants AD AXL [200] Hypogonadotropic hypogonadism with or without anosmia Sequence variants AD BMP15 #300510 Ovarian dysgenesis, primary amenorrhea, POF4 Sequence variants, deletions X-linked BUB1B #176430 Premature chromatid separation, recurrent pregnancy loss Sequence variants AD CCDC141 [116] Kallmann syndrome Sequence variants AR CPEB1 [201, 202] Primary ovarian insufficiency Deletions AD DIAPH2 #300511 Primary or secondary amenorrhea, POF2A Sequence variants, deletions X-linked DUSP6 #615269 Hypogonadotropic hypogonadism HH19 with or without anosmia Sequence variants AD EIF4ENIF1 [115] Primary ovarian insufficiency Sequence variants AD ERCC6 #616946 Primary ovarian insufficiency, POF11 Sequence variants AD ESR1 #615363 Estrogen resistance, primary amenorrhea Sequence variants AR ESR2 #618187 Ovarian dysgenesis, ODG8, primary amenorrhea Sequence variants AD F2 #614390 Susceptibility to recurrent pregnancy loss, RPRGL2 Sequence variants AD F5 #614389 Susceptibility to recurrent pregnancy loss, RPRGL1 Sequence variants AD FEZF1 #616030 Hypogonadotropic hypogonadism HH22 with or without anosmia Sequence variants AR FGF17 #615270 Hypogonadotropic hypogonadism HH20 with or without anosmia Sequence variants AD FGF8 #612702 Hypogonadotropic hypogonadism HH6 with or without anosmia Sequence variants AD FGFR1 #147950 Hypogonadotropic hypogonadism HH2 with or without anosmia Sequence variants AD FIGLA #612310 Ovarian insufficiency, POF6 Sequence variants AD, AR FLRT3 #615271 Hypogonadotropic hypogonadism HH21 with or without anosmia Sequence variants AD FMR1 #311360#300869 Primary ovarian insufficiency, POF1 Expansion of trinucleotide (CCG)n repeats, duplication X-linked FSHB #229070 Ovarian insufficiency, hypogonadotropic hypogonadism HH24 Sequence variants AR/AD FSHR #233300 Ovarian dysgenesis, primary amenorrhea, ODG1 Sequence variants AR GDF9 #618014 Primary ovarian insufficiency, POF14 Sequence variants, duplication AR, digenic GNRH1 #614837 Hypogonadotropic hypogonadism HH12 with or without anosmia Sequence variants AR GNRHR #146110 Hypogonadotropic hypogonadism HH7, primary amenorrhea Sequence variants AR HAX1 [203] Primary ovarian insufficiency, short stature Sequence variants AD HFM1 #615724 Ovarian dysgenesis, ovarian insufficiency, POF9 Sequence variants AR HS6ST1 #614880 Hypogonadotropic hypogonadism HH15 with or without anosmia Sequence variants AD IL17RD #615267 Hypogonadotropic hypogonadism HH18 with or without anosmia Sequence variants AD, AR KHDC3L [166] Preimplantation embryonic lethality Sequence variants AR KISS1 #614842 Hypogonadotropic hypogonadism HH13 with or without anosmia Sequence variants AR KISS1R #614837 Hypogonadotropic hypogonadism, HH8, primary amenorrhea Sequence variants AR LHB #228300 Hypogonadotropic hypogonadism, HH23 Sequence variants AR LHCGR #238320 Luteinizing hormone resistance, amenorrhea, and infertility Sequence variants AR LHX8 [99] Ovarian dysgenesis, ovarian insufficiency Sequence variants AD MCM8 #236700 Ovarian dysgenesis, primary amenorrhea, POF10 Sequence variants AR MCM9 #616185 Ovarian dysgenesis, primary amenorrhea, ODG4 Sequence variants AR MRPS22 #618117 Ovarian dysgenesis, ODG7, primary amenorrhea Sequence variants AR MSH5 #617442 Primary ovarian insufficiency, POF13 Sequence variants AR NLRP2, NLRP5 [160] Primary infertility, Oocyte maturation defect, Sequence variants AR NOBOX #611548 Ovarian dysgenesis, primary ovarian insufficiency, POF5 Sequence variants AD, AR 554 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 Ta b l e 1Continued Gene Phenotype MIM/[ref] Phenotype, reproductive outcomes Genetic defects Mode of inheritance NR5A1 #612964 Gonadal dysgenesis, ovotesticular DSD, secondary ovarian insufficiency, POF7 Del/dup, Sequence variants AD NSMF #614838 Hypogonadotropic hypogonadism HH9 with or without anosmia Sequence variants AD NUP107 #618078 Ovarian dysgenesis, ODG6, primary amenorrhea Sequence variants AR PADI6 #617234 Preimplantation embryonic lethality, PREMBL12 Sequence variants AR PATL2 #617743 Primary infertility, oocyte maturation defect, OOMD4, early embryonic arrest Sequence variants AR POF1B #300604 Primary ovarian insufficiency, POF2B Sequence variants X-linked POLR3H [204] Primary ovarian insufficiency Sequence variants AR PROK2 #610628 Hypogonadotropic hypogonadism HH4 with or without anosmia Sequence variants AD PROKR2 #244200 Hypogonadotropic hypogonadism HH3 with or without anosmia Sequence variants AD PRLR #615555 Hyperprolactinemia, amenorrhea Sequence variants AR PSMC3IP #614324 Ovarian dysgenesis, ODG3, primary amenorrhea Sequence variants AR REC8 [99] Ovarian dysgenesis, ovarian insufficiency Sequence variants unknown SALL4 [205] Primary ovarian insufficiency Sequence variants AD SEMA3A #614897 Hypogonadotropic hypogonadism HH16 with or without anosmia Sequence variants AD SGO2 [58] Primary ovarian insufficiency Sequence variants AR SLC29A3 #602782 Histiocytosis-lymphadenopathy plus syndrome Sequence variants AR SMC1B [99] Ovarian dysgenesis, ovarian insufficiency Sequence variants unknown SOHLH1 #617690 Ovarian dysgenesis, ODG5, primary ovarian insufficiency Sequence variants AR SOHLH2 #616066 Primary ovarian insufficiency Sequence variants AD SOX8 [192] Ovarian dysgenesis, primary or secondary amenorrhea Sequence variants AD SPIDR [206] Ovarian dysgenesis Sequence variants AR SPRY4 #615266 Hypogonadotropic hypogonadism HH17 with or without anosmia Sequence variants AD STAG3 #615723 Ovarian dysgenesis, ovarian insufficiency, POF8 Sequence variants AR SYCE1 #616947 Ovarian dysgenesis, ovarian insufficiency POF12 Sequence variants AR SYCP3 #270960 Susceptibility to recurrent pregnancy loss, RPRGL4 Sequence variants, deletions AD TAC3 #614839 Hypogonadotropic hypogonadism HH10 with or without anosmia Sequence variants AR TACR3 #614840 Hypogonadotropic hypogonadism HH11 with or without anosmia Sequence variants AR TLE6 #616814 Preimplantation embryonic lethality, PREMBL1 Sequence variants AR TUBB8 #616780 Primary infertility, oocyte maturation defect, OOMD2 Sequence variants (de novo, paternal transmission) AD, AR WDR11 #614858 Hypogonadotropic hypogonadism HH14 with or without anosmia Sequence variants AD WEE2 #617996 Primary infertility, oocyte maturation defect, OOMD5 Sequence variants AR WNT4 #158330 M ¨ullerian aplasia, hyperandrogenism, primary amenorrhea Sequence variants AD ZP1 #615774 Primary infertility, oocyte maturation defect, OOMD1 Sequence variants AR ZP2 [150] Primary infertility Sequence variants AR, digenic ZP3 #617712 Primary infertility, oocyte maturation defect, OOMD3 Sequence variants (de novo, paternal transmission) AD also correlate with antral follicle count [ 86]. The mechanisms of MCM8 and MCM9 action are currently under intensive study. Both MCM8 and MCM9 are members of a complex that is rapidly re- cruited at the DNA damage sites to resolve double-strand breaks [87]. During meiosis, programmed double-strand breaks are gen- erated to attain accurate segregation and crossover of homologous chromosomes. To maintain germ cells’ genomic integrity and pre- vent DNA from possible damage, breaks must be repaired via a homologous recombination-mediated mechanism. Inability to effi- ciently repair chromosomal ends activates cell apoptosis, leading to depletion of germ cells and dramatic loss of ovarian reserves [ 75, 87]. It is possible that deficiency in MCM8/MCM9 leads to oocyte loss due to unrepaired double-strand DNA breaks. Abnormal folliculogenesis and female infertility The number of primordial follicles at birth, their quality, and the rate of germ cell depletion are the factors that define reproductive lifespan in women [88–91]. In some women, oocyte depletion occurs during the fetal stages of oogenesis, endowing them with a relatively Genetics female infertility, 2019, Vol. 101, No. 3 555 Ta b l e 2 .Syndromic conditions accompanied by ovarian insufficiency and female infertility. Gene Phenotype MIM/[ref] Phenotype, reproductive outcomes Genetic defects Mode of inheritance AARS2 #615889 Leukoencephalopathy, progressive, with ovarian failure Sequence variants AR AIRE #340300 Autoimmune polyendocrinopathy syndrome Sequence variants AD, AR ANOS1 #308700 Kallmann syndrome Sequence variants, deletions X-linked AR #300068 Androgen insensitivity, ovarian dysgenesis, XY females Sequence variants, deletions X-linked ATM #208900 Ataxia-telangiectasia Sequence variants, deletions AR BRCA2 [73] Ovarian dysgenesis, microcephaly, cancer predisposition Sequence variants AR CCDC39 #613807 Ciliary dyskinesia, recurrent respiratory infections, infertility Sequence variants AR, digenic CHD7 #214800 CHARGE syndrome, hypogonadotropic hypogonadism, ovarian dysgenesis Sequence variants, deletions AD CLPP #614129 Perrault syndrome 3, progressive hearing loss, primary ovarian insufficiency Sequence variants AR CYP17A1 #202110 Congenital adrenal hyperplasia due to 17-hydroxylase deficiency Sequence variants, deletions AR CYP21A2 #201910 Congenital adrenal hyperplasia due to 21-hydroxylase deficiency Sequence variants, deletions AR DCAF17 #241080 Hypogonadism, ovarian dysgenesis, partial alopecia, Woodhouse–Sakati syndrome Sequence variants AR DDX11 #613398 Warsaw breakage syndrome Sequence variants AR EIF2B1/B2/B3/B4/B5 #603896 Leukoencephalopathy with vanishing white matter, gonadal dysgenesis, primary or secondary amenorrhea Sequence variants AR ERAL1 #617565 Perrault syndrome 6, progressive hearing loss, primary ovarian insufficiency Sequence variants AR ERCC2 #601675 Trichothiodystrophy 1, brittle, sulfur-deficient hair, ichthyosis, developmental disabilities, decreased fertility, short stature, infections Sequence variants AR ERCC3 #616390 Trichothiodystrophy 2 Sequence variants AR FANCA, multiple #227650 Fanconi anemia Sequence variants, deletions AR FOXL2 #110100 Blepharophimosis with ovarian dysgenesis, POF3 Sequence variants, deletions, duplications, translocations AD GALT #230400 Galactosemia, hypoglycemia, renal tubular dysfunction, muscle hypotonia, hypogonadism Sequence variants, deletions AR H6PD #604931 Cortisone reductase deficiency 1, hirsutism, oligomenorrhea, infertility Sequence variants AR HARS2 #614926 Perrault syndrome 2, sensorineural deafness, primary amenorrhea, streak gonads Sequence variants AR HSD11B1 #614662 Cortisone reductase deficiency 2, oligomenorrhea, infertility Sequence variants AD HSD17B4 #233400 Perrault syndrome 1, progressive hearing loss, primary ovarian insufficiency Sequence variants AR KHDC3L #614293 Hydatidiform mole, recurrent pregnancy loss, gestational trophoblastic neoplasia Sequence variants AR LARS2 #615300 Perrault syndrome 4, progressive hearing loss, primary ovarian insufficiency Sequence variants AR LMNA #151660 Lipodystrophy, familial partial, type 2, abnormal distribution of subcutaneous fat, polycystic ovary syndrome, infertility, spontaneous abortions, fetal death Sequence variants AD LMNA #212112 Malouf syndrome, cardiomyopathy, skeletal anomalies, amenorrhea Sequence variants AD MEI1 [172] Hydatidiform mole, recurrent pregnancy loss, gestational trophoblastic neoplasia MKKS #236700 McKusick-Kaufman syndrome, hypogenitalism, hydrometrocolpos Sequence variants AR NBN #251260 Nijmegen breakage syndrome, primary amenorrhea, microcephaly and immunodeficiency Sequence variants AR NLRP7 #231090 Hydatidiform mole, recurrent pregnancy loss, gestational trophoblastic neoplasia Sequence variants, deletions AR NR0B1 #300018 46,XY sex reversal 2, dosage-sensitive Duplications X-linked NR3C1 #615962 Glucocorticoid resistance, infertility, male-pattern baldness, hirsutism, and menstrual irregularities Sequence variants AD 556 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 Ta b l e 2Continued Gene Phenotype MIM/[ref] Phenotype, reproductive outcomes Genetic defects Mode of inheritance PGM1 #614921 Congenital disorder of glycosylation, cleft lip and bifid uvula, hepatopathy, hypoglycemia, short stature, and exercise intolerance, rhabdomyolysis, dilated cardiomyopathy, and hypogonadotropic hypogonadism Sequence variants AR PMM2 #212065 Ovarian dysgenesis, cerebellar atrophy, mental impairment, pigmentary retinopathy Sequence variants AR PNPLA6 #215470 Boucher-Neuhauser syndrome, pinocerebellar ataxia, hypogonadotropic hypogonadism, visual impairment Sequence variants AR POLG #157640 Progressive external ophthalmoplegia, amenorrhea Sequence variants AD PPARG #151660 Lipodystrophy, familial partial, type 3, insulin-resistant diabetes mellitus, infertility Sequence variants AD PROP1 #262600 Combined pituitary hormone deficiency, growth failure Sequence variants, deletions AR RCBTB1 [119] Retinitis pigmentosa, goiter, primary ovarian insufficiency, intellectual disability Sequence variants AR REC114 [172] Hydatidiform mole, recurrent pregnancy loss, gestational trophoblastic neoplasia Sequence variants AR RECQL2 #210900 Werner syndrome Sequence variants AR RECQL3 #277700 Bloom syndrome Sequence variants AR RNF216 #212840 Cerebellar ataxia and hypogonadotropic hypogonadism Sequence variants, digenic AR SLC29A3 #602782 Histiocytosis-lymphadenopathy plus syndrome, joint deformities, sensorineural hearing loss, and subsequent development of generalized lymphadenopathy and swellings in the eyelids, hypogonadotropic hypogonadism, amenorrhea Sequence variants AR SOX10 [117] Kallmann syndrome, deafness Sequence variants AD TOP6BL [172] Hydatidiform mole, recurrent pregnancy loss, gestational trophoblastic neoplasia Sequence variants AR TP63 #603543 Limb-mammary syndrome, hand/foot anomalies and hypoplasia/aplasia of the mammary gland, amenorrhea, absence of the uterus and ovaries Sequence variants AD TRIM37 #253250 Mulibrey nanism, gonadal dysgenesis Sequence variants AR TWNK #616138 Perrault syndrome 5, progressive hearing loss, primary ovarian insufficiency Sequence variants AR WT1 #256370 Nephrotic syndrome, gonadal dysgenesis Sequence variants AD XPA, XPC, ERCC2, ERCC3, ERCC4 #278700 Xeroderma pigmentosum Sequence variants AR XRCC4 #616541 Short stature, microcephaly, and endocrine dysfunction, ataxia Sequence variants AR small pool by the time of puberty. In others, there is accelerated loss of oocytes after the establishment of the primordial follicle pool, and can be due to environmental exposures such as cigarette smoking. Elimination of oocytes is accelerated with maternal age and is re- lated to deterioration of oocyte quality. In fact, about 40–60% of oocytes from women of 40 years are aneuploid [92], while the oocyte quality of younger women with diminished ovarian reserve are unknown. Diminished ovarian reserve (DOR) is another term used for women with a relatively low antral follicle count on ultrasound ex- aminations ( <8) and low AMH levels and affects 10% of women undergoing infertility treatment [ 93]. DOR is more prevalent than POI but, in biological reality, may very well have similar genetic underpinnings, except that less penetrant mutations may be more frequent in DOR individuals. Folliculogenesis involves proliferation and differentiation of granulosa cells and maturation of the oocyte. This process includes a series of signaling events between the cells within the follicle. Al- though the granulosa and theca cells have long been implicated in ovarian function, animal studies have revealed a synergistic effect of GDF9 and BMP15 on granulosa cell differentiation, folliculo- genesis, and ovulation [ 94–97]. BMP15 and GDF9, members of the transforming growth factor- beta (TGFβ) superfamily, are ex- pressed in oocytes through folliculogenesis and are involved in a cascade of regulatory processes directing granulosa cell prolifera- tion, steroidogenesis, cumulus expansion, and apoptosis. GDF9 has been implicated in the transition from the primary to the secondary follicle stage [ 96]. BMP15 stimulates follicle maturation during the gonadotropin-independent phase and regulates granulosa cell sen- sitivity to follicle-stimulating hormone (FSH). It has been observed that a high level of GDF9 and BMP15 in the follicle is associated with good embryo morphology. Human GDF9 and BMP15 gene variants, mostly heterozygous, have been detected in up to 10% of women with hypergonadotropic ovarian failure, POI, primary and secondary amenorrhea, and polycystic ovary syndrome [ 94, 98, 99]. However, more convincing functional studies are needed to deter- mine the true role of these variants in female infertility. Multiple oocyte-specific transcription factors, including FIGLA, NOBOX, LHX8, SOHLH1, and SOHLH2, also control follic- ular development and play a role in cytoplasmic maturation by Genetics female infertility, 2019, Vol. 101, No. 3 557 regulating the expression of maternal-effect factors essential for future embryonic activation [ 28, 30, 100]. Heterozygous and homozygous sequence variants in these transcription factors were found to be associated with ovarian dysgenesis and POI [ 37, 101–109]. In mice, these genes cause phenotypes in autosomal recessive fashion, and this is likely to be true in humans, but further studies are necessary to determine whether heterozygous variants in these genes can cause pathology. Pleotropic and syndromic conditions resulting in POI and female infertility Expansion of CGG repeats in the 5’UTR of the FMR1 gene is an- other genetic condition strongly associated with diminished ovarian reserve and primary ovarian insufficiency, as the FMR1 protein is ex- pressed in neurons and granulosa cells. FMR1 is an mRNA-binding protein that also regulates translation. Fragile X intellectual disabil- ity syndrome is caused by inactivation of the FMR1 gene, located on the X chromosome, mainly due to DNA hypermethylation of the promoter and the CGG repeat region when the repeat size exceeds 200. Full mutation alleles arise from premutation alleles with 55– 200 CGG repeats that demonstrate a repeat length instability and a tendency to expand from one generation to the next. The prevalence of premutation alleles in the general population is estimated to be 1 per 150–300 females, with the highest incidence, 1 per 100 females, in Colombia and Israel [ 110, 111]. Interestingly, women with 55–200 CGG repeats (premutation al- leles) have elevated mRNA expression and decreased protein level posing a significant risk for premature ovarian aging and infertility, while females with over 200 CGG repeats, full-mutation carriers, do not have any form of ovarian dysfunction. FMR1 premutation alleles confer risk for adult neurodegenerative disorder in males as well as POI in females. A phenomenon in which a pathogenic variant is the cause of multiple apparently unrelated conditions has been defined as pleiotropy. Such effects can be explained by the failure of a single molecular function involved in multiple biological processes, vari- ably affecting cellular physiology. Premutation carriers were noted to have significantly elevated FSH correlated with increasing CGG repeats and decreased inhibin B/A, progesterone, and antim ¨ulerian hormone levels. The exact mechanism of oocyte toxicity in women with FMR1 premutation alleles is unknown, although mRNA overexpression may result in RNA/protein gain-of-function disorder affecting follic- ular maturation, as well as increasing the degree of follicular apopto- sis [112–114]. Another gene, eukaryotic translation initiation factor 4E nuclear import factor 1 ( EIF4ENIF1) has been recently impli- cated in ovarian germ cell development and primary ovarian insuffi- ciency [115]. Defects in eIF4ENIF1 may result in decreased mRNA degradation and increased mRNA stability, similar to the mechanism of fragile X primary ovarian insufficiency. A significant number of syndromic conditions, such Perrault syndrome ( HSD17B4, HARS2, CLPP, LARS2, C10ORF2 ), ble- pharophimosis with ptosis, and epicanthus inversus syndrome (FOXL2); leukoencephalopathy with vanishing white matter due to pathogenic variants in the EIF2B1, EIF2B2, EIF2B4, and EIF2B5 genes; galactosemia and carbohydrate-deficient glycoprotein syndromes with pathogenic variants in GALT and PMM2 genes, and many others (Table 2) have ovarian dysge- nesis and/or primary ovarian insufficiency among their clinical manifestations [116–122]. Mitochondria and female infertility Defects in the POLG gene, which encodes polymerase gamma, re- sponsible for mitochondrial DNA synthesis, are the cause of pro- gressive external ophthalmoplegia and are also reported in multiple women with POI [ 123–124]. The association of nuclear and mito- chondrial genes responsible for mitochondria function with female oogenesis and fertility is not surprising as the number of mitochon- dria increases at least 1000-fold in human oocytes [ 125–126]. Mito- chondria are essential for multiple processes during oogenesis, such as ATP production, apoptosis, and calcium homeostasis [ 127]. Mi- tochondrial dysfunction can lead to defects in oocyte maturation, impaired spindle assembly and chromosome missegregation, poor pre-implantation development, and implantation failure [ 124]. Mi- tochondrial inheritance is exclusively maternal and is considered to be a key determinant of female reproductive aging and infertility [128]. In addition to POLG, a number of nuclear genes regulate mitochondrial function, such as the genes responsible for Perrault syndrome (HARS2, HSD17B4, LARS2 ). The clinical significance of other genes involved in mitochondrial DNA maintenance, repair, and response to oxidative stress, such as RAD51, RAD51C,a n d XRCC3, remains to be established. Deficiencies in hormonal signaling and response in female infertility The gonadotropins, follicle-stimulating hormone (FSH) and luteiniz- ing hormone (LH), play a pivotal role in regulation of follicu- logenesis, oocyte maturation, ovulation, implantation, and estra- diol synthesis [ 129–130]. Recruitment of primordial follicles is a gonadotropin-independent process, while subsequent follicle growth is gonadotropin-sensitive. Upon binding to the receptors, go- nadotropins stimulate oocyte development from early antral stage to maturation and convert androgen to estrogen in granulosa cells. FSH is a pituitary glycoprotein hormone composed of an alpha chain (CGA) that is similar to other pituitary glycoproteins such as LH, chorionic gonadotropin, and thyroid-stimulating hormone, and a beta chain encoded by FSHB. Patients with pathogenic variants in the FSHB (FSH beta subunit) and the FSHR (FSH receptor) genes develop follicles up to preantral stages, but further maturation is blocked. Defects in FSHB lead to a low production of FSH, resulting in hypogonadotropic hypogonadism [ 131, 132], whereas changes in FSHR cause hypergonadotropic hypogonadism [ 133–135]. The severity of the phenotype in patients with variations in the FSHR gene correlates with the effect of the genetic change in the FSHR function. Pathogenic variants in FSHR, specifically the c.566C > T change, are more frequent in a Finnish population [ 133], but are rare in other ethnic groups [ 133, 134]. Hypogonadotropic hypogonadism is a rare disorder character- ized by the deficiency of gonadotropin-releasing hormone (GnRH) due to its impaired production, secretion, or function. It often man- ifests as incomplete or absent puberty and infertility. GnRH acts via the GnRH receptor, which affects both synthesis and release of LH and FSH, which in turn, control gonadal maturation, which then feeds back via Inhibin and Activin to complete the hypothalamic– pituitary–gonadal axis [ 136]. A considerable proportion of patients affected by hypogonadotropic hypogonadism have genetic changes leading to an isolated or a syndromic condition with X-linked, au- tosomal recessive, and autosomal dominant inheritance. Hypogo- nadotropic hypogonadism [ 137] is a genetically heterogeneous con- dition with both sporadic and familial cases and more than 25 558 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 causative genes identified to date (Table 2). The most commonly affected genes are ANOS1 (KAL1), SOX10, IL17RD, FGFR1, CHD7, PROKR2, GNRHR, and TACR3, while pathogenic vari- ants in AXL, DMXL2, FEZF1, FGF17, FGF8, GNRH1, HESX1, HS6ST1, KISS1, KISS1R, LEP, LEPR, NR0B1, NSMF, OTUD4, PCSK1, PNPLA6, PROK2, RNF216, SEMA3A, SEMA7A, TAC3, and WDR11 are less frequent. Kallmann syndrome is characterized by a defective sense of smell in approximately 50% of patients with hypogonadotropic hypogonadism. The presence of anosmia should alert clinicians for a possible congenital hypogonadotropic hypogo- nadism and facilitate the search for a definitive molecular diagnosis in affected patients. Genetics of polycystic ovaries Development of preovulatory follicles depends greatly on regulation by a number of factors, such as FSH, oocyte-secreted proteins, and insulin-like growth factors [ 130]. Mature oocytes are surrounded by granulosa and thecal cells, which produce an androgen substrate, a prerequisite for ovarian estrogen biosynthesis. Importantly, hor- monal imbalances related to ovarian factors or to systemic hormonal dysregulation is a frequent cause of POI as well as polycystic ovary syndrome (PCOS). PCOS is the most common form of female infertility, affecting about 10% of women of reproductive age, and is associated with poor conception rates and high (30–50%) pregnancy loss rates. Mul- tiple studies have established a strong correlation between enlarged polycystic ovaries and obesity, hirsutism, and amenorrhea. Hyper- plasia of theca and stromal cells, elevated testosterone and LH, and low estradiol and FSH levels have been observed in women with hirsutism and oligomenorrhea [ 137–140]. Genomic changes in the FSHR, BMP15, and LHCGR genes as well as noncoding RNAs have been proposed to disturb a fine balance between LH and FSH hor- monal stimulation of the thecal and granulosa cell compartments, leading to excessive androstenedione and low estradiol concentra- tion in follicles. LHCGR is a receptor for both LH and chorionic gonadotropin. LHCGR variants have been described in patients with non- syndromic infertility, cystic ovaries, empty follicles, and primary and secondary amenorrhea [ 141, 142]. The LHCGR c.935G > A poly- morphism, promoter hypomethylation, and reduced expression of LHCGR were observed in women with PCOS; however, the molec- ular causes of PCOS remain to be elucidated. Genome wide association studies (GWAS) studies have been very helpful in identifying novel loci that play important roles in human reproduction. A GWAS among Han Chinese women with PCOS identified eight highly significant loci. Among these are three novel loci that contain LH–choriogonadotropin receptor ( LHCGR), thyroid adenoma-associated gene ( THADA), and differentially ex- pressed in normal and neoplastic cells (DENN)–MAPK activating death domain (MADD) containing 1A ( DENND1A). THADA and DENND1A loci associations have been replicated in some of the Eu- ropean cohorts [ 139]. Eight additional loci were identified in a fol- low up study [ 143]. Further functional studies are necessary to fully understand the role of these genes in human reproductive pathology. Other endocrine disturbances Endocrine disorders such as diabetes, hypothyroidism, conditions affecting the adrenal glands, or hypothalamus are frequently accompanied by delay or lack of development of secondary sexual characteristics, irregular menstrual cycles, or amenorrhea. Females affected by congenital endocrinopathies, such hyperprolactinemia (PRLR), combined pituitary hormone deficiency ( PROP1); autoim- mune polyendocrinopathy syndrome ( AIRE) frequently present with primary ovarian insufficiency and infertility (Table 2). It is also important to point out that pathogenic variants in DNA damage repair genes can affect endocrine function, and sometimes endocrine dysfunction can be the first presenting sign of a more serious condition such as Fanconi anemia [ 144]. Genetic causes of fertilization failure Ovulation in mammals is regulated by a cyclic secretion of LH. Eggs surrounded by the zona pellucida and somatic cumulus cells may be fertilized by a capacitated sperm. Sperm capacitation is a complex process, comprising structural and molecular changes to the acrosome, that happen to the spermatozoa residing within the female reproductive tract [ 145]. Female-specific factors that affect sperm capacitation and the sperm’s ability to recognize and penetrate through the cumulus cell layer and zona pellucida are unknown, but genetic defects in genes responsible for this process are likely to cause fertilization failure and infertility. These patients present with primary infertility despite normal ovarian reserve, regular menstrual cycles, and normal sex hormone levels. The zona pellucida, an extracellular matrix surrounding the de- veloping oocyte, contains four types of glycoprotein, ZP1, ZP2, ZP3, and ZP4. During oocyte development, the zona pellucida can be first observed in primary follicles as an external coat that physi- cally separates the oocyte from the surrounding granulosa cells. The zona pellucida plays an important role in protection of the growing oocyte, oocyte-follicle cell interactions, and sperm recognition and binding [146]. Right after fertilization, the zona pellucida undergoes changes which prevent polyspermy, support blastocyst development, and preclude premature implantation. Nucleotide sequence variations in the human zona pellucida genes may cause defects in the formation of zona pellucida and subsequent infertility. An autosomal recessive inheritance has been documented in women with loss-of-function ZP1 variants and ab- sence of the zona pellucida around their oocytes [ 147]a n di n women with pathogenic sequence variants in ZP2 and dysfunctional zona pellucida [ 148]. Empty follicle syndrome has been reported to cause infertility in women with heterozygous pathogenic variants in the ZP3 gene [ 149]. Oocyte retrieval after an IVF treatment cycle showed a highly disorganized follicle content with absent or degen- erated oocytes lacking zonae pellucidae. ZP3 expression is limited to oocytes; thus, the infertile phenotype is limited to females who have a de novo alterations or inherit pathogenic heterozygous variants from their fathers [ 148]. Digenic inheritance of two inherited vari- ants in the ZP2 and ZP3 genes can also result in oocyte maturation defects and infertility [ 150]. In humans, ZP2, ZP3, and ZP4 glycoproteins form long fila- ments connected by ZP1, providing an interactive oocyte-granulosa cell environment. In the absence of a functional zona pellucida, intercellular interaction between the developing oocyte and gran- ulosa cells is interrupted, resulting in oocyte degeneration prior to ovulation. During fertilization, the zona pellucida glycoproteins serve as sperm receptors, providing highly species-specific cell recognition and binding. To date, female-specific alterations leading to inability of sperm binding are unknown, but males with defects in zona pellucida binding proteins may produce dysmorphic sper- matozoa with incompetence or reduced ability to penetrate the zona Genetics female infertility, 2019, Vol. 101, No. 3 559 pellucida. Successful interaction between a single sperm and the zona pellucida as well as the activation of a blocking mechanism that prevents the fusion of additional sperm with the egg is critical for fertilization. Polyspermic fertilization, which leads to the formation of non-viable polyploid embryos, is prevented by the release of zinc ions and an enzyme ovastacin (MIM ∗ 608860) stored in egg cortical granules to immobilize egg-surrounding spermatozoa immediately after fertilization and to cleave the sperm-binding region within ZP2 [ 151]. Defects in genes implicated in the polyspermy blockage have yet to be discovered in humans. Maternal effect genes and post-fertilization preimplantation embryonic failure Identification of human genes that result in early embryonic lethality, before implantation, is challenging. Recent evidence from assisted reproductive technologies and embryo research suggests that idio- pathic infertility may be caused by very early forms of embryonic lethality. It is estimated that up to 70% of human embryos pro- duced in IVF are viable, while the remaining embryos arrest during cleavage-stage embryogenesis [ 152]. After fertilization, the zygotic genome is transcriptionally silent, and the first few cell divisions of the embryo depend on maternally derived cytoplasmic factors ac- cumulated during oocyte maturation. These maternal components include RNA, proteins, subcellular organelles and macromolecules that are encoded by maternal-effect genes [ 153–155]. Maternal factors play three major roles in preimplantation devel- opment: remodeling of condensed maternal and paternal genomes to form the diploid cell upon fertilization; gradual degradation of maternal RNA and proteins; and transcriptional activation of the embryonic genome. Failure in any of these biological processes leads to female infertility. Based on animal models, many genes are in- volved in early, post-fertilization cleavage events, including chro- matin remodeling, epigenetic reprogramming, embryo genome acti- vation, and cell specification. A few of these genes have been identified in humans very re- cently, in part due to application of whole exome sequencing to phenotypically well-characterized clinical cases. Women with recur- rent unsuccessful attempts of in vitro fertilization combined with intracytoplasmic sperm injection are diagnosed with primary female infertility as the majority of their embryos fail to develop. Biallelic al- terations in PADI6 [156], PATL2 [157, 158], TLE6 [159], NLRP2 [160], NLRP5 [160], and WEE2 [161], as well as heterozygous and homozygous pathogenic variants in TUBB8 [162, 163], were found to be responsible for a spectrum of early embryonic lethality phe- notypes, including oocyte maturation arrest, failure of fertilization, early embryonic arrest at the blastomere stage, and implantation failure [164]. The subcortical maternal complex, uniquely expressed in mammalian oocytes is composed of at least four proteins: OOEP, NLRP5, TLE6 ,a n d KHDC3L [165]. Maternal nucleotide variants in the TLE6 or NLRP5 gene have been linked to arrested embryo development on Day 3 and failure to form blastocysts [ 159, 160]. Pathogenic variants in KHDC3L are associated with recurrent hy- datidiform mole and have also been found in patients with embryos arrested at the morula stage [ 166]. Loss-of-function variants of PATL2 were reported in infertile females affected by oocyte germinal vesicle arrest and primary infertility [ 157, 158]. PATL2 encodes an RNA-binding protein that acts as a translational repressor and is highly expressed in human germinal vesicle and polar body MI oocytes. The exact role of PATL2 and mechanism leading to oocyte maturation arrest is unknown, although PATL2 deficiency may impact canonical translational repression and further hinder protein synthesis. Defects in WEE2 result in female infertility due to inability of the oocyte to exit metaphase II [ 161]. Oocytes from women with loss-of-function pathogenic variants in WEE2 fail to complete MII exit and form pronuclei, resulting in fertilization failure. The TUBB8 gene encodes a beta-tubulin subunit that is essential for oocyte meiotic spindle assembly. Oocytes of patients with pathogenic variants in TUBB8 show no spindle formation or disorganized spindle and commonly fail to extrude a polar body. In some instances, the first polar body can be extruded in the absence of a properly assembled spindle; however, those embryos still fail to progress beyond the 4- or 8-cell stage [167]. Post-implantation pathologies: hydatidiform moles Triploid human pregnancy, a conception that contains an additional set of chromosomes resulting in a total of 69 chromosomes in each cell, is an aberrant condition characterized by early embryonic ar- rest or first trimester miscarriage. Triploid embryos resulting from fertilization by two spermatozoa are detected in ∼1–2% of concep- tions [ 168] and are thought to be caused by inefficient polyspermy blockage by the zona pellucida. Triploidy may also occur from the fertilization of a diploid egg by a haploid sperm (digynic triploidy) or fertilization of a haploid egg by a diploid sperm (diandric triploidy or partial hydatidiform mole). Although rare, recurrent familial cases of triploidy due to a failure to extrude the polar body in meiosis suggest a genetic predisposition [ 169–171]. Such oocyte maturation defects may lead to an early miscarriage of an unrecognized preg- nancy and a diagnosis of primary infertility. Complete hydatidiform mole, a pregnancy formed by the fusion of two sperm or by dupli- cation of a paternal haploid genome in the absence of a maternal counterpart, can be caused by biallelic pathogenic variants in the MEI1, REC114,o r TOP6BL genes [172]. These genes are involved in double-strand DNA break formation, and defects can lead to ex- trusion of chromosomes from oocytes into the first polar body, cre- ating an “empty” egg. Women with such a meiotic abnormality may suffer infertility, recurrent miscarriage, complete hydatidiform mole with excessive trophoblastic proliferation, and failure of embryonic development. Another cause of a highly recurrent hydatidiform mole with a diploid (normal) set of chromosomes is autosomal recessive inheritance of pathogenic variants in the NLRP7 and KHDC3L genes [173–175]. Interestingly, hydatidiform moles in this condition are not androgenetic but have biparental genomic complements. In such condition, a normal haploid oocyte has been fertilized by a haploid spermatozoon; however, the maternal genome failed to establish a maternally-specific gene expression profile, resulting in trophoblastic proliferation and embryonic failure attributed to abnormal imprinting similar to a phenotype seen in androgenetic molar conceptuses. To date, NLRP7 biallelic sequence variants, genomic deletions, and complex rearrangements have been observed in affected females [ 173]. In addition, a heterozygous carrier of a pathogenic variant in the NLRP7 gene with a history of recurrent miscarriages and abnormal imprinting in two deceased children has been reported [ 176]. Although women who carry biallelic variants in these genes are healthy, they have a risk of reproductive failure and a possible impact on the fetal imprinting profile and 560 S.A. Yatsenko and A. Rajkovic, 2019, Vol. 101, No. 3 viability. Other genes involved in establishing a female-specific DNA methylation pattern in mature oocytes remain to be elucidated. Female infertility due to recurrent miscarriages and fetal lethality Recurrent miscarriages, defined as two or more consecutive preg- nancy losses, may be attributed to maternal, placental or fetal causes of infertility. It has been estimated that up to 60% of all concep- tions will abort spontaneously within the first 12 weeks of gesta- tion [66]. Beside fetal aneuploidy, which is responsible for 50–60% of overall fetal losses, many risk factors have been recognized to cause recurrent miscarriages, including anatomical uterine anoma- lies, polycystic ovary syndrome, antiphospholipid syndrome (up to 15%), endocrinological abnormalities ( ∼10%), unbalanced prod- ucts of parental balanced translocations (2–4%), X chromosome aberrations, thrombophilic conditions such as heterozygous vari- ants in the coagulation factor II gene ( F2) and coagulation factor Vg e n e( F5), placental insufficiency due to defects in the placental anticoagulant protein annexin A5 ( ANXA5), as well as other genetic conditions affecting fetal viability [177–180]. Numerous studies have investigated the role of polymorphisms in the methylenetetrahydro- folate reductase gene ( MTHFR) in the risk of recurrent miscarriages [181]; however, the associations are inconsistent, and MTHFR geno- typing has no clinical utility at the present. Whole exome and genome sequencing approaches may help in defining the monogenic and com- plex genetic causes of diminished fetal viability in couples with re- current miscarriages and stillborn children. Infertility due to developmental defects in female reproductive organs Genetic factors play a crucial role in organogenesis of the reproduc- tive system. Beside gonadal dysgenesis, malformation of fallopian tubes and uterus, uterine leiomyoma, or endometriosis, can lead to infertility, miscarriage, premature labor, or fetal death [ 123, 182]. Abnormalities in the female reproductive tract occur in up to 3.0% of live births in humans and include agenesis, atresia, and abnormal septation of the oviducts (fallopian tubes), uterus, cervix, or vagina, which embryologically arise from the M ¨ullerian duct. Heterozygous sequence variants, deletions, and expansion in the polyadenosine tail of the HOXA13 gene are well-known causes of hand-foot-genital syndrome characterized by fusion of M ¨ullerian structures and uter- ine malformations [ 183, 184]. Rare variants in other Homeobox A family genes ( HOXA10 and HOXA11)a sw e l la sa b e r r a n te x - pression of HOXA11-AS1 antisense RNA (a long noncoding RNA) have been identified in individuals with sporadic uterine malforma- tions and infertile women with endometriosis [ 185, 186]. Heterozy- gous pathogenic variants in HNF1B, LHX1, WNT4, WNT7A, and WNT9B have been reported in individuals with syndromic and vari- able uterine malformations, including Mayer-Rokitansky-K ¨uster- Hauser syndrome [ 187]. Less solid evidence links PAX2, PAX8 , and EMX2 genes with female reproductive tract malformations [188]. Concluding remarks and future studies Mammalian reproduction is regulated by a large number of genes that are essential for differentiation of highly specialized cells, com- plex intra- and intercellular signaling, and dynamic tissue-specific and organ-specific interactions among hypothalamus, pituitary, and the reproductive tract. Studies conducted on animal models have identified thousands of candidate genes and micro RNAs that might be involved in fertility [ 189]. Pathogenic alterations in a subset of these genes have been found to cause ovarian dysfunction and infer- tility in humans. With the advances of exome and genome sequencing and careful phenotyping, substantial progress has been made in the discovery of genes causing isolated and syndromic forms of primary ovarian insufficiency, preimplantation development [ 153, 190], and germline-limited alterations associated with lethal X-linked and au- tosomal phenotypes. It is evident that ovarian function depends on the level and timing of transcription of many genes and is controlled by non-coding regulatory DNA sequences [ 191–193], microRNAs [194–196], and small non-coding RNA molecules [ 197] that will be the focus of future studies. For many genes, ovarian dysgenesis and primary ovarian insuffi- ciency are the result of homozygous or biallelic pathogenic variants; however, little is known about the consequences of heterozygous pathogenic variants in these genes. Digenic and oligogenic inheri- tance in several affected patients points toward a possible polygenic cause of POI and infertility resulting from the cumulative effects of less harmful alterations among genes involved in a highly com- plex functional interplay. Heterozygous changes in key genes may confer susceptibility loci, while genomic changes in other loci push the effects of any damage above a “threshold.” Polygenic inheri- tance may explain developmental failure in embryos resulting from poor-quality oocytes related to female factors of infertility or incom- petence of an embryo or fetus to develop postfertilization due to a biparental polygenic burden. Additional studies, including whole genome sequencing and high-throughput functional assays will con- tinue to enlarge our understanding of the interplay between genomic and reproductive health. Furthermore, a possibility of in vitro ga- metogenesis from stem cells [ 198], may offer opportunities to edit pathogenic variants prior to fertilization, and restore fertility in af- fected individuals.

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Whole-exome sequencing reveals SALL4 variants in pre- mature ovarian insufficiency: an update on genotype-phenotype correla- tions. Hum Genet 2019; 138:83–92. 206. Smirin-Yosef P, Zuckerman-Levin N, Tzur S, Granot Y, Cohen L, Sach- senweger J, Borck G, Lagovsky I, Salmon-Divon M, Wiesm ¨uller L, Basel- Vanagaite L. A biallelic mutation in the homologous recombination re- pair gene SPIDR is associated with human gonadal dysgenesis. J Clin Endocrinol Metab 2017; 102:681–688. Aleksandar Rajkovic is Chief Genomics Of- ficer, Stuart Lindsay Distinguished Professor in Experimental Pathology I and the Direc- tor of the Genomic Medicine Initiative at the University of California, San Francisco. He investigates the genetic underpinnings of the formation and differentiation of gametes and reproductive tract, their role of these genes in human disease, embryo lethality and ori- gin of heritable human disorders. His labora- tory discovered numerous transcriptional regulators such as Sohlh1, Sohlh2, Lhx8,a n d Nobox that regulate gamete development and reproductive tract development. These transcription factors are nec- essary to drive oocyte growth, and synthesis of maternal effect genes that are essential for early embryogenesis and are likely involved in setting of epigenetic marks. Mutations in these oocyte-specific tran- scriptional regulators associate with human condition of premature ovarian insufficiency and infertility, emphasizing the importance of these pathways to women’s health. His laboratory also discovered that DNA damage repair genes such as MCM8 and MCM9 are mu- tated in women with infertility and the lab is exploring the link between DNA damage repair genes with infertility phenotypes and accelerated overall aging, as well as the effect of these genes on the overall health of offspring and genesis of structural birth defects. These and other studies indicate that many of the reproductive dis- orders are developmental in origin. Svetlana Yatsenko is the Associated Profes- sor at the Departments of Pathology, Obstet- rics, Gynecology & Reproductive Sciences, and Magee Womens Research Institute, Di- rector of Cytogenetics and Genomics Labora- tories, at the University of Pittsburgh Medical Center, Pittsburgh. Her main research inter- ests are centered on study of molecular bases and mechanisms of genomic alterations and complex chromosomal rearrangements, as well as gene dosage effect that lead to congenital disorders, birth defects, preimplantation de- velopment and prenatal phenotypes, fetal viability, malformations of reproductive system, gonadal dysgenesis and dysfunction, and disor- ders of sexual development. Her laboratory is also focused on study of alterations involving the X chromosome and their contribution to premature ovarian insufficiency, fetal lethality, sexual dimorphism and femalepredominant diseases.